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Herrmann D, Meng S, Yang H, Mansky LM, Saad JS. The Assembly of HTLV-1-How Does It Differ from HIV-1? Viruses 2024; 16:1528. [PMID: 39459862 PMCID: PMC11512237 DOI: 10.3390/v16101528] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2024] [Revised: 09/21/2024] [Accepted: 09/25/2024] [Indexed: 10/28/2024] Open
Abstract
Retroviral assembly is a highly coordinated step in the replication cycle. The process is initiated when the newly synthesized Gag and Gag-Pol polyproteins are directed to the inner leaflet of the plasma membrane (PM), where they facilitate the budding and release of immature viral particles. Extensive research over the years has provided crucial insights into the molecular determinants of this assembly step. It is established that Gag targeting and binding to the PM is mediated by interactions of the matrix (MA) domain and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). This binding event, along with binding to viral RNA, initiates oligomerization of Gag on the PM, a process mediated by the capsid (CA) domain. Much of the previous studies have focused on human immunodeficiency virus type 1 (HIV-1). Although the general steps of retroviral replication are consistent across different retroviruses, comparative studies revealed notable differences in the structure and function of viral components. In this review, we present recent findings on the assembly mechanisms of Human T-cell leukemia virus type 1 and highlight key differences from HIV-1, focusing particularly on the molecular determinants of Gag-PM interactions and CA assembly.
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Affiliation(s)
- Dominik Herrmann
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA;
| | - Shuyu Meng
- Institute for Molecular Virology, University of Minnesota–Twin Cities, Minneapolis, MN 55455, USA; (S.M.); (H.Y.); (L.M.M.)
- Molecular Pharmacology and Therapeutics Graduate Program, University of Minnesota–Twin Cities, Minneapolis, MN 55455, USA
| | - Huixin Yang
- Institute for Molecular Virology, University of Minnesota–Twin Cities, Minneapolis, MN 55455, USA; (S.M.); (H.Y.); (L.M.M.)
| | - Louis M. Mansky
- Institute for Molecular Virology, University of Minnesota–Twin Cities, Minneapolis, MN 55455, USA; (S.M.); (H.Y.); (L.M.M.)
- Molecular Pharmacology and Therapeutics Graduate Program, University of Minnesota–Twin Cities, Minneapolis, MN 55455, USA
- Department of Diagnostic and Biological Sciences, University of Minnesota–Twin Cities, Minneapolis, MN 55455, USA
- Masonic Cancer Center, University of Minnesota–Twin Cities, Minneapolis, MN 55455, USA
- Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota–Twin Cities, Minneapolis, MN 55455, USA
| | - Jamil S. Saad
- Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA;
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2
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Li M, Yang R, Chen X, Wang H, Ghirlando R, Dimitriadis EK, Craigie R. HIV-1 Integrase Assembles Multiple Species of Stable Synaptic Complex Intasomes That Are Active for Concerted DNA Integration In vitro. J Mol Biol 2024; 436:168557. [PMID: 38582148 PMCID: PMC11134455 DOI: 10.1016/j.jmb.2024.168557] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/01/2024] [Revised: 03/28/2024] [Accepted: 03/28/2024] [Indexed: 04/08/2024]
Abstract
Retroviral DNA integration is mediated by nucleoprotein complexes (intasomes) in which a pair of viral DNA ends are bridged by a multimer of integrase (IN). Most of the high-resolution structures of HIV-1 intasomes are based on an HIV-1 IN with an Sso7d protein domain fused to the N-terminus. Sso7d-IN aggregates much less than wild-type IN and has been critical for structural studies of HIV-1 intasomes. Unexpectedly, these structures revealed that the common core architecture that mediates catalysis could be assembled in various ways, giving rise to both tetrameric and dodecameric intasomes, together with other less well-characterized species. This differs from related retroviruses that assemble unique multimeric intasomes, although the number of protomers in the intasome varies between viruses. The question of whether the additional Sso7d domain contributes to the heterogeneity of HIV-1 intasomes is therefore raised. We have addressed this by biochemical and structural studies of intasomes assembled with wild-type HIV-1 IN. Negative stain and cryo-EM reveal a similar range of multimeric intasome species as with Sso7d-IN with the same common core architecture. Stacks of intasomes resulting from domain swapping are also seen with both wild-type and Sso7d-IN intasomes. The propensity to assemble multimeric intasome species is, therefore, an intrinsic property of HIV-1 IN and is not conferred by the presence of the Sso7d domain. The recently solved intasome structures of different retroviral species, which have been reported to be tetrameric, octameric, dodecameric, and hexadecameric, highlight how a common intasome core architecture can be assembled in different ways for catalysis.
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Affiliation(s)
- Min Li
- Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA.
| | - Renbin Yang
- Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA
| | - Xuemin Chen
- Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA
| | - Huaibin Wang
- Laboratory of Cell and Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA
| | - Rodolfo Ghirlando
- Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA
| | - Emilios K Dimitriadis
- Laboratory of Bioengineering and Physical Science, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD 20892, USA
| | - Robert Craigie
- Laboratory of Molecular Biology, NIDDK, National Institutes of Health, Bethesda, MD 20892, USA.
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3
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Grandgenett DP, Engelman AN. Brief Histories of Retroviral Integration Research and Associated International Conferences. Viruses 2024; 16:604. [PMID: 38675945 PMCID: PMC11054761 DOI: 10.3390/v16040604] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2024] [Revised: 04/05/2024] [Accepted: 04/10/2024] [Indexed: 04/28/2024] Open
Abstract
The field of retroviral integration research has a long history that started with the provirus hypothesis and subsequent discoveries of the retroviral reverse transcriptase and integrase enzymes. Because both enzymes are essential for retroviral replication, they became valued targets in the effort to discover effective compounds to inhibit HIV-1 replication. In 2007, the first integrase strand transfer inhibitor was licensed for clinical use, and subsequently approved second-generation integrase inhibitors are now commonly co-formulated with reverse transcriptase inhibitors to treat people living with HIV. International meetings specifically focused on integrase and retroviral integration research first convened in 1995, and this paper is part of the Viruses Special Issue on the 7th International Conference on Retroviral Integration, which was held in Boulder Colorado in the summer of 2023. Herein, we overview key historical developments in the field, especially as they pertain to the development of the strand transfer inhibitor drug class. Starting from the mid-1990s, research advancements are presented through the lens of the international conferences. Our overview highlights the impact that regularly scheduled, subject-specific international meetings can have on community-building and, as a result, on field-specific collaborations and scientific advancements.
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Affiliation(s)
- Duane P. Grandgenett
- Department of Molecular Microbiology and Immunology, School of Medicine, Saint Louis University, St. Louis, MO 63104, USA
| | - Alan N. Engelman
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
- Department of Medicine, Harvard Medical School, Boston, MA 02115, USA
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4
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Renzi G, Carta F, Supuran CT. The Integrase: An Overview of a Key Player Enzyme in the Antiviral Scenario. Int J Mol Sci 2023; 24:12187. [PMID: 37569561 PMCID: PMC10419282 DOI: 10.3390/ijms241512187] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2023] [Revised: 07/23/2023] [Accepted: 07/26/2023] [Indexed: 08/13/2023] Open
Abstract
Integration of a desossiribonucleic acid (DNA) copy of the viral ribonucleic acid (RNA) into host genomes is a fundamental step in the replication cycle of all retroviruses. The highly conserved virus-encoded Integrase enzyme (IN; EC 2.7.7.49) catalyzes such a process by means of two consecutive reactions named 3'-processing (3-P) and strand transfer (ST). The Authors report and discuss the major discoveries and advances which mainly contributed to the development of Human Immunodeficiency Virus (HIV) -IN targeted inhibitors for therapeutic applications. All the knowledge accumulated over the years continues to serve as a valuable resource for the design and development of effective antiretroviral drugs.
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Affiliation(s)
| | - Fabrizio Carta
- Neuroscienze, Psicologia, Area del Farmaco e Salute del Bambino (NEUROFARBA) Department, Sezione di Scienze Farmaceutiche e Nutraceutiche, University of Florence, Via Ugo Schiff 6, Sesto Fiorentino, 50019 Florence, Italy; (G.R.); (C.T.S.)
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5
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Lannes L, Furman CM, Hickman AB, Dyda F. Zinc-finger BED domains drive the formation of the active Hermes transpososome by asymmetric DNA binding. Nat Commun 2023; 14:4470. [PMID: 37491363 PMCID: PMC10368747 DOI: 10.1038/s41467-023-40210-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/17/2022] [Accepted: 07/18/2023] [Indexed: 07/27/2023] Open
Abstract
The Hermes DNA transposon is a member of the eukaryotic hAT superfamily, and its transposase forms a ring-shaped tetramer of dimers. Our investigation, combining biochemical, crystallography and cryo-electron microscopy, and in-cell assays, shows that the full-length Hermes octamer extensively interacts with its transposon left-end through multiple BED domains of three Hermes protomers contributed by three dimers explaining the role of the unusual higher-order assembly. By contrast, the right-end is bound to no BED domains at all. Thus, this work supports a model in which Hermes multimerizes to gather enough BED domains to find its left-end among the abundant genomic DNA, facilitating the subsequent interaction with the right-end.
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Affiliation(s)
- Laurie Lannes
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Christopher M Furman
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Alison B Hickman
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892, USA
| | - Fred Dyda
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892, USA.
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6
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Richetta C, Tu NQ, Delelis O. Different Pathways Conferring Integrase Strand-Transfer Inhibitors Resistance. Viruses 2022; 14:v14122591. [PMID: 36560595 PMCID: PMC9785060 DOI: 10.3390/v14122591] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2022] [Revised: 11/17/2022] [Accepted: 11/19/2022] [Indexed: 11/23/2022] Open
Abstract
Integrase Strand Transfer Inhibitors (INSTIs) are currently used as the most effective therapy in the treatment of human immunodeficiency virus (HIV) infections. Raltegravir (RAL) and Elvitegravir (EVG), the first generation of INSTIs used successfully in clinical treatment, are susceptible to the emergence of viral resistance and have a high rate of cross-resistance. To counteract these resistant mutants, second-generation INSTI drugs have been developed: Dolutegravir (DTG), Cabotegravir (CAB), and Bictegravir (BIC). However, HIV is also able to develop resistance mechanisms against the second-generation of INSTIs. This review describes the mode of action of INSTIs and then summarizes and evaluates some typical resistance mutations, such as substitution and insertion mutations. The role of unintegrated viral DNA is also discussed as a new pathway involved in conferring resistance to INSTIs. This allows us to have a more detailed understanding of HIV resistance to these inhibitors, which may contribute to the development of new INSTIs in the future.
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Bhanja SK, Rath PK, Goel A, Mehra M, Dhara SK, Paswan VK, Attia YA, Alqhtani AH, Ali ABA, Shehata AM. In ovo nano-silver and nutrient supplementation improves immunity and resistance against Newcastle disease virus challenge in broiler chickens. Front Vet Sci 2022; 9:948069. [PMID: 36187823 PMCID: PMC9523696 DOI: 10.3389/fvets.2022.948069] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2022] [Accepted: 08/22/2022] [Indexed: 11/13/2022] Open
Abstract
Silver nanoparticles (AgNPs) interact with the microbes and host immune system to protect against diseases. Fertile broiler eggs (n = 900) were allotted to six groups: un-injected control, sham (sterile water), AgNPs (50 μg), AgNPs+Amino acids (Methionine-10 mg + Arginine-25 mg), AgNPs+Vitamins (Vit B1-72μg + Vit B6-140μg), and AgNPs+Trace Elements (Zn-80 μg and Se-0.3 μg) and incubated for 18 days. On 18th embryonic day, 0.6 ml test solution was injected at the broad end of egg using 25 mm needle and transferred to hatcher. Post-hatch, half of the chicks from each group were vaccinated with Newcastle disease (ND) vaccine, and the other half were kept as unvaccinated unit and reared for 42 d with standard management practices. Hatchability, 1st and 42nd d body weight, feed intake, and feed conversion ratio were similar between treatment groups in both vaccinated and unvaccinated units. The relative weight of bursa Fabricius and thymus was similar, but spleen weight was higher (P ≤ 0.05) in AgNPs, AgNPs+Vits, and AgNPs+TEs chicks than control group. Cellular immune response (against mitogen phytohemagglutinin-P) was higher (P ≤ 0.05) in AgNPs+TEs chicks, whereas HA titer against sheep red blood cells antigen, serum IgG, IgM, and HI titer against ND vaccine was apparently higher in AgNPs+Vits group chicks than control. No clinical symptoms were observed in the vaccinated groups except for a few control birds 6 days postchallenge (PC). Three days PC, unvaccinated birds show depression, off feed, greenish diarrhea, and nasal discharge and the control group started dying. The highest cumulative infection (CI) was observed in sham (79.17%) and un-injected control (75%), but lowest in AgNPs+AAs birds (58.33%) on 3rd dpi. The CI reached 100% on 5th dpi in control groups and AgNPs, and 91.67% and 93.75% in AgNPs+TEs and AgNPs+AAs group, respectively. The AgNPs+TEs and AgNPs+AAs group birds lived for more than 90 h compared to 75 h in control groups and also had higher IL-6 and IL-2 gene expressions at 24 h PC. It was concluded that 50 μg/egg AgNPs with vitamins (B1 and B6) and trace elements (Zn and Se) improved performance, but AgNPs with trace elements and amino acids enhanced immune response and resistance against vND virus challenge in broilers.
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Affiliation(s)
- Subrat Kumar Bhanja
- ICAR-Central Avian Research Institute, Bareilly, UP, India
- *Correspondence: Subrat Kumar Bhanja
| | | | - Akshat Goel
- ICAR-Central Avian Research Institute, Bareilly, UP, India
| | - Manish Mehra
- ICAR-Central Avian Research Institute, Bareilly, UP, India
| | - Sujoy K. Dhara
- ICAR-Indian Veterinary Research Institute, Bareilly, UP, India
| | - Vinod K. Paswan
- Department of Dairy Science and Food Technology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, India
| | - Youssef A. Attia
- Department of Animal and Poultry Production, Faculty of Agriculture, Damanhour University, Damanhour, Egypt
| | - Abdulmohsen Hussen Alqhtani
- Animal Production Department, Food and Agriculture Sciences College, King Saud University, Riyadh, Saudi Arabia
| | - Ahmed B. A. Ali
- Department of Animal and Veterinary Science, Clemson University, Clemson, SC, United States
| | - Abdelrazeq M. Shehata
- Department of Animal Production, Faculty of Agriculture, Al-Azhar University, Cairo, Egypt
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8
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Jóźwik IK, Li W, Zhang DW, Wong D, Grawenhoff J, Ballandras-Colas A, Aiyer S, Cherepanov P, Engelman A, Lyumkis D. B-to-A transition in target DNA during retroviral integration. Nucleic Acids Res 2022; 50:8898-8918. [PMID: 35947647 PMCID: PMC9410886 DOI: 10.1093/nar/gkac644] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2022] [Revised: 07/06/2022] [Accepted: 07/19/2022] [Indexed: 01/21/2023] Open
Abstract
Integration into host target DNA (tDNA), a hallmark of retroviral replication, is mediated by the intasome, a multimer of integrase (IN) assembled on viral DNA (vDNA) ends. To ascertain aspects of tDNA recognition during integration, we have solved the 3.5 Å resolution cryo-EM structure of the mouse mammary tumor virus (MMTV) strand transfer complex (STC) intasome. The tDNA adopts an A-like conformation in the region encompassing the sites of vDNA joining, which exposes the sugar-phosphate backbone for IN-mediated strand transfer. Examination of existing retroviral STC structures revealed conservation of A-form tDNA in the analogous regions of these complexes. Furthermore, analyses of sequence preferences in genomic integration sites selectively targeted by six different retroviruses highlighted consistent propensity for A-philic sequences at the sites of vDNA joining. Our structure additionally revealed several novel MMTV IN-DNA interactions, as well as contacts seen in prior STC structures, including conserved Pro125 and Tyr149 residues interacting with tDNA. In infected cells, Pro125 substitutions impacted the global pattern of MMTV integration without significantly altering local base sequence preferences at vDNA insertion sites. Collectively, these data advance our understanding of retroviral intasome structure and function, as well as factors that influence patterns of vDNA integration in genomic DNA.
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Affiliation(s)
- Ilona K Jóźwik
- The Salk Institute for Biological Studies, La Jolla, CA 92037, USA
| | - Wen Li
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Center, Boston, MA 02215, USA,Department of Medicine, Harvard Medical School, Boston, MA 02115, USA
| | - Da-Wei Zhang
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Center, Boston, MA 02215, USA,Department of Medicine, Harvard Medical School, Boston, MA 02115, USA,Institute of Bioinformatics and Medical Engineering, School of Electrical and Information Engineering, Jiangsu University of Technology, Changzhou 213001, China
| | - Doris Wong
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Center, Boston, MA 02215, USA
| | - Julia Grawenhoff
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Center, Boston, MA 02215, USA
| | | | - Sriram Aiyer
- The Salk Institute for Biological Studies, La Jolla, CA 92037, USA
| | - Peter Cherepanov
- Chromatin Structure and Mobile DNA Laboratory, The Francis Crick Institute, London NW1 1AT, UK,Department of Infectious Disease, St-Mary's Campus, Imperial College London, London W2 1PG, UK
| | - Alan N Engelman
- Correspondence may also be addressed to Alan N. Engelman. Tel: +1 617 632 4361; Fax: +1 617 632 4338;
| | - Dmitry Lyumkis
- To whom correspondence should be addressed. Tel: +1 858 453 4100 (Ext 1155);
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9
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Engelman AN, Kvaratskhelia M. Multimodal Functionalities of HIV-1 Integrase. Viruses 2022; 14:926. [PMID: 35632668 PMCID: PMC9144474 DOI: 10.3390/v14050926] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2022] [Revised: 04/20/2022] [Accepted: 04/26/2022] [Indexed: 01/11/2023] Open
Abstract
Integrase is the retroviral protein responsible for integrating reverse transcripts into cellular genomes. Co-packaged with viral RNA and reverse transcriptase into capsid-encased viral cores, human immunodeficiency virus 1 (HIV-1) integrase has long been implicated in reverse transcription and virion maturation. However, the underlying mechanisms of integrase in these non-catalytic-related viral replication steps have remained elusive. Recent results have shown that integrase binds genomic RNA in virions, and that mutational or pharmacological disruption of integrase-RNA binding yields eccentric virion particles with ribonucleoprotein complexes situated outside of the capsid shell. Such viruses are defective for reverse transcription due to preferential loss of integrase and viral RNA from infected target cells. Parallel research has revealed defective integrase-RNA binding and eccentric particle formation as common features of class II integrase mutant viruses, a phenotypic grouping of viruses that display defects at steps beyond integration. In light of these new findings, we propose three new subclasses of class II mutant viruses (a, b, and c), all of which are defective for integrase-RNA binding and particle morphogenesis, but differ based on distinct underlying mechanisms exhibited by the associated integrase mutant proteins. We also assess how these findings inform the role of integrase in HIV-1 particle maturation.
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Affiliation(s)
- Alan N. Engelman
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA
- Department of Medicine, Harvard Medical School, Boston, MA 02115, USA
| | - Mamuka Kvaratskhelia
- Division of Infectious Diseases, Anschutz Medical Campus, University of Colorado School of Medicine, Aurora, CO 80045, USA
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10
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Passos DO, Li M, Craigie R, Lyumkis D. Retroviral integrase: Structure, mechanism, and inhibition. Enzymes 2021; 50:249-300. [PMID: 34861940 DOI: 10.1016/bs.enz.2021.06.007] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Abstract
The retroviral protein Integrase (IN) catalyzes concerted integration of viral DNA into host chromatin to establish a permanent infection in the target cell. We learned a great deal about the mechanism of catalytic integration through structure/function studies over the previous four decades of IN research. As one of three essential retroviral enzymes, IN has also been targeted by antiretroviral drugs to treat HIV-infected individuals. Inhibitors blocking the catalytic integration reaction are now state-of-the-art drugs within the antiretroviral therapy toolkit. HIV-1 IN also performs intriguing non-catalytic functions that are relevant to the late stages of the viral replication cycle, yet this aspect remains poorly understood. There are also novel allosteric inhibitors targeting non-enzymatic functions of IN that induce a block in the late stages of the viral replication cycle. In this chapter, we will discuss the function, structure, and inhibition of retroviral IN proteins, highlighting remaining challenges and outstanding questions.
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Affiliation(s)
| | - Min Li
- National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, United States
| | - Robert Craigie
- National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, United States
| | - Dmitry Lyumkis
- The Salk Institute for Biological Studies, La Jolla, CA, United States; The Scripps Research Institute, La Jolla, CA, United States.
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Studies towards the Design and Synthesis of Novel 1,5-Diaryl-1 H-imidazole-4-carboxylic Acids and 1,5-Diaryl-1 H-imidazole-4-carbohydrazides as Host LEDGF/p75 and HIV-1 Integrase Interaction Inhibitors. Molecules 2021; 26:molecules26206203. [PMID: 34684786 PMCID: PMC8540437 DOI: 10.3390/molecules26206203] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2021] [Revised: 10/07/2021] [Accepted: 10/11/2021] [Indexed: 11/23/2022] Open
Abstract
Two targeted sets of novel 1,5-diaryl-1H-imidazole-4-carboxylic acids 10 and carbohydrazides 11 were designed and synthesized from their corresponding ester intermediates 17, which were prepared via cycloaddition of ethyl isocyanoacetate 16 and diarylimidoyl chlorides 15. Evaluation of these new target scaffolds in the AlphaScreenTM HIV-1 IN-LEDGF/p75 inhibition assay identified seventeen compounds exceeding the pre-defined 50% inhibitory threshold at 100 µM concentration. Further evaluation of these compounds in the HIV-1 IN strand transfer assay at 100 μM showed that none of the compounds (with the exception of 10a, 10l, and 11k, with marginal inhibitory percentages) were actively bound to the active site, indicating that they are selectively binding to the LEDGF/p75-binding pocket. In a cell-based HIV-1 antiviral assay, compounds 11a, 11b, 11g, and 11h exhibited moderate antiviral percentage inhibition of 33–45% with cytotoxicity (CC50) values of >200 µM, 158.4 µM, >200 µM, and 50.4 µM, respectively. The antiviral inhibitory activity displayed by 11h was attributed to its toxicity. Upon further validation of their ability to induce multimerization in a Western blot gel assay, compounds 11a, 11b, and 11h appeared to increase higher-order forms of IN.
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12
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Kumar D, Sharma P, Shabu, Kaur R, Lobe MMM, Gupta GK, Ntie-Kang F. In search of therapeutic candidates for HIV/AIDS: rational approaches, design strategies, structure-activity relationship and mechanistic insights. RSC Adv 2021; 11:17936-17964. [PMID: 35480193 PMCID: PMC9033207 DOI: 10.1039/d0ra10655k] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2020] [Accepted: 04/19/2021] [Indexed: 12/23/2022] Open
Abstract
The HIV/AIDS pandemic is a serious threat to the health and development of mankind, which has affected about 37.9 million people worldwide. The increasing negative health, economic and social impacts of this disease have led to the search for new therapeutic candidates for the mitigation of AIDS/HIV. However, to date, there is still no treatment that can cure this disease. Furthermore, the clinically available drugs have numerous severe side effects. Hence, the synthesis of novel agents from natural leads is one of the rational approaches to obtain new drugs in modern medicinal chemistry. This review article is an effort to summarize recent developments with regards to the discovery of novel analogs with promising biological potential against HIV/AIDS. Herein, we also aim to discuss prospective directions on the progress of more credible and specific analogues. Besides presenting design strategies, the present communication also highlights the structure-activity relationship together with the structural features of the most promising molecules, their IC50 values, mechanistic insights and some interesting key findings revealed during their biological evaluation. The interactions with the amino acid residues of the enzymes responsible for HIV-1 inhibition are also discussed. This collection will be of great interest for researchers working in this area.
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Affiliation(s)
- Dinesh Kumar
- Sri Sai College of Pharmacy Manawala Amritsar-143001 Punjab India +91-9988902489
| | - Pooja Sharma
- Sri Sai College of Pharmacy Manawala Amritsar-143001 Punjab India +91-9988902489
- Department of Pharmaceutical Sciences and Drug Research, Punjabi University Patiala India
| | - Shabu
- Indian Institute of Integrative Medicine (CSIR-IIIM) Canal Road Jammu 180001 India
| | - Ramandeep Kaur
- Sri Sai College of Pharmacy Manawala Amritsar-143001 Punjab India +91-9988902489
| | - Maloba M M Lobe
- Department of Chemistry, Faculty of Science, University of Buea P. O. Box 63 Buea Cameroon +237 685625811
| | - Girish K Gupta
- Department of Pharmaceutical Chemistry, Sri Sai College of Pharmacy Badhani Pathankot-145001 Punjab India
| | - Fidele Ntie-Kang
- Department of Chemistry, Faculty of Science, University of Buea P. O. Box 63 Buea Cameroon +237 685625811
- Institute for Pharmacy, Martin-Luther-Universität Halle-Wittenberg Kurt-Mothes-Str. 3 06120 Halle (Saale) Germany +49 3455525043
- Institute of Botany, Technical University of Dresden Zellescher Weg 20b 01062 Dresden Germany
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13
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Engelman AN. HIV Capsid and Integration Targeting. Viruses 2021; 13:125. [PMID: 33477441 PMCID: PMC7830116 DOI: 10.3390/v13010125] [Citation(s) in RCA: 25] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2020] [Revised: 01/13/2021] [Accepted: 01/15/2021] [Indexed: 12/20/2022] Open
Abstract
Integration of retroviral reverse transcripts into the chromosomes of the cells that they infect is required for efficient viral gene expression and the inheritance of viral genomes to daughter cells. Before integration can occur, retroviral reverse transcription complexes (RTCs) must access the nuclear environment where the chromosomes reside. Retroviral integration is non-random, with different types of virus-host interactions impacting where in the host chromatin integration takes place. Lentiviruses such as HIV efficiently infect interphase cells because their RTCs have evolved to usurp cellular nuclear import transport mechanisms, and research over the past decade has revealed specific interactions between the HIV capsid protein and nucleoporin (Nup) proteins such as Nup358 and Nup153. The interaction of HIV capsid with cleavage and polyadenylation specificity factor 6 (CPSF6), which is a component of the cellular cleavage and polyadenylation complex, helps to dictate nuclear import as well as post-nuclear RTC invasion. In the absence of the capsid-CPSF6 interaction, RTCs are precluded from reaching nuclear speckles and gene-rich regions of chromatin known as speckle-associated domains, and instead mis-target lamina-associated domains out at the nuclear periphery. Highlighting this area of research, small molecules that inhibit capsid-host interactions important for integration site targeting are highly potent antiviral compounds.
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Affiliation(s)
- Alan N. Engelman
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; ; Tel.: +1-617-632-4361
- Department of Medicine, Harvard Medical School, Boston, MA 02115, USA
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14
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Kim J, Lee GE, Shin CG. Foamy Virus Integrase in Development of Viral Vector for Gene Therapy. J Microbiol Biotechnol 2020; 30:1273-1281. [PMID: 32699199 PMCID: PMC9728412 DOI: 10.4014/jmb.2003.03046] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/23/2020] [Revised: 06/29/2020] [Accepted: 07/14/2020] [Indexed: 12/15/2022]
Abstract
Due to the broad host suitability of viral vectors and their high gene delivery capacity, many researchers are focusing on viral vector-mediated gene therapy. Among the retroviruses, foamy viruses have been considered potential gene therapy vectors because of their non-pathogenicity. To date, the prototype foamy virus is the only retrovirus that has a high-resolution structure of intasomes, nucleoprotein complexes formed by integrase, and viral DNA. The integration of viral DNA into the host chromosome is an essential step for viral vector development. This process is mediated by virally encoded integrase, which catalyzes unique chemical reactions. Additionally, recent studies on foamy virus integrase elucidated the catalytic functions of its three distinct domains and their effect on viral pathogenicity. This review focuses on recent advancements in biochemical, structural, and functional studies of foamy virus integrase for gene therapy vector research.
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Affiliation(s)
- Jinsun Kim
- Department of Systems Biotechnology, Chung-Ang University, Anseong 17546, Republic of Korea
| | - Ga-Eun Lee
- Department of Systems Biotechnology, Chung-Ang University, Anseong 17546, Republic of Korea
| | - Cha-Gyun Shin
- Department of Systems Biotechnology, Chung-Ang University, Anseong 17546, Republic of Korea,Corresponding author Phone: +82-31-670-3067 Fax: +82-31-675-3108 E-mail:
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15
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Elliott JL, Kutluay SB. Going beyond Integration: The Emerging Role of HIV-1 Integrase in Virion Morphogenesis. Viruses 2020; 12:E1005. [PMID: 32916894 PMCID: PMC7551943 DOI: 10.3390/v12091005] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/26/2020] [Revised: 09/03/2020] [Accepted: 09/07/2020] [Indexed: 12/22/2022] Open
Abstract
The HIV-1 integrase enzyme (IN) plays a critical role in the viral life cycle by integrating the reverse-transcribed viral DNA into the host chromosome. This function of IN has been well studied, and the knowledge gained has informed the design of small molecule inhibitors that now form key components of antiretroviral therapy regimens. Recent discoveries unveiled that IN has an under-studied yet equally vital second function in human immunodeficiency virus type 1 (HIV-1) replication. This involves IN binding to the viral RNA genome in virions, which is necessary for proper virion maturation and morphogenesis. Inhibition of IN binding to the viral RNA genome results in mislocalization of the viral genome inside the virus particle, and its premature exposure and degradation in target cells. The roles of IN in integration and virion morphogenesis share a number of common elements, including interaction with viral nucleic acids and assembly of higher-order IN multimers. Herein we describe these two functions of IN within the context of the HIV-1 life cycle, how IN binding to the viral genome is coordinated by the major structural protein, Gag, and discuss the value of targeting the second role of IN in virion morphogenesis.
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Affiliation(s)
| | - Sebla B. Kutluay
- Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, MO 63110, USA;
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16
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Abstract
Retroviral integration, the process of covalently inserting viral DNA into the host genome, is a point of no return in the replication cycle. Yet, strand transfer is intrinsically iso-energetic and it is not clear how efficient integration can be achieved. Here we investigate the dynamics of strand transfer and demonstrate that consecutive nucleoprotein intermediates interacting with a supercoiled target are increasingly stable, resulting in a net forward rate. Multivalent target interactions at discrete auxiliary interfaces render target capture irreversible, while allowing dynamic site selection. Active site binding is transient but rapidly results in strand transfer, which in turn rearranges and stabilizes the intasome in an allosteric manner. We find the resulting strand transfer complex to be mechanically stable and extremely long-lived, suggesting that a resolving agent is required in vivo.
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17
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Wilson MD, Renault L, Maskell DP, Ghoneim M, Pye VE, Nans A, Rueda DS, Cherepanov P, Costa A. Retroviral integration into nucleosomes through DNA looping and sliding along the histone octamer. Nat Commun 2019; 10:4189. [PMID: 31519882 PMCID: PMC6744463 DOI: 10.1038/s41467-019-12007-w] [Citation(s) in RCA: 28] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2019] [Accepted: 08/08/2019] [Indexed: 01/02/2023] Open
Abstract
Retroviral integrase can efficiently utilise nucleosomes for insertion of the reverse-transcribed viral DNA. In face of the structural constraints imposed by the nucleosomal structure, integrase gains access to the scissile phosphodiester bonds by lifting DNA off the histone octamer at the site of integration. To clarify the mechanism of DNA looping by integrase, we determined a 3.9 Å resolution structure of the prototype foamy virus intasome engaged with a nucleosome core particle. The structural data along with complementary single-molecule Förster resonance energy transfer measurements reveal twisting and sliding of the nucleosomal DNA arm proximal to the integration site. Sliding the nucleosomal DNA by approximately two base pairs along the histone octamer accommodates the necessary DNA lifting from the histone H2A-H2B subunits to allow engagement with the intasome. Thus, retroviral integration into nucleosomes involves the looping-and-sliding mechanism for nucleosomal DNA repositioning, bearing unexpected similarities to chromatin remodelers. Retroviral integrases catalyze the insertion of viral DNA into the host cell DNA and can use nucleosomes as substrates for integration. Here the authors present the 3.9 Å cryo-EM structure of prototype foamy virus integrase after strand transfer into nucleosomal DNA, which together with single-molecule FRET measurements provides evidence for a DNA looping and sliding mechanism of integrases.
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Affiliation(s)
- Marcus D Wilson
- Macromolecular Machines Laboratory, The Francis Crick Institute, NW1 1AT, London, UK.,Wellcome Centre for Cell Biology, University of Edinburgh, Edinburgh, EH9 3JR, UK
| | - Ludovic Renault
- Macromolecular Machines Laboratory, The Francis Crick Institute, NW1 1AT, London, UK.,NeCEN, University of Leiden, 2333CC, Leiden, Netherlands
| | - Daniel P Maskell
- Chromatin structure and mobile DNA Laboratory, The Francis Crick Institute, London, NW1 1AT, UK.,Faculty of Biological Sciences, Leeds, LS2 9JT, UK
| | - Mohamed Ghoneim
- Single Molecule Imaging Group, MRC London Institute for Medical Science, London, W12 0NN, UK.,Molecular Virology, Department of Medicine, Imperial College London, London, W12 0NN, UK
| | - Valerie E Pye
- Chromatin structure and mobile DNA Laboratory, The Francis Crick Institute, London, NW1 1AT, UK
| | - Andrea Nans
- Structural Biology Science Technology Platform, The Francis Crick Institute, London, NW1 1AT, UK
| | - David S Rueda
- Single Molecule Imaging Group, MRC London Institute for Medical Science, London, W12 0NN, UK. .,Molecular Virology, Department of Medicine, Imperial College London, London, W12 0NN, UK.
| | - Peter Cherepanov
- Chromatin structure and mobile DNA Laboratory, The Francis Crick Institute, London, NW1 1AT, UK. .,Department of Medicine, Imperial College London, St-Mary's Campus, Norfolk Place, London, W2 1PG, UK.
| | - Alessandro Costa
- Macromolecular Machines Laboratory, The Francis Crick Institute, NW1 1AT, London, UK.
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18
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Engelman AN. Multifaceted HIV integrase functionalities and therapeutic strategies for their inhibition. J Biol Chem 2019; 294:15137-15157. [PMID: 31467082 DOI: 10.1074/jbc.rev119.006901] [Citation(s) in RCA: 55] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Antiretroviral inhibitors that are used to manage HIV infection/AIDS predominantly target three enzymes required for virus replication: reverse transcriptase, protease, and integrase. Although integrase inhibitors were the last among this group to be approved for treating people living with HIV, they have since risen to the forefront of treatment options. Integrase strand transfer inhibitors (INSTIs) are now recommended components of frontline and drug-switch antiretroviral therapy formulations. Integrase catalyzes two successive magnesium-dependent polynucleotidyl transferase reactions, 3' processing and strand transfer, and INSTIs tightly bind the divalent metal ions and viral DNA end after 3' processing, displacing from the integrase active site the DNA 3'-hydroxyl group that is required for strand transfer activity. Although second-generation INSTIs present higher barriers to the development of viral drug resistance than first-generation compounds, the mechanisms underlying these superior barrier profiles are incompletely understood. A separate class of HIV-1 integrase inhibitors, the allosteric integrase inhibitors (ALLINIs), engage integrase distal from the enzyme active site, namely at the binding site for the cellular cofactor lens epithelium-derived growth factor (LEDGF)/p75 that helps to guide integration into host genes. ALLINIs inhibit HIV-1 replication by inducing integrase hypermultimerization, which precludes integrase binding to genomic RNA and perturbs the morphogenesis of new viral particles. Although not yet approved for human use, ALLINIs provide important probes that can be used to investigate the link between HIV-1 integrase and viral particle morphogenesis. Herein, I review the mechanisms of retroviral integration as well as the promises and challenges of using integrase inhibitors for HIV/AIDS management.
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Affiliation(s)
- Alan N Engelman
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, Massachusetts 02215 Department of Medicine, Harvard Medical School, Boston, Massachusetts 02115
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19
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Borrenberghs D, Zurnic I, De Wit F, Acke A, Dirix L, Cereseto A, Debyser Z, Hendrix J. Post-mitotic BET-induced reshaping of integrase quaternary structure supports wild-type MLV integration. Nucleic Acids Res 2019; 47:1195-1210. [PMID: 30445610 PMCID: PMC6379647 DOI: 10.1093/nar/gky1157] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/12/2018] [Revised: 10/28/2018] [Accepted: 10/30/2018] [Indexed: 12/29/2022] Open
Abstract
The Moloney murine leukemia virus (MLV) is a prototype gammaretrovirus requiring nuclear disassembly before DNA integration. In the nucleus, integration site selection towards promoter/enhancer elements is mediated by the host factor bromo- and extraterminal domain (BET) proteins (bromodomain (Brd) proteins 2, 3 and 4). MLV-based retroviral vectors are used in gene therapy trials. In some trials leukemia occurred through integration of the MLV vector in close proximity to cellular oncogenes. BET-mediated integration is poorly understood and the nature of integrase oligomers heavily debated. Here, we created wild-type infectious MLV vectors natively incorporating fluorescent labeled IN and performed single-molecule intensity and Förster resonance energy transfer experiments. The nuclear localization of the MLV pre-integration complex neither altered the IN content, nor its quaternary structure. Instead, BET-mediated interaction of the MLV intasome with chromatin in the post-mitotic nucleus reshaped its quaternary structure.
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Affiliation(s)
- Doortje Borrenberghs
- Laboratory for Photochemistry and Spectroscopy, Department of Chemistry, KU Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium.,Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium
| | - Irena Zurnic
- Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium
| | - Flore De Wit
- Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium
| | - Aline Acke
- Laboratory for Photochemistry and Spectroscopy, Department of Chemistry, KU Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium
| | - Lieve Dirix
- Laboratory for Photochemistry and Spectroscopy, Department of Chemistry, KU Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium.,Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium
| | - Anna Cereseto
- Center for Integrative Biology (CIBIO), University of Trento, I-38123 Trento, Italy
| | - Zeger Debyser
- Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Kapucijnenvoer 33, B-3000 Leuven, Flanders, Belgium
| | - Jelle Hendrix
- Laboratory for Photochemistry and Spectroscopy, Department of Chemistry, KU Leuven, Celestijnenlaan 200F, B-3001 Leuven, Belgium.,Dynamic Bioimaging Lab, Advanced Optical Microscopy Centre and Biomedical Research Institute (BIOMED), Hasselt University, Agoralaan C, B-3590 Diepenbeek, Belgium
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20
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Barski MS, Minnell JJ, Maertens GN. Inhibition of HTLV-1 Infection by HIV-1 First- and Second-Generation Integrase Strand Transfer Inhibitors. Front Microbiol 2019; 10:1877. [PMID: 31474960 PMCID: PMC6705210 DOI: 10.3389/fmicb.2019.01877] [Citation(s) in RCA: 25] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2019] [Accepted: 07/30/2019] [Indexed: 12/21/2022] Open
Abstract
More than 10 million people worldwide are infected with the retrovirus human T-cell lymphotropic virus type 1 (HTLV-1). Infection phenotypes can range from asymptomatic to severe adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy. HTLV-1, like human immunodeficiency virus type 1 (HIV-1), is a blood-borne pathogen and viral infection happens in a similar fashion, with the major mode of transmission through breastfeeding. There is a strong correlation between time of infection and disease development, with a higher incidence of ATLL in patients infected during childhood. There is no successful therapeutic or preventative regimen for HTLV-1. It is therefore essential to develop therapies to inhibit transmission or block the onset/development of HTLV-1 associated diseases. Recently, we have seen the overwhelming success of integrase strand transfer inhibitors (INSTIs) in the treatment of HIV-1. Previously, raltegravir was shown to inhibit HTLV-1 infection. Here, we tested FDA-approved and two Phase II HIV-1 INSTIs in vitro and in a cell-to-cell infection model and show that they are highly active in blocking HTLV-1 infection, with bictegravir (EC50 = 0.30 ± 0.17 nM) performing best overall. INSTIs, in particular bictegravir, are more potent in blocking HTLV-1 transmission than tenofovir disproxil fumarate (TDF), an RT inhibitor. Our data suggest that HIV-1 INSTIs could present a good clinical strategy in HTLV-1 management and justifies the inclusion of INSTIs in clinical trials.
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Affiliation(s)
- Michał S Barski
- Division of Infectious Diseases, Section of Molecular Virology, Department of Medicine, St Mary's Hospital, Imperial College London, London, United Kingdom
| | - Jordan J Minnell
- Division of Infectious Diseases, Section of Molecular Virology, Department of Medicine, St Mary's Hospital, Imperial College London, London, United Kingdom
| | - Goedele N Maertens
- Division of Infectious Diseases, Section of Molecular Virology, Department of Medicine, St Mary's Hospital, Imperial College London, London, United Kingdom
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21
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Richetta C, Thierry S, Thierry E, Lesbats P, Lapaillerie D, Munir S, Subra F, Leh H, Deprez E, Parissi V, Delelis O. Two-long terminal repeat (LTR) DNA circles are a substrate for HIV-1 integrase. J Biol Chem 2019; 294:8286-8295. [PMID: 30971426 DOI: 10.1074/jbc.ra118.006755] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2018] [Revised: 04/08/2019] [Indexed: 02/01/2023] Open
Abstract
Integration of the HIV-1 DNA into the host genome is essential for viral replication and is catalyzed by the retroviral integrase. To date, the only substrate described to be involved in this critical reaction is the linear viral DNA produced in reverse transcription. However, during HIV-1 infection, two-long terminal repeat DNA circles (2-LTRcs) are also generated through the ligation of the viral DNA ends by the host cell's nonhomologous DNA end-joining pathway. These DNAs contain all the genetic information required for viral replication, but their role in HIV-1's life cycle remains unknown. We previously showed that both linear and circular DNA fragments containing the 2-LTR palindrome junction can be efficiently cleaved in vitro by recombinant integrases, leading to the formation of linear 3'-processed-like DNA. In this report, using in vitro experiments with purified proteins and DNAs along with DNA endonuclease and in vivo integration assays, we show that this circularized genome can also be efficiently used as a substrate in HIV-1 integrase-mediated integration both in vitro and in eukaryotic cells. Notably, we demonstrate that the palindrome cleavage occurs via a two-step mechanism leading to a blunt-ended DNA product, followed by a classical 3'-processing reaction; this cleavage leads to integrase-dependent integration, highlighted by a 5-bp duplication of the host genome. Our results suggest that 2-LTRc may constitute a reserve supply of HIV-1 genomes for proviral integration.
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Affiliation(s)
- Clémence Richetta
- Laboratoire de Biologie et Pharmacologie Appliquée, Centre National de la Recherche Scientifique UMR8113, ENS-Cachan, 94235 Cachan
| | - Sylvain Thierry
- Laboratoire de Biologie et Pharmacologie Appliquée, Centre National de la Recherche Scientifique UMR8113, ENS-Cachan, 94235 Cachan
| | - Eloise Thierry
- Laboratoire de Biologie et Pharmacologie Appliquée, Centre National de la Recherche Scientifique UMR8113, ENS-Cachan, 94235 Cachan
| | - Paul Lesbats
- Laboratoire de Microbiologie Fondamentale et Pathogénicité, Centre National de la Recherche Scientifique UMR5234, Université Victor Segalen Bordeaux 2, 33076 Bordeaux, France
| | - Delphine Lapaillerie
- Laboratoire de Microbiologie Fondamentale et Pathogénicité, Centre National de la Recherche Scientifique UMR5234, Université Victor Segalen Bordeaux 2, 33076 Bordeaux, France
| | - Soundasse Munir
- Laboratoire de Biologie et Pharmacologie Appliquée, Centre National de la Recherche Scientifique UMR8113, ENS-Cachan, 94235 Cachan
| | - Frédéric Subra
- Laboratoire de Biologie et Pharmacologie Appliquée, Centre National de la Recherche Scientifique UMR8113, ENS-Cachan, 94235 Cachan
| | - Hervé Leh
- Laboratoire de Biologie et Pharmacologie Appliquée, Centre National de la Recherche Scientifique UMR8113, ENS-Cachan, 94235 Cachan
| | - Eric Deprez
- Laboratoire de Biologie et Pharmacologie Appliquée, Centre National de la Recherche Scientifique UMR8113, ENS-Cachan, 94235 Cachan
| | - Vincent Parissi
- Laboratoire de Microbiologie Fondamentale et Pathogénicité, Centre National de la Recherche Scientifique UMR5234, Université Victor Segalen Bordeaux 2, 33076 Bordeaux, France
| | - Olivier Delelis
- Laboratoire de Biologie et Pharmacologie Appliquée, Centre National de la Recherche Scientifique UMR8113, ENS-Cachan, 94235 Cachan.
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22
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Bera S, Pandey KK, Aihara H, Grandgenett DP. Differential assembly of Rous sarcoma virus tetrameric and octameric intasomes is regulated by the C-terminal domain and tail region of integrase. J Biol Chem 2018; 293:16440-16452. [PMID: 30185621 DOI: 10.1074/jbc.ra118.004768] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2018] [Revised: 08/28/2018] [Indexed: 01/07/2023] Open
Abstract
Retrovirus integrase (IN) catalyzes the concerted integration of linear viral DNA ends into chromosomes. The atomic structures of five different retrovirus IN-DNA complexes, termed intasomes, have revealed varying IN subunit compositions ranging from tetramers to octamers, dodecamers, and hexadecamers. Intasomes containing two IN-associated viral DNA ends capable of concerted integration are termed stable synaptic complexes (SSC), and those formed with a viral/target DNA substrate representing the product of strand-transfer reactions are strand-transfer complexes (STC). Here, we investigated the mechanisms associated with the assembly of the Rous sarcoma virus SSC and STC. C-terminal truncations of WT IN (286 residues) indicated a role of the last 18 residues ("tail" region) in assembly of the tetrameric and octameric SSC, physically stabilized by HIV-1 IN strand-transfer inhibitors. Fine mapping through C-terminal truncations and site-directed mutagenesis suggested that at least three residues (Asp-268-Thr-270) past the last β-strand in the C-terminal domain (CTD) are necessary for assembly of the octameric SSC. In contrast, the assembly of the octameric STC was independent of the last 18 residues of IN. Single-site substitutions in the CTD affected the assembly of the SSC, but not necessarily of the STC, suggesting that STC assembly may depend less on specific interactions of the CTD with viral DNA. Additionally, we demonstrate that trans-communication between IN dimer-DNA complexes facilitates the association of native long-terminal repeat (LTR) ends with partially defective LTR ends to produce a hybrid octameric SSC. The differential assembly of the tetrameric and octameric SSC improves our understanding of intasomes.
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Affiliation(s)
- Sibes Bera
- From the Department of Molecular Microbiology and Immunology, Institute for Molecular Virology, Saint Louis University Health Sciences Center, Saint Louis, Missouri 63104 and
| | - Krishan K Pandey
- From the Department of Molecular Microbiology and Immunology, Institute for Molecular Virology, Saint Louis University Health Sciences Center, Saint Louis, Missouri 63104 and
| | - Hideki Aihara
- the Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455
| | - Duane P Grandgenett
- From the Department of Molecular Microbiology and Immunology, Institute for Molecular Virology, Saint Louis University Health Sciences Center, Saint Louis, Missouri 63104 and
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23
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Craigie R. Nucleoprotein Intermediates in HIV-1 DNA Integration: Structure and Function of HIV-1 Intasomes. Subcell Biochem 2018; 88:189-210. [PMID: 29900498 DOI: 10.1007/978-981-10-8456-0_9] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
Abstract
Integration of a DNA copy of the viral genome into host DNA is an essential step in the replication cycle of HIV-1 and other retroviruses and is an important therapeutic target for drugs. DNA integration is catalyzed by the viral integrase protein and proceeds through a series of stable nucleoprotein complexes of integrase, viral DNA ends and target DNA. These nucleoprotein complexes are collectively called intasomes. Retroviral intasomes undergo a series of transitions between initial formation and catalysis of the DNA cutting and joining steps of DNA integration. Intasomes, rather than free integrase protein, are the target of currently approved drugs that target HIV-1 DNA integration. High-resolution structures of HIV-1 intasomes are needed to understand their detailed mechanism of action and how HIV-1 may escape by developing resistance. Here, we focus on our current knowledge of the structure and function of HIV-1 intasomes, with reference to related systems as required to put this knowledge in context.
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Affiliation(s)
- Robert Craigie
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.
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24
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Abstract
Integration of the reverse-transcribed viral cDNA into the host's genome is a critical step in the lifecycle of all retroviruses. Retrovirus integration is carried out by integrase (IN), a virus-encoded enzyme that forms an oligomeric 'intasome' complex with both ends of the linear viral DNA to catalyze their concerted insertions into the backbones of the host's DNA. IN also forms a complex with host proteins, which guides the intasome to the host's chromosome. Recent structural studies have revealed remarkable diversity as well as conserved features among the architectures of the intasome assembly from different genera of retroviruses. This chapter will review how IN oligomerizes to achieve its function, with particular focus on alpharetrovirus including the avian retrovirus Rous sarcoma virus. Another chapter (Craigie) will focus on the structure and function of IN from HIV-1.
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Affiliation(s)
- Duane P Grandgenett
- Saint Louis University Health Sciences Center, Department of Microbiology and Immunology, Institute for Molecular Virology, Doisy Research Center, St. Louis, MO, USA
| | - Hideki Aihara
- Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
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25
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Lopez MA, Mackler RM, Altman MP, Yoder KE. Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase. J Vis Exp 2017. [PMID: 29286489 DOI: 10.3791/56605] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.
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Affiliation(s)
- Miguel A Lopez
- Department of Cancer Biology and Genetics, The Ohio State University College of Medicine
| | - Randi M Mackler
- Department of Cancer Biology and Genetics, The Ohio State University College of Medicine
| | - Matthew P Altman
- Department of Cancer Biology and Genetics, The Ohio State University College of Medicine
| | - Kristine E Yoder
- Department of Cancer Biology and Genetics, The Ohio State University College of Medicine;
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26
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Engelman AN, Cherepanov P. Retroviral intasomes arising. Curr Opin Struct Biol 2017; 47:23-29. [PMID: 28458055 PMCID: PMC5660667 DOI: 10.1016/j.sbi.2017.04.005] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2017] [Revised: 04/10/2017] [Accepted: 04/11/2017] [Indexed: 01/26/2023]
Abstract
Retroviral DNA integration takes place in the context of the intasome nucleoprotein complex. X-ray crystal structures of functional spumaviral intasomes were previously revealed to harbor a homotetramer of integrase, and it was generally believed that integrase tetramers catalyzed the integration of other retroviruses. The elucidation of new structures from four different retroviruses over the past year has however revealed this is not the case. The number of integrase molecules required to construct the conserved intasome core structure differs between viral species. While four subunits suffice for spumaviruses, α- and β-retroviruses require eight and the lentiviruses use up to sixteen. Herein we described these alternative architectures, highlighting both evolutionary and structural constraints that result in the different integrase-DNA stoichiometries across Retroviridae.
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Affiliation(s)
- Alan N Engelman
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Department of Medicine, Harvard Medical School, Boston, MA 02115, USA.
| | - Peter Cherepanov
- Chromatin Structure and Mobile DNA, The Francis Crick Institute, London NW1 1AT, UK; Department of Medicine, Imperial College London, St-Mary's Campus, Norfolk Place, London W2 1PG, UK.
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Abstract
My laboratory investigations have been driven by an abiding interest in understanding the consequences of genetic rearrangement in evolution and disease, and in using viruses to elucidate fundamental mechanisms in biology. Starting with bacteriophages and moving to the retroviruses, my use of the tools of genetics, molecular biology, biochemistry, and biophysics has spanned more than half a century-from the time when DNA structure was just discovered to the present day of big data and epigenetics. Both riding and contributing to the successive waves of technology, my laboratory has elucidated fundamental mechanisms in DNA replication, repair, and recombination. We have made substantial contributions in the area of retroviral oncogenesis, delineated mechanisms that control retroviral gene expression, and elucidated critical details of the structure and function of the retroviral enzymes-reverse transcriptase, protease, and integrase-and have had the satisfaction of knowing that the fundamental knowledge gained from these studies contributed important groundwork for the eventual development of antiviral drugs to treat AIDS. While pursuing laboratory research as a principal investigator, I have also been a science administrator-moving from laboratory head to department chair and, finally, to institute director. In addition, I have undertaken a number of community service, science-related "extracurricular" activities during this time. Filling all of these roles, while being a wife and mother, has required family love and support, creative management, and, above all, personal flexibility-with not too much long-term planning. I hope that this description of my journey, with various roles, obstacles, and successes, will be both interesting and informative, especially to young female scientists.
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Affiliation(s)
- Anna Marie Ann Skalka
- Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111;
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28
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Zhao XZ, Smith SJ, Maskell DP, Métifiot M, Pye VE, Fesen K, Marchand C, Pommier Y, Cherepanov P, Hughes SH, Burke TR. Structure-Guided Optimization of HIV Integrase Strand Transfer Inhibitors. J Med Chem 2017; 60:7315-7332. [PMID: 28737946 PMCID: PMC5601359 DOI: 10.1021/acs.jmedchem.7b00596] [Citation(s) in RCA: 44] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2017] [Indexed: 12/16/2022]
Abstract
Integrase mutations can reduce the effectiveness of the first-generation FDA-approved integrase strand transfer inhibitors (INSTIs), raltegravir (RAL) and elvitegravir (EVG). The second-generation agent, dolutegravir (DTG), has enjoyed considerable clinical success; however, resistance-causing mutations that diminish the efficacy of DTG have appeared. Our current findings support and extend the substrate envelope concept that broadly effective INSTIs can be designed by filling the envelope defined by the DNA substrates. Previously, we explored 1-hydroxy-2-oxo-1,2-dihydro-1,8-naphthyridine-3-carboxamides as an INSTI scaffold, making a limited set of derivatives, and concluded that broadly effective INSTIs can be developed using this scaffold. Herein, we report an extended investigation of 6-substituents as well the first examples of 7-substituted analogues of this scaffold. While 7-substituents are not well-tolerated, we have identified novel substituents at the 6-position that are highly effective, with the best compound (6p) retaining better efficacy against a broad panel of known INSTI resistant mutants than any analogues we have previously described.
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Affiliation(s)
- Xue Zhi Zhao
- Chemical
Biology Laboratory and HIV Dynamics and Replication Program, Center
for Cancer Research, National Cancer Institute,
National Institutes of Health, Frederick, Maryland 21702, United States
| | - Steven J. Smith
- Chemical
Biology Laboratory and HIV Dynamics and Replication Program, Center
for Cancer Research, National Cancer Institute,
National Institutes of Health, Frederick, Maryland 21702, United States
| | - Daniel P. Maskell
- Chromatin
Structure and Mobile DNA, The Francis Crick
Institute, London NW1 1AT, United Kingdom
| | - Mathieu Métifiot
- Developmental
Therapeutics Branch and Laboratory of Molecular Pharmacology, Center
for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland 20892, United States
| | - Valerie E. Pye
- Chromatin
Structure and Mobile DNA, The Francis Crick
Institute, London NW1 1AT, United Kingdom
| | - Katherine Fesen
- Developmental
Therapeutics Branch and Laboratory of Molecular Pharmacology, Center
for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland 20892, United States
| | - Christophe Marchand
- Developmental
Therapeutics Branch and Laboratory of Molecular Pharmacology, Center
for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland 20892, United States
| | - Yves Pommier
- Developmental
Therapeutics Branch and Laboratory of Molecular Pharmacology, Center
for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland 20892, United States
| | - Peter Cherepanov
- Chromatin
Structure and Mobile DNA, The Francis Crick
Institute, London NW1 1AT, United Kingdom
- Imperial
College London, St-Mary’s
Campus, Norfolk Place, London W2 1PG, United Kingdom
| | - Stephen H. Hughes
- Developmental
Therapeutics Branch and Laboratory of Molecular Pharmacology, Center
for Cancer Research, National Cancer Institute,
National Institutes of Health, Bethesda, Maryland 20892, United States
| | - Terrence R. Burke
- Chemical
Biology Laboratory and HIV Dynamics and Replication Program, Center
for Cancer Research, National Cancer Institute,
National Institutes of Health, Frederick, Maryland 21702, United States
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Passos DO, Li M, Yang R, Rebensburg SV, Ghirlando R, Jeon Y, Shkriabai N, Kvaratskhelia M, Craigie R, Lyumkis D. Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome. Science 2017; 355:89-92. [PMID: 28059769 DOI: 10.1126/science.aah5163] [Citation(s) in RCA: 140] [Impact Index Per Article: 17.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2016] [Accepted: 12/02/2016] [Indexed: 12/25/2022]
Abstract
Like all retroviruses, HIV-1 irreversibly inserts a viral DNA (vDNA) copy of its RNA genome into host target DNA (tDNA). The intasome, a higher-order nucleoprotein complex composed of viral integrase (IN) and the ends of linear vDNA, mediates integration. Productive integration into host chromatin results in the formation of the strand transfer complex (STC) containing catalytically joined vDNA and tDNA. HIV-1 intasomes have been refractory to high-resolution structural studies. We used a soluble IN fusion protein to facilitate structural studies, through which we present a high-resolution cryo-electron microscopy (cryo-EM) structure of the core tetrameric HIV-1 STC and a higher-order form that adopts carboxyl-terminal domain rearrangements. The distinct STC structures highlight how HIV-1 can use the common retroviral intasome core architecture to accommodate different IN domain modules for assembly.
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Affiliation(s)
- Dario Oliveira Passos
- Laboratory of Genetics and Helmsley Center for Genomic Medicine, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Min Li
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Renbin Yang
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Stephanie V Rebensburg
- Center for Retrovirus Research and College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
| | - Rodolfo Ghirlando
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Youngmin Jeon
- Laboratory of Genetics and Helmsley Center for Genomic Medicine, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
| | - Nikoloz Shkriabai
- Center for Retrovirus Research and College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
| | - Mamuka Kvaratskhelia
- Center for Retrovirus Research and College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA
| | - Robert Craigie
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA
| | - Dmitry Lyumkis
- Laboratory of Genetics and Helmsley Center for Genomic Medicine, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA.
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30
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Grawenhoff J, Engelman AN. Retroviral integrase protein and intasome nucleoprotein complex structures. World J Biol Chem 2017; 8:32-44. [PMID: 28289517 PMCID: PMC5329712 DOI: 10.4331/wjbc.v8.i1.32] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/01/2016] [Revised: 12/24/2016] [Accepted: 01/14/2017] [Indexed: 02/05/2023] Open
Abstract
Retroviral replication proceeds through the integration of a DNA copy of the viral RNA genome into the host cellular genome, a process that is mediated by the viral integrase (IN) protein. IN catalyzes two distinct chemical reactions: 3’-processing, whereby the viral DNA is recessed by a di- or trinucleotide at its 3’-ends, and strand transfer, in which the processed viral DNA ends are inserted into host chromosomal DNA. Although IN has been studied as a recombinant protein since the 1980s, detailed structural understanding of its catalytic functions awaited high resolution structures of functional IN-DNA complexes or intasomes, initially obtained in 2010 for the spumavirus prototype foamy virus (PFV). Since then, two additional retroviral intasome structures, from the α-retrovirus Rous sarcoma virus (RSV) and β-retrovirus mouse mammary tumor virus (MMTV), have emerged. Here, we briefly review the history of IN structural biology prior to the intasome era, and then compare the intasome structures of PFV, MMTV and RSV in detail. Whereas the PFV intasome is characterized by a tetrameric assembly of IN around the viral DNA ends, the newer structures harbor octameric IN assemblies. Although the higher order architectures of MMTV and RSV intasomes differ from that of the PFV intasome, they possess remarkably similar intasomal core structures. Thus, retroviral integration machineries have adapted evolutionarily to utilize disparate IN elements to construct convergent intasome core structures for catalytic function.
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31
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Fuller JR, Rice PA. Target DNA bending by the Mu transpososome promotes careful transposition and prevents its reversal. eLife 2017; 6. [PMID: 28177285 PMCID: PMC5357137 DOI: 10.7554/elife.21777] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2016] [Accepted: 02/07/2017] [Indexed: 12/19/2022] Open
Abstract
The transposition of bacteriophage Mu serves as a model system for understanding DDE transposases and integrases. All available structures of these enzymes at the end of the transposition reaction, including Mu, exhibit significant bends in the transposition target site DNA. Here we use Mu to investigate the ramifications of target DNA bending on the transposition reaction. Enhancing the flexibility of the target DNA or prebending it increases its affinity for transpososomes by over an order of magnitude and increases the overall reaction rate. This and FRET confirm that flexibility is interrogated early during the interaction between the transposase and a potential target site, which may be how other DNA binding proteins can steer selection of advantageous target sites. We also find that the conformation of the target DNA after strand transfer is involved in preventing accidental catalysis of the reverse reaction, as conditions that destabilize this conformation also trigger reversal. DOI:http://dx.doi.org/10.7554/eLife.21777.001 Pieces of DNA called transposons can move or copy themselves around the genome. Some viruses – such as HIV and Mu (a virus that infects bacteria) – act as transposons to hide their DNA by inserting it into their host’s genome. Mu, HIV and many transposons all work in the same, somewhat unusual way. Like many chemical reactions, joining DNAs together needs a source of energy to make it happen, yet these viruses and transposons do not need high energy inputs to work. In addition, they do not look for a specific DNA sequence to insert their DNA into. This gives them the advantage of inserting copies of their DNA anywhere in the host’s genome, but also means that multiple copies might mistakenly insert into each other. Visualizations of the insertion process show that the DNA that the viruses insert their DNA into is always bent like a U-turn. Why does this bending occur? It may be that the bending helps the virus to choose where in the DNA to insert and acts as a way to power the chemical reaction that joins the DNA. To investigate this possibility, Fuller and Rice performed experiments using purified fragments of DNA and the enzyme from Mu that does the DNA joining chemistry. The results revealed that making the insertion site DNA easier to bend made the insertion much faster. Furthermore, a mutant enzyme that struggled to bend the DNA had trouble keeping the chemistry going, and so the viral DNA would accidentally pop back out after it was joined. Thus the insertion site DNA is like a spring: the enzyme puts a lot of energy into bending it, but once the viral DNA has been inserted that energy is released to power the reaction to completion. Fuller and Rice conclude that if other proteins were to pre-bend or otherwise make the DNA more flexible, this would tell the DNA-joining enzyme where to insert, which helps explain the roles of known targeting proteins for Mu and HIV. Further work is now needed to investigate whether these other targeting proteins exist for other viruses and transposons, and to identify them. DOI:http://dx.doi.org/10.7554/eLife.21777.002
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Affiliation(s)
- James R Fuller
- Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, United States
| | - Phoebe A Rice
- Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, United States
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32
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Thierry E, Deprez E, Delelis O. Different Pathways Leading to Integrase Inhibitors Resistance. Front Microbiol 2017; 7:2165. [PMID: 28123383 PMCID: PMC5225119 DOI: 10.3389/fmicb.2016.02165] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2016] [Accepted: 12/23/2016] [Indexed: 12/20/2022] Open
Abstract
Integrase strand-transfer inhibitors (INSTIs), such as raltegravir (RAL), elvitegravir, or dolutegravir (DTG), are efficient antiretroviral agents used in HIV treatment in order to inhibit retroviral integration. By contrast to RAL treatments leading to well-identified mutation resistance pathways at the integrase level, recent clinical studies report several cases of patients failing DTG treatment without clearly identified resistance mutation in the integrase gene raising questions for the mechanism behind the resistance. These compounds, by impairing the integration of HIV-1 viral DNA into the host DNA, lead to an accumulation of unintegrated circular viral DNA forms. This viral DNA could be at the origin of the INSTI resistance by two different ways. The first one, sustained by a recent report, involves 2-long terminal repeat circles integration and the second one involves expression of accumulated unintegrated viral DNA leading to a basal production of viral particles maintaining the viral information.
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Affiliation(s)
- Eloïse Thierry
- Laboratoire de Biologie et Pharmacologie Appliquée, CNRS UMR8113, Ecole Normale Supérieure de Cachan, Université Paris-Saclay Cachan, France
| | - Eric Deprez
- Laboratoire de Biologie et Pharmacologie Appliquée, CNRS UMR8113, Ecole Normale Supérieure de Cachan, Université Paris-Saclay Cachan, France
| | - Olivier Delelis
- Laboratoire de Biologie et Pharmacologie Appliquée, CNRS UMR8113, Ecole Normale Supérieure de Cachan, Université Paris-Saclay Cachan, France
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33
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Balasubramanian S, Rajagopalan M, Bojja RS, Skalka AM, Andrake MD, Ramaswamy A. The conformational feasibility for the formation of reaching dimer in ASV and HIV integrase: a molecular dynamics study. J Biomol Struct Dyn 2016; 35:3469-3485. [PMID: 27835934 DOI: 10.1080/07391102.2016.1257955] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/08/2023]
Abstract
Retroviral integrases are reported to form alternate dimer assemblies like the core-core dimer and reaching dimer. The core-core dimer is stabilized predominantly by an extensive interface between two catalytic core domains. The reaching dimer is stabilized by N-terminal domains that reach to form intermolecular interfaces with the other subunit's core and C-terminal domains (CTD), as well as CTD-CTD interactions. In this study, molecular dynamics (MD), Brownian dynamics (BD) simulations, and free energy analyses, were performed to elucidate determinants for the stability of the reaching dimer forms of full-length Avian Sarcoma Virus (ASV) and Human Immunodeficiency Virus (HIV) IN, and to examine the role of the C-tails (the last ~16-18 residues at the C-termini) in their structural dynamics. The dynamics of an HIV reaching dimer derived from small angle X-ray scattering and protein crosslinking data, was compared with the dynamics of a core-core dimer model derived from combining the crystal structures of two-domain fragments. The results showed that the core domains in the ASV reaching dimer express free dynamics, whereas those in the HIV reaching dimer are highly stable. BD simulations suggest a higher rate of association for the HIV core-core dimer than the reaching dimer. The predicted stability of these dimers was therefore ranked in the following order: ASV reaching dimer < HIV reaching dimer < composite core-core dimer. Analyses of MD trajectories have suggested residues that are critical for intermolecular contacts in each reaching dimer. Tests of these predictions and insights gained from these analyses could reveal a potential pathway for the association and dissociation of full-length IN multimers.
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Affiliation(s)
- Sangeetha Balasubramanian
- a Centre for Bioinformatics, School of Life Sciences , Pondicherry University , Puducherry 605014 , India
| | - Muthukumaran Rajagopalan
- a Centre for Bioinformatics, School of Life Sciences , Pondicherry University , Puducherry 605014 , India
| | - Ravi Shankar Bojja
- b Institute for Cancer Research , Fox Chase Cancer Center , Philadelphia , PA 19111 , USA
| | - Anna Marie Skalka
- b Institute for Cancer Research , Fox Chase Cancer Center , Philadelphia , PA 19111 , USA
| | - Mark D Andrake
- b Institute for Cancer Research , Fox Chase Cancer Center , Philadelphia , PA 19111 , USA
| | - Amutha Ramaswamy
- a Centre for Bioinformatics, School of Life Sciences , Pondicherry University , Puducherry 605014 , India
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34
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Borrenberghs D, Dirix L, De Wit F, Rocha S, Blokken J, De Houwer S, Gijsbers R, Christ F, Hofkens J, Hendrix J, Debyser Z. Dynamic Oligomerization of Integrase Orchestrates HIV Nuclear Entry. Sci Rep 2016; 6:36485. [PMID: 27830755 PMCID: PMC5103197 DOI: 10.1038/srep36485] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2016] [Accepted: 10/04/2016] [Indexed: 11/16/2022] Open
Abstract
Nuclear entry is a selective, dynamic process granting the HIV-1 pre-integration complex (PIC) access to the chromatin. Classical analysis of nuclear entry of heterogeneous viral particles only yields averaged information. We now have employed single-virus fluorescence methods to follow the fate of single viral pre-integration complexes (PICs) during infection by visualizing HIV-1 integrase (IN). Nuclear entry is associated with a reduction in the number of IN molecules in the complexes while the interaction with LEDGF/p75 enhances IN oligomerization in the nucleus. Addition of LEDGINs, small molecule inhibitors of the IN-LEDGF/p75 interaction, during virus production, prematurely stabilizes a higher-order IN multimeric state, resulting in stable IN multimers resistant to a reduction in IN content and defective for nuclear entry. This suggests that a stringent size restriction determines nuclear pore entry. Taken together, this work demonstrates the power of single-virus imaging providing crucial insights in HIV replication and enabling mechanism-of-action studies.
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Affiliation(s)
- Doortje Borrenberghs
- Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, 3000, Belgium.,Laboratory for Photochemistry and Spectroscopy, Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Heverlee, 3001, Belgium
| | - Lieve Dirix
- Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, 3000, Belgium.,Laboratory for Photochemistry and Spectroscopy, Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Heverlee, 3001, Belgium
| | - Flore De Wit
- Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, 3000, Belgium
| | - Susana Rocha
- Laboratory for Photochemistry and Spectroscopy, Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Heverlee, 3001, Belgium
| | - Jolien Blokken
- Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, 3000, Belgium
| | - Stéphanie De Houwer
- Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, 3000, Belgium
| | - Rik Gijsbers
- Laboratory for Viral Vector Technology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, 3000, Belgium
| | - Frauke Christ
- Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, 3000, Belgium
| | - Johan Hofkens
- Laboratory for Photochemistry and Spectroscopy, Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Heverlee, 3001, Belgium
| | - Jelle Hendrix
- Laboratory for Photochemistry and Spectroscopy, Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Heverlee, 3001, Belgium
| | - Zeger Debyser
- Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, 3000, Belgium
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35
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Kessl JJ, Sharma A, Kvaratskhelia M. Methods for the Analyses of Inhibitor-Induced Aberrant Multimerization of HIV-1 Integrase. Methods Mol Biol 2016; 1354:149-64. [PMID: 26714710 DOI: 10.1007/978-1-4939-3046-3_10] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/22/2023]
Abstract
HIV-1 integrase (IN) is an important therapeutic target as its function is essential for the viral lifecycle. The discovery of multifunctional allosteric IN inhibitors or ALLINIs, which potently impair viral replication by promoting aberrant, higher order IN multimerization as well as inhibit IN interactions with its cellular cofactor, LEDGF/p75, has opened new venues to exploit IN multimerization as a therapeutic target. Furthermore, the recent discovery of multimerization selective IN inhibitors or MINIs, has provided new investigational probes to study the direct effects of aberrant IN multimerization in vitro and in infected cells. Here we describe three complementary methods designed to detect and quantify the effects of these new classes of inhibitors on IN multimerization. These methods include a homogenous time-resolved fluorescence-based assay which allows for measuring EC50 values for the inhibitor-induced aberrant IN multimerization, a dynamic light scattering-based assay which allows for monitoring the formation and sizes of oligomeric IN particles in a time-dependent manner, and a chemical cross-linking-based assay of interacting IN subunits which allows for the determination of IN oligomers in viral particles.
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Affiliation(s)
- Jacques J Kessl
- Center for Retrovirus Research and Comprehensive Cancer Center, College of Pharmacy, The Ohio State University, Columbus, OH, 43210, USA.
| | - Amit Sharma
- Center for Retrovirus Research and Comprehensive Cancer Center, College of Pharmacy, The Ohio State University, Columbus, OH, 43210, USA.,Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA, 98109, USA
| | - Mamuka Kvaratskhelia
- Center for Retrovirus Research and Comprehensive Cancer Center, College of Pharmacy, The Ohio State University, Columbus, OH, 43210, USA
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36
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The Multifaceted Contributions of Chromatin to HIV-1 Integration, Transcription, and Latency. INTERNATIONAL REVIEW OF CELL AND MOLECULAR BIOLOGY 2016; 328:197-252. [PMID: 28069134 DOI: 10.1016/bs.ircmb.2016.08.006] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
The capacity of the human immunodeficiency virus (HIV-1) to establish latent infections constitutes a major barrier to the development of a cure for HIV-1. In latent infection, replication competent HIV-1 provirus is integrated within the host genome but remains silent, masking the infected cells from the activity of the host immune response. Despite the progress in elucidating the molecular players that regulate HIV-1 gene expression, the mechanisms driving the establishment and maintenance of latency are still not fully understood. Transcription from the HIV-1 genome occurs in the context of chromatin and is subjected to the same regulatory mechanisms that drive cellular gene expression. Much like in eukaryotic genes, the nucleosomal landscape of the HIV-1 promoter and its position within genomic chromatin are determinants of its transcriptional activity. Understanding the multilayered chromatin-mediated mechanisms that underpin HIV-1 integration and expression is of utmost importance for the development of therapeutic strategies aimed at reducing the pool of latently infected cells. In this review, we discuss the impact of chromatin structure on viral integration, transcriptional regulation and latency, and the host factors that influence HIV-1 replication by regulating chromatin organization. Finally, we describe therapeutic strategies under development to target the chromatin-HIV-1 interplay.
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37
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Suttiprapa S, Rinaldi G, Tsai IJ, Mann VH, Dubrovsky L, Yan HB, Holroyd N, Huckvale T, Durrant C, Protasio AV, Pushkarsky T, Iordanskiy S, Berriman M, Bukrinsky MI, Brindley PJ. HIV-1 Integrates Widely throughout the Genome of the Human Blood Fluke Schistosoma mansoni. PLoS Pathog 2016; 12:e1005931. [PMID: 27764257 PMCID: PMC5072744 DOI: 10.1371/journal.ppat.1005931] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2016] [Accepted: 09/13/2016] [Indexed: 11/18/2022] Open
Abstract
Schistosomiasis is the most important helminthic disease of humanity in terms of morbidity and mortality. Facile manipulation of schistosomes using lentiviruses would enable advances in functional genomics in these and related neglected tropical diseases pathogens including tapeworms, and including their non-dividing cells. Such approaches have hitherto been unavailable. Blood stream forms of the human blood fluke, Schistosoma mansoni, the causative agent of the hepatointestinal schistosomiasis, were infected with the human HIV-1 isolate NL4-3 pseudotyped with vesicular stomatitis virus glycoprotein. The appearance of strong stop and positive strand cDNAs indicated that virions fused to schistosome cells, the nucleocapsid internalized and the RNA genome reverse transcribed. Anchored PCR analysis, sequencing HIV-1-specific anchored Illumina libraries and Whole Genome Sequencing (WGS) of schistosomes confirmed chromosomal integration; >8,000 integrations were mapped, distributed throughout the eight pairs of chromosomes including the sex chromosomes. The rate of integrations in the genome exceeded five per 1,000 kb and HIV-1 integrated into protein-encoding loci and elsewhere with integration bias dissimilar to that of human T cells. We estimated ~ 2,100 integrations per schistosomulum based on WGS, i.e. about two or three events per cell, comparable to integration rates in human cells. Accomplishment in schistosomes of post-entry processes essential for HIV-1replication, including integrase-catalyzed integration, was remarkable given the phylogenetic distance between schistosomes and primates, the natural hosts of the genus Lentivirus. These enigmatic findings revealed that HIV-1 was active within cells of S. mansoni, and provided the first demonstration that HIV-1 can integrate into the genome of an invertebrate.
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Affiliation(s)
- Sutas Suttiprapa
- Department of Microbiology, Immunology & Tropical Medicine, and Research Center for Neglected Diseases of Poverty, School of Medicine and Health Sciences, The George Washington University, Washington, DC, United States of America
- Department of Microbiology, Faculty of Science, Mahidol University, Phyathai, Rachthewee, Bangkok
- Department of Pathology, Faculty of Medicine, Khon Kaen University, Muang Khon Kaen, Thailand
| | - Gabriel Rinaldi
- Department of Microbiology, Immunology & Tropical Medicine, and Research Center for Neglected Diseases of Poverty, School of Medicine and Health Sciences, The George Washington University, Washington, DC, United States of America
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom
| | - Isheng J. Tsai
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom
- Biodiversity Research Center, Academia Sinica, Taipei, Taiwan
| | - Victoria H. Mann
- Department of Microbiology, Immunology & Tropical Medicine, and Research Center for Neglected Diseases of Poverty, School of Medicine and Health Sciences, The George Washington University, Washington, DC, United States of America
| | - Larisa Dubrovsky
- Department of Microbiology, Immunology & Tropical Medicine, and Research Center for Neglected Diseases of Poverty, School of Medicine and Health Sciences, The George Washington University, Washington, DC, United States of America
| | - Hong-bin Yan
- Department of Microbiology, Immunology & Tropical Medicine, and Research Center for Neglected Diseases of Poverty, School of Medicine and Health Sciences, The George Washington University, Washington, DC, United States of America
- Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 1, Lanzhou, Gansu, The People's Republic of China
| | - Nancy Holroyd
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom
| | - Thomas Huckvale
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom
| | - Caroline Durrant
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom
| | - Anna V. Protasio
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom
| | - Tatiana Pushkarsky
- Department of Microbiology, Immunology & Tropical Medicine, and Research Center for Neglected Diseases of Poverty, School of Medicine and Health Sciences, The George Washington University, Washington, DC, United States of America
| | - Sergey Iordanskiy
- Department of Microbiology, Immunology & Tropical Medicine, and Research Center for Neglected Diseases of Poverty, School of Medicine and Health Sciences, The George Washington University, Washington, DC, United States of America
- National Center for Biodefense and Infectious Diseases, School of Systems Biology, George Mason University, Manassas, Virginia, United States of America
| | - Matthew Berriman
- Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom
| | - Michael I. Bukrinsky
- Department of Microbiology, Immunology & Tropical Medicine, and Research Center for Neglected Diseases of Poverty, School of Medicine and Health Sciences, The George Washington University, Washington, DC, United States of America
| | - Paul J. Brindley
- Department of Microbiology, Immunology & Tropical Medicine, and Research Center for Neglected Diseases of Poverty, School of Medicine and Health Sciences, The George Washington University, Washington, DC, United States of America
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Varadarajan J, McWilliams MJ, Mott BT, Thomas CJ, Smith SJ, Hughes SH. Drug resistant integrase mutants cause aberrant HIV integrations. Retrovirology 2016; 13:71. [PMID: 27682062 PMCID: PMC5041404 DOI: 10.1186/s12977-016-0305-6] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2016] [Accepted: 09/19/2016] [Indexed: 12/21/2022] Open
Abstract
Background
HIV-1 integrase is the target for three FDA-approved drugs, raltegravir, elvitegravir, and dolutegravir. All three drugs bind at the active site of integrase and block the strand transfer step of integration. We previously showed that sub-optimal doses of the anti-HIV drug raltegravir can cause aberrant HIV integrations that are accompanied by a variety of deletions, duplications, insertions and inversions of the adjacent host sequences. Results We show here that a second drug, elvitegravir, also causes similar aberrant integrations. More importantly, we show that at least two of the three clinically relevant drug resistant integrase mutants we tested, N155H and G140S/Q148H, which reduce the enzymatic activity of integrase, can cause the same sorts of aberrant integrations, even in the absence of drugs. In addition, these drug resistant mutants have an elevated IC50 for anti-integrase drugs, and concentrations of the drugs that would be optimal against the WT virus are suboptimal for the mutants. Conclusions We previously showed that suboptimal doses of a drug that binds to the HIV enzyme integrase and blocks the integration of a DNA copy of the viral genome into host DNA can cause aberrant integrations that involve rearrangements of the host DNA. We show here that suboptimal doses of a second anti-integrase drug can cause similar aberrant integrations. We also show that drug-resistance mutations in HIV integrase can also cause aberrant integrations, even in the absence of an anti-integrase drug. HIV DNA integrations in the oncogenes BACH2 and MKL2 that do not involve rearrangements of the viral or host DNA can stimulate the proliferation of infected cells. Based on what is known about the association of DNA rearrangements and the activation of oncogenes in human tumors, it is possible that some of the deletions, duplications, insertions, and inversions of the host DNA that accompany aberrant HIV DNA integrations could increase the chances that HIV integrations could lead to the development of a tumor. Electronic supplementary material The online version of this article (doi:10.1186/s12977-016-0305-6) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Janani Varadarajan
- HIV Dynamics and Replication Program, Vector Design and Replication Section, National Cancer Institute-Frederick, 1050 Boyles Street, Bldg. 539, Room 130A, Frederick, MD, 21702, USA.,Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA
| | - Mary Jane McWilliams
- HIV Dynamics and Replication Program, Vector Design and Replication Section, National Cancer Institute-Frederick, 1050 Boyles Street, Bldg. 539, Room 130A, Frederick, MD, 21702, USA
| | - Bryan T Mott
- Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA
| | - Craig J Thomas
- Division of Preclinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA
| | - Steven J Smith
- HIV Dynamics and Replication Program, Vector Design and Replication Section, National Cancer Institute-Frederick, 1050 Boyles Street, Bldg. 539, Room 130A, Frederick, MD, 21702, USA
| | - Stephen H Hughes
- HIV Dynamics and Replication Program, Vector Design and Replication Section, National Cancer Institute-Frederick, 1050 Boyles Street, Bldg. 539, Room 130A, Frederick, MD, 21702, USA.
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Abstract
INTRODUCTION Since the last revision of both European and American guidelines (EACS and DHHS), new data from clinical trials and cohort studies, as well as experience in clinical practice, have prompted significant changes to the list of recommended/preferred options for the treatment of HIV infected patients, highlighted the role of INSTI-based regimens. Dolutegravir (DTG) in combination with abacavir/lamivudine (ABC/3TC) is one of these preferred regimens in multiple clinical scenarios, including treatment-naive and treatment-experienced patients. AREAS COVERED In this article we describe the coformulation of ABC/3TC/DTG in a fixed-dose combination (FDC) approved in September 2014 for the treatment of HIV infection. We focused our research on the efficacy and safety data resulting from phase 2 and 3 clinical study, particularly on the results of both SPRING (1 and 2) and SINGLE studies. EXPERT OPINION Triple combination therapy with ABC/3TC/DTG should be considered among the initial options for treatment-naive patients, being effective, well tolerated, with a high genetic barrier to resistance along with a convenient once-daily administration. In treatment-experienced patients the single-tablet regimen (STR) based on ABC/3TC/DTG could be used as simplification strategy in subjects with sustained viral suppression, as the high genetic barrier of DTG should ensure a safe switch from both NNRTI or PI based regimens.
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Affiliation(s)
- Laura Comi
- a USC Malattie Infettive , Azienda Ospedaliera Papa Giovanni XXIII Ringgold standard institution , Bergamo , Italy
| | - Franco Maggiolo
- a USC Malattie Infettive , Azienda Ospedaliera Papa Giovanni XXIII Ringgold standard institution , Bergamo , Italy
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40
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Agharbaoui FE, Hoyte AC, Ferro S, Gitto R, Buemi MR, Fuchs JR, Kvaratskhelia M, De Luca L. Computational and synthetic approaches for developing Lavendustin B derivatives as allosteric inhibitors of HIV-1 integrase. Eur J Med Chem 2016; 123:673-683. [PMID: 27517812 DOI: 10.1016/j.ejmech.2016.07.077] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/26/2016] [Revised: 07/25/2016] [Accepted: 07/31/2016] [Indexed: 01/26/2023]
Abstract
Through structure-based virtual screening and subsequent activity assays of selected natural products, Lavendustin B was previously identified as an inhibitor of HIV-1 integrase (IN) interaction with its cognate cellular cofactor, lens epithelium-derived growth factor (LEDGF/p75). In order to improve the inhibitory potency we have employed in silico-based approaches. Particularly, a series of new analogues was designed and docked into the LEDGF/p75 binding pocket of HIV-1 IN. To identify promising leads we used the Molecular Mechanics energies combined with the Generalized Born and Surface Area continuum solvation (MM-GBSA) method, molecular dynamics simulations and analysis of hydrogen bond occupancies. On the basis of these studies, six analogues of Lavendustine B, containing the benzylamino-hydroxybenzoic scaffold, were selected for synthesis and structure activity-relationship (SAR) studies. Our results demonstrated a good correlation between computational and experimental data, and all six analogues displayed an improved potency for inhibiting IN binding to LEDGF/p75 in vitro to respect to the parent compound Lavendustin B. Additionally, these analogs show to inhibit weakly LEDGF/p75-independent IN catalytic activity suggesting a multimodal allosteric mechanism of action. Nevertheless, for the synthesized compounds similar profiles for HIV-1 inhibition and cytoxicity were highlighted. Taken together, our studies elucidated the mode of action of Lavendustin B analogs and provided a path for their further development as a new promising class of HIV-1 integrase inhibitors.
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Affiliation(s)
- Fatima E Agharbaoui
- Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche e Ambientali (CHIBIOFARAM), Polo Universitario SS. Annunziata, Università di Messina, Viale Annunziata, I-98168, Messina, Italy; Center for Retrovirus Research and College of Pharmacy, The Ohio State University, Columbus, OH, 43210, USA; Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, 43210, USA.
| | - Ashley C Hoyte
- Center for Retrovirus Research and College of Pharmacy, The Ohio State University, Columbus, OH, 43210, USA
| | - Stefania Ferro
- Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche e Ambientali (CHIBIOFARAM), Polo Universitario SS. Annunziata, Università di Messina, Viale Annunziata, I-98168, Messina, Italy
| | - Rosaria Gitto
- Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche e Ambientali (CHIBIOFARAM), Polo Universitario SS. Annunziata, Università di Messina, Viale Annunziata, I-98168, Messina, Italy
| | - Maria Rosa Buemi
- Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche e Ambientali (CHIBIOFARAM), Polo Universitario SS. Annunziata, Università di Messina, Viale Annunziata, I-98168, Messina, Italy
| | - James R Fuchs
- Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, The Ohio State University, Columbus, OH, 43210, USA
| | - Mamuka Kvaratskhelia
- Center for Retrovirus Research and College of Pharmacy, The Ohio State University, Columbus, OH, 43210, USA
| | - Laura De Luca
- Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche e Ambientali (CHIBIOFARAM), Polo Universitario SS. Annunziata, Università di Messina, Viale Annunziata, I-98168, Messina, Italy.
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41
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Lopez MA, Mackler RM, Yoder KE. Removal of nuclease contamination during purification of recombinant prototype foamy virus integrase. J Virol Methods 2016; 235:134-138. [PMID: 27269588 DOI: 10.1016/j.jviromet.2016.06.002] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2016] [Revised: 05/31/2016] [Accepted: 06/04/2016] [Indexed: 10/21/2022]
Abstract
Retroviral infection requires integration of the viral genome into the host genome. Recombinant integrase proteins may be purified following bacterial expression. A bulk biochemical assay of integrase function relies on the conversion of supercoiled plasmids to linear or relaxed circles. Single molecule molecular tweezer assays of integrase also evaluate the conversion of supercoiled DNA to nicked and broken species. A bacterial nuclease that co-purifies with retroviral integrase may affect the quantitation of integration activity in bulk or single molecule assays. During purification of retroviral integrase from bacteria, fractions may be screened for contaminating nuclease activity. In order to efficiently separate the nuclease from integrase, the binding affinities of each protein must differ. We find that a co-purifying nuclease may be efficiently separated from integrase based on heparin affinity, but not ionic affinity.
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Affiliation(s)
- Miguel A Lopez
- Molecular Virology, Immunology, and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210, United States
| | - Randi M Mackler
- Molecular Virology, Immunology, and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210, United States
| | - Kristine E Yoder
- Molecular Virology, Immunology, and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210, United States.
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42
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Feng L, Dharmarajan V, Serrao E, Hoyte A, Larue RC, Slaughter A, Sharma A, Plumb MR, Kessl JJ, Fuchs JR, Bushman FD, Engelman AN, Griffin PR, Kvaratskhelia M. The Competitive Interplay between Allosteric HIV-1 Integrase Inhibitor BI/D and LEDGF/p75 during the Early Stage of HIV-1 Replication Adversely Affects Inhibitor Potency. ACS Chem Biol 2016; 11:1313-21. [PMID: 26910179 DOI: 10.1021/acschembio.6b00167] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
Abstract
Allosteric HIV-1 integrase inhibitors (ALLINIs) have recently emerged as a promising class of antiretroviral agents and are currently in clinical trials. In infected cells, ALLINIs potently inhibit viral replication by impairing virus particle maturation but surprisingly exhibit a reduced EC50 for inhibiting HIV-1 integration in target cells. To better understand the reduced antiviral activity of ALLINIs during the early stage of HIV-1 replication, we investigated the competitive interplay between a potent representative ALLINI, BI/D, and LEDGF/p75 with HIV-1 integrase. While the principal binding sites of BI/D and LEDGF/p75 overlap at the integrase catalytic core domain dimer interface, we show that the inhibitor and the cellular cofactor induce markedly different multimerization patterns of full-length integrase. LEDGF/p75 stabilizes an integrase tetramer through the additional interactions with the integrase N-terminal domain, whereas BI/D induces protein-protein interactions in C-terminal segments that lead to aberrant, higher-order integrase multimerization. We demonstrate that LEDGF/p75 binds HIV-1 integrase with significantly higher affinity than BI/D and that the cellular protein is able to reverse the inhibitor induced aberrant, higher-order integrase multimerization in a dose-dependent manner in vitro. Consistent with these observations, alterations of the cellular levels of LEDGF/p75 markedly affected BI/D EC50 values during the early steps of HIV-1 replication. Furthermore, genome-wide sequencing of HIV-1 integration sites in infected cells demonstrate that LEDGF/p75-dependent integration site selection is adversely affected by BI/D treatment. Taken together, our studies elucidate structural and mechanistic details of the interplay between LEDGF/p75 and BI/D during the early stage of HIV-1 replication.
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Affiliation(s)
- Lei Feng
- Center for Retrovirus Research and College
of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
| | - Venkatasubramanian Dharmarajan
- Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, Florida 33458, United States
| | - Erik Serrao
- Department
of Cancer Immunology and Virology, Dana-Farber Cancer Institute and
Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, United States
| | - Ashley Hoyte
- Center for Retrovirus Research and College
of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
| | - Ross C. Larue
- Center for Retrovirus Research and College
of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
| | - Alison Slaughter
- Center for Retrovirus Research and College
of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
| | - Amit Sharma
- Center for Retrovirus Research and College
of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
| | - Matthew R. Plumb
- Center for Retrovirus Research and College
of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
| | - Jacques J. Kessl
- Center for Retrovirus Research and College
of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
| | - James R. Fuchs
- Division of Medicinal Chemistry and Pharmacognosy,
College of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
| | - Frederic D. Bushman
- Perelman School of Medicine, Department of Microbiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States
| | - Alan N. Engelman
- Department
of Cancer Immunology and Virology, Dana-Farber Cancer Institute and
Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, United States
| | - Patrick R. Griffin
- Department of Molecular Therapeutics, The Scripps Research Institute, Scripps Florida, Jupiter, Florida 33458, United States
| | - Mamuka Kvaratskhelia
- Center for Retrovirus Research and College
of Pharmacy, The Ohio State University, Columbus, Ohio 43210, United States
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43
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Abstract
The integration of a DNA copy of the viral RNA genome into host chromatin is the defining step of retroviral replication. This enzymatic process is catalyzed by the virus-encoded integrase protein, which is conserved among retroviruses and LTR-retrotransposons. Retroviral integration proceeds via two integrase activities: 3'-processing of the viral DNA ends, followed by the strand transfer of the processed ends into host cell chromosomal DNA. Herein we review the molecular mechanism of retroviral DNA integration, with an emphasis on reaction chemistries and architectures of the nucleoprotein complexes involved. We additionally discuss the latest advances on anti-integrase drug development for the treatment of AIDS and the utility of integrating retroviral vectors in gene therapy applications.
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Affiliation(s)
- Paul Lesbats
- Clare Hall Laboratories, The Francis Crick Institute , Blanche Lane, South Mimms, EN6 3LD, U.K
| | - Alan N Engelman
- Department of Cancer Immunology and Virology, Dana-Farber Cancer Institute and Department of Medicine, Harvard Medical School , 450 Brookline Avenue, Boston, Massachusetts 02215 United States
| | - Peter Cherepanov
- Clare Hall Laboratories, The Francis Crick Institute , Blanche Lane, South Mimms, EN6 3LD, U.K.,Imperial College London , St-Mary's Campus, Norfolk Place, London, W2 1PG, U.K
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44
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Jones ND, Lopez MA, Hanne J, Peake MB, Lee JB, Fishel R, Yoder KE. Retroviral intasomes search for a target DNA by 1D diffusion which rarely results in integration. Nat Commun 2016; 7:11409. [PMID: 27108531 PMCID: PMC4848512 DOI: 10.1038/ncomms11409] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2015] [Accepted: 03/23/2016] [Indexed: 12/13/2022] Open
Abstract
Retroviruses must integrate their linear viral cDNA into the host genome for a productive infection. Integration is catalysed by the retrovirus-encoded integrase (IN), which forms a tetramer or octamer complex with the viral cDNA long terminal repeat (LTR) ends termed an intasome. IN removes two 3'-nucleotides from both LTR ends and catalyses strand transfer of the recessed 3'-hydroxyls into the target DNA separated by 4-6 bp. Host DNA repair restores the resulting 5'-Flap and single-stranded DNA (ssDNA) gap. Here we have used multiple single molecule imaging tools to determine that the prototype foamy virus (PFV) retroviral intasome searches for an integration site by one-dimensional (1D) rotation-coupled diffusion along DNA. Once a target site is identified, the time between PFV strand transfer events is 470 ms. The majority of PFV intasome search events were non-productive. These observations identify new dynamic IN functions and suggest that target site-selection limits retroviral integration.
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Affiliation(s)
- Nathan D Jones
- Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University Medical Center, Columbus, Ohio 43210, USA.,The Interdisciplinary Biophysics Graduate Program, The Ohio State University, Columbus, Ohio 43210, USA
| | - Miguel A Lopez
- Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University Medical Center, Columbus, Ohio 43210, USA
| | - Jeungphill Hanne
- Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University Medical Center, Columbus, Ohio 43210, USA
| | - Mitchell B Peake
- Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University Medical Center, Columbus, Ohio 43210, USA
| | - Jong-Bong Lee
- Department of Physics, Pohang University of Science and Technology (POSTECH), Pohang 790-784, Korea.,Interdisciplinary Bioscience and Bioengineering, POSTECH, Pohang 790-784, Korea
| | - Richard Fishel
- Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University Medical Center, Columbus, Ohio 43210, USA.,The Interdisciplinary Biophysics Graduate Program, The Ohio State University, Columbus, Ohio 43210, USA.,Department of Physics, The Ohio State University, Columbus, Ohio 43210, USA
| | - Kristine E Yoder
- Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University Medical Center, Columbus, Ohio 43210, USA
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45
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Serrao E, Cherepanov P, Engelman AN. Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites. J Vis Exp 2016. [PMID: 27023428 DOI: 10.3791/53840] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.
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Affiliation(s)
- Erik Serrao
- Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute
| | | | - Alan N Engelman
- Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute;
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46
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Cryo-EM reveals a novel octameric integrase structure for betaretroviral intasome function. Nature 2016; 530:358-61. [PMID: 26887496 PMCID: PMC4908968 DOI: 10.1038/nature16955] [Citation(s) in RCA: 73] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2015] [Accepted: 12/30/2015] [Indexed: 12/26/2022]
Abstract
Retroviral integrase catalyses the integration of viral DNA into host target DNA, which is an essential step in the life cycle of all retroviruses. Previous structural characterization of integrase-viral DNA complexes, or intasomes, from the spumavirus prototype foamy virus revealed a functional integrase tetramer, and it is generally believed that intasomes derived from other retroviral genera use tetrameric integrase. However, the intasomes of orthoretroviruses, which include all known pathogenic species, have not been characterized structurally. Here, using single-particle cryo-electron microscopy and X-ray crystallography, we determine an unexpected octameric integrase architecture for the intasome of the betaretrovirus mouse mammary tumour virus. The structure is composed of two core integrase dimers, which interact with the viral DNA ends and structurally mimic the integrase tetramer of prototype foamy virus, and two flanking integrase dimers that engage the core structure via their integrase carboxy-terminal domains. Contrary to the belief that tetrameric integrase components are sufficient to catalyse integration, the flanking integrase dimers were necessary for mouse mammary tumour virus integrase activity. The integrase octamer solves a conundrum for betaretroviruses as well as alpharetroviruses by providing critical carboxy-terminal domains to the intasome core that cannot be provided in cis because of evolutionarily restrictive catalytic core domain-carboxy-terminal domain linker regions. The octameric architecture of the intasome of mouse mammary tumour virus provides new insight into the structural basis of retroviral DNA integration.
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47
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Recognition of the nucleosome by chromatin factors and enzymes. Curr Opin Struct Biol 2016; 37:54-61. [PMID: 26764865 DOI: 10.1016/j.sbi.2015.11.014] [Citation(s) in RCA: 96] [Impact Index Per Article: 10.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2015] [Revised: 11/23/2015] [Accepted: 11/28/2015] [Indexed: 11/23/2022]
Abstract
Dynamic expression of the genome requires coordinated binding of chromatin factors and enzymes that carry out genome-templated processes. Until recently, the molecular mechanisms governing how these factors and enzymes recognize and act on the fundamental unit of chromatin, the nucleosome core particle, have remained a mystery. A small, yet growing set of structures of the nucleosome in complex with chromatin factors and enzymes highlights the importance of multivalency in defining nucleosome binding and specificity. Many such interactions include an arginine anchor motif, which targets a unique acidic patch on the nucleosome surface. These emerging paradigms for chromatin recognition will be discussed, focusing on several recent structural breakthroughs.
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48
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Thierry E, Deprez E, Delelis O. Different Pathways Leading to Integrase Inhibitors Resistance. Front Microbiol 2016. [PMID: 28123383 DOI: 10.3389/fmicb.2016.02165/bibtex] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/27/2023] Open
Abstract
Integrase strand-transfer inhibitors (INSTIs), such as raltegravir (RAL), elvitegravir, or dolutegravir (DTG), are efficient antiretroviral agents used in HIV treatment in order to inhibit retroviral integration. By contrast to RAL treatments leading to well-identified mutation resistance pathways at the integrase level, recent clinical studies report several cases of patients failing DTG treatment without clearly identified resistance mutation in the integrase gene raising questions for the mechanism behind the resistance. These compounds, by impairing the integration of HIV-1 viral DNA into the host DNA, lead to an accumulation of unintegrated circular viral DNA forms. This viral DNA could be at the origin of the INSTI resistance by two different ways. The first one, sustained by a recent report, involves 2-long terminal repeat circles integration and the second one involves expression of accumulated unintegrated viral DNA leading to a basal production of viral particles maintaining the viral information.
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Affiliation(s)
- Eloïse Thierry
- Laboratoire de Biologie et Pharmacologie Appliquée, CNRS UMR8113, Ecole Normale Supérieure de Cachan, Université Paris-Saclay Cachan, France
| | - Eric Deprez
- Laboratoire de Biologie et Pharmacologie Appliquée, CNRS UMR8113, Ecole Normale Supérieure de Cachan, Université Paris-Saclay Cachan, France
| | - Olivier Delelis
- Laboratoire de Biologie et Pharmacologie Appliquée, CNRS UMR8113, Ecole Normale Supérieure de Cachan, Université Paris-Saclay Cachan, France
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Li M, Lin S, Craigie R. Outer domains of integrase within retroviral intasomes are dispensible for catalysis of DNA integration. Protein Sci 2015; 25:472-8. [PMID: 26537415 DOI: 10.1002/pro.2837] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2015] [Revised: 10/28/2015] [Accepted: 10/30/2015] [Indexed: 12/16/2022]
Abstract
Retroviral DNA integration is mediated by nucleoprotein complexes (intasomes) comprising a pair of viral DNA ends synapsed by a tetramer of integrase. Current integrase inhibitors act on intasomes rather than free integrase protein. Structural and functional studies of intasomes are essential to understand their mechanism of action and how the virus can escape by mutation. To date, prototype foamy virus (PFV) is the only retrovirus for which high-resolution structures of intasomes have been determined. In the PFV intasome structure, only the core domains of the outer subunits are ordered; the N-terminal domain, C-terminal domain, and N-terminal extension domain are disordered. Are these "missing domains" required for function or are they dispensable? We have devised a strategy to assemble "hetero-intasomes" in which the outer domains are not present as a tool to assess the functional role of the missing domains for catalysis of integration. We find that the disordered domains of outer subunits are not required for intasome assembly or catalytic activity as catalytic core domains can substitute for the outer subunits in the case of both PFV and HIV-1 intasomes.
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Affiliation(s)
- Min Li
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, 20892
| | - Shiqiang Lin
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, 20892
| | - Robert Craigie
- Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, 20892
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Abstract
The retroviral integrases are virally encoded, specialized recombinases that catalyze the insertion of viral DNA into the host cell's DNA, a process that is essential for virus propagation. We have learned a great deal since the existence of an integrated form of retroviral DNA (the provirus) was first proposed by Howard Temin in 1964. Initial studies focused on the genetics and biochemistry of avian and murine virus DNA integration, but the pace of discovery increased substantially with advances in technology, and an influx of investigators focused on the human immunodeficiency virus. We begin with a brief account of the scientific landscape in which some of the earliest discoveries were made, and summarize research that led to our current understanding of the biochemistry of integration. A more detailed account of recent analyses of integrase structure follows, as they have provided valuable insights into enzyme function and raised important new questions.
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Affiliation(s)
- Mark D Andrake
- Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111; ,
| | - Anna Marie Skalka
- Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111; ,
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