Herpesviruses require membrane fusion for entry and spread, a process facilitated by the fusion glycoprotein B (gB) and the regulatory factor gH/gL. The human cytomegalovirus (HCMV) gH/gL can be modified by the accessory protein gO, or the set of proteins UL128, UL130, and UL131. While the binding of the gH/gL/gO and gH/gL/UL128-131 complexes to cellular receptors, including PDGFRα and NRP2, has been well-characterized structurally, the specific role of receptor engagements by the gH/gL/gO and gH/gL/UL128-131 in regulation of fusion has remained unclear. We describe a cell-cell fusion assay that can quantitatively measure fusion on a timescale of minutes and demonstrate that binding of gH/gL/gO to PDGFRα dramatically enhances gB-mediated cell-cell fusion. In contrast, gH/gL/pUL128-131-regulated fusion is significantly slower, and gH/gL alone cannot promote gB fusion activity within this timescale. The genetic diversity of gO influenced the observed cell-cell fusion rates, correlating with previously reported effects on HCMV infectivity. Mutations in gL that had no effect on the formation of gH/gL/gO or binding to PDGFRa dramatically reduced the cell-cell fusion rate, suggesting that gL plays a critical role in linking the gH/gL/gO-PDGFRa receptor binding to activation of gB. Several neutralizing human monoclonal antibodies were found to potently block gH/gL/gO-PDGFRa-regulated cell-cell fusion, suggesting this mechanism as a therapeutic target.

Development of vaccines and therapeutics targeting the fusion apparatus of human cytomegalovirus (HCMV) has been limited by the lack of an in vitro cell-cell fusion assay that faithfully models the receptor-dependent fusion characteristic of HCMV entry. The cell-cell fusion assay described here demonstrated that the binding of gH/gL/gO to its receptor, PDGFRα, serves to regulate the activity of the fusion protein gB, and this is specifically vulnerable to inhibition by neutralizing antibodies. Moreover, the measurement of fusion kinetics allows for mutational studies of the fusion mechanism, assessing the influence of genetic diversity among the viral glycoproteins and studying the mechanism of neutralizing antibodies.

Human cytomegalovirus (hCMV) poses a severe threat to fetuses, newborns, and immunocompromised individuals. No approved vaccines and limited treatment options are current medical challenges. Here, we analyze the human B cell responses to glycoprotein B (gB) in three top hCMV neutralizers from a cohort of 283 individuals with latent-infected hCMV. By single-cell amplification of memory B cells, we identify a cluster of potent neutralizing monoclonal antibodies (nAbs) that competitively recognize an unknown vulnerable site on gB antigenic domain 5 (AD-5). This cluster of nAbs functionally outperforms the nAbs utilized in clinical trials. Cryoelectron microscopy (cryo-EM) unveils the structural basis of the neutralization mechanism of an antibody directly targeting the fusion subdomain on AD-5. Moreover, immunological analyses of human and mouse sera have preliminarily validated the potential superiority of AD-5-focused immune responses. Overall, our results will support the development of optimized gB-based vaccines and provide promising prophylactic and therapeutic antibody candidates against hCMV infection.

Human cytomegalovirus (HCMV) is a critical pathogen in immunocompromised populations, such as organ transplant recipients as well as congenitally infected neonates with immature immune systems. Despite decades of research and the growing financial burden associated with the management of HCMV, there is no licensed vaccine to date. In this review, we aim to outline the complexity of HCMV and the antigens it presents and the journey and challenges of developing an effective HCMV vaccine, as well as further highlight the recent analyses of the most successful vaccine candidate so far-gB/MF59.

Human cytomegalovirus (HCMV) is a β-herpesvirus that establishes asymptomatic infections in immunocompetent individuals but can cause severe or even life-threatening symptoms in immunocompromised patients. HCMV can replicate in a wide variety of cells through the engagement of diverse cell factors with the viral envelope protein gH/gL/gO (trimer) or gH/gL/UL128/UL130/UL131a (pentamer), allowing for systemic spread within the human host. This study explores HCMV infection tropism and dynamics in human microglia, demonstrating the susceptibility of microglia to both clinical and laboratory HCMV strains, albeit with lower efficacy for the laboratory strain, implying that both the gH/gL-trimer and -pentamer can mediate virus entry in microglia. The importance of the gH/gL pentamer for virus entry was demonstrated by the inhibition of virus infection upon pre-incubation with a soluble neuropilin-2 (NRP-2) entry factor. Further, we demonstrated that HCMV infection can be effectively inhibited by monoclonal antibodies specific for the gH/gL complexes and HCMV hyperimmunoglobulin. Lastly, we report that microglia infection can be prevented by newly characterized chemical entry inhibitors. Altogether, these findings underscore the potential of microglia as valuable models for studying HCMV neurotropism and strategies to block virus infection in cells that can impact neurological disorders.

Fetal infection with human cytomegalovirus (hCMV) can cause sensorineural hearing loss and vestibular impairment, yet its pathogenesis remains unclear. This study aims to identify potential target cell types of hCMV in the human fetal inner ear. Viral particles use several envelope glycoproteins to enter target cells, including the pentameric complex, the trimeric complex and glycoprotein B. Platelet-derived growth factor receptor alpha (PDGFRA) serves as the receptor in fibroblasts, neuropilin-2 (NRP2) in epithelial, endothelial and dendritic cells as well as in leukocytes. Upon binding of these glycoproteins, glycoprotein B initiates membrane fusion which is proposed to be mediated by EGFR. When and where these proteins are expressed in the fetal inner ear during development is unknown. To address this, expression patterns of PDGFRA, NRP2 and EGFR were investigated in human fetal inner ear tissue using single-nucleus RNA sequencing data (first trimester: N = 2) and immunohistochemistry (first trimester: N = 6, second trimester: N = 5). PDGFRA gene and protein expression was detected in mesenchymal cells, NRP2 protein expression in epithelial cells and endothelial cells, and EGFR gene and protein expression in both epithelial cells and mesenchymal cells. Notably, all three receptors were present in tissue from the first and second trimesters. In conclusion, hCMV host receptors PDGFRA, NRP2 and EGFR are expressed in mesenchymal, epithelial and endothelial cells within the cochlea and vestibular organs during the first and second trimesters. These cell types may serve as targets for hCMV infection of the fetal inner ear.

Corneal endotheliitis is an inflammatory process, most commonly of viral etiology, that manifests clinically with features including corneal edema, keratic precipitates, and a mild anterior chamber reaction. Several studies have implicated human herpesviruses from the Herpesviridae family as primary causes of corneal endotheliitis, including cytomegalovirus (CMV), varicella zoster virus (VZV), and herpes simplex viruses 1 and 2 (HSV-1 and HSV-2). This review critically evaluates the present literature surrounding herpesvirus infections of the corneal endothelium.

Intercellular communication is fundamental to multicellular life and a core determinant of outcomes during viral infection, where the common goals of virus and host for persistence and replication are generally at odds. Hosts rely on encoded innate and adaptive immune responses to detect and clear viral pathogens, while viruses can exploit or disrupt these pathways and other intercellular communication processes to enhance their spread and promote pathogenesis. While virus-induced signaling can result in systemic changes to the host, striking alterations are observed within the cellular microenvironment directly surrounding a site of infection, termed the virus microenvironment (VME). Mechanisms employed by viruses to condition their VMEs are emerging and are critical for understanding the biology and pathologies of viral infections. Recent advances in experimental approaches, including proteomic methods, have enabled study of the VME in unprecedented detail. In this review article, we provide a primer on proteomic approaches used to study how viral infections alter intercellular communication, highlighting the ways in which these approaches have been implemented and the exciting biology they have uncovered. First, we consider the different molecules secreted by an infected cell, including proteins, either soluble or contained within extracellular vesicles, and metabolites. We further discuss the modalities of interactions facilitated by alteration at the cell surface of infected cells, including immunopeptide presentation and interactions with the extracellular matrix. Finally, we review spatial profiling approaches that have allowed distinguishing how specific subpopulations of cells within a VME respond to infection and alter their protein composition, discussing valuable insights these methods have offered.

Elephant endotheliotropic herpesvirus (EEHV), a persistent threat, has caused significant mortality among young Asian elephants (Elephas maximus), raising serious concerns for the conservation of this endangered species. Given the urgent need for protective measures, research into EEHV vaccine development has become increasingly critical. This study evaluated the immune response in mice following immunization with an EEHV1A-glycoprotein B (gB) subunit vaccine. The vaccine incorporated gBF1 and gBF2, corresponding to segments of the gB ectodomains I and IV, respectively, along with emulsion adjuvants Montanide™ ISA 206 VG and incomplete Freund's adjuvant (IFA). The findings revealed that both gBF1 and gBF2, when paired with these adjuvants, were capable of inducing strong humoral immune responses against EEHV-gB, as demonstrated by the ability of sera from immunized mice to detect EEHV-gB ex vivo. Additionally, in terms of cellular immunity, the vaccine formulations predominantly activated CD4+ T cells, including both Th1 (IFN-γ+) and Th2 (IL-4+) cells, with no significant activation of CD8+ T cells. If also applicable in elephants, gB-based vaccines would be a significant step forward in the fight against EEHV.

Cervical cancer remains a significant health concern worldwide and the primary cause of cancerous cervical lesions is the infection with high-risk human papillomavirus (HR-HPV). However, emerging evidence suggests that HR-HPV infection alone is insufficient for cancer development, and other co-factors may contribute to cervical carcinogenesis. Human cytomegalovirus (HCMV), a common herpesvirus frequently detected in cervical cancer samples, has demonstrated oncogenic potential.

This review aims to explore the molecular interactions between HR-HPV and HCMV in promoting cervical cancer progression.

A comprehensive search was conducted in PubMed and Google Scholar, focusing on articles examining the role of HCMV in cervical tissues and/or cells, selected based on relevance and significance.

The reviewed literature indicates that HCMV and HR-HPV share several oncogenic mechanisms that could drive cervical cell transformation.

Both viruses may synergistically promote cervical epithelial transformation and tumor progression in multiple ways. HR-HPV may facilitate HCMV entry by increasing host cell receptors essential for viral attachment. Additionally, HR-HPV and HCMV may cooperatively disrupt cellular processes, enhancing carcinogenesis. Both viruses may also modulate the local immune environment, enabling immune evasion and lesion persistence. However, further in vitro and in vivo studies are required to validate these hypotheses.

Herpesviruses require membrane fusion for entry and spread, a process facilitated by the fusion glycoprotein B (gB) and the regulatory factor gH/gL. The human cytomegalovirus (HCMV) gH/gL can be modified by the accessory protein gO, or the set of proteins UL128, UL130 and UL131. While the binding of the gH/gL/gO and gH/gL/UL128-131 complexes to cellular receptors including PDFGRα and NRP2 has been well-characterized structurally, the specific role of receptor engagements by the gH/gL/gO and gH/gL/UL128-131 in regulation of fusion has remained unclear. We describe a cell-cell fusion assay that can quantitatively measure fusion on a timescale of minutes and demonstrate that binding of gH/gL/gO to PDGFRα dramatically enhances gB-mediated cell-cell fusion. In contrast, gH/gL/pUL128-131-regulated fusion is significantly slower and gH/gL alone cannot promote gB fusion activity within this timescale. The genetic diversity of gO influenced the observed cell-cell fusion rates, correlating with previously reported effects on HCMV infectivity. Mutations in gL that had no effect on formation of gH/gL/gO or binding to PDGFRα dramatically reduced the cell-cell fusion rate, suggesting that gL plays a critical role in linking the gH/gL/gO-PDGFRα receptor-binding to activation of gB. Several neutralizing human monoclonal antibodies were found to potently block gH/gL/gO-PDGFRα regulated cell-cell fusion, suggesting this mechanism as a therapeutic target.

Sodium taurocholate cotransporting polypeptide (NTCP) has been identified as the cellular receptor for HBV. However, hepatocytes expressing NTCP exhibit varying susceptibilities to HBV infection. This study aimed to investigate whether other host factors modulate the process of HBV infection.

Liver biopsy samples obtained from children with hepatitis B were used for single-cell sequencing and susceptibility analysis. Primary human hepatocytes, HepG2-NTCP cells, and human liver chimeric mice were used to analyze the effect of candidate host factors on HBV infection.

Single-cell sequencing and susceptibility analysis revealed a positive correlation between neuropilin-1 (NRP1) expression and HBV infection. In the HBV-infected cell model, NRP1 overexpression before HBV inoculation significantly enhanced viral attachment and internalization, and promoted viral infection in the presence of NTCP. Mechanistic studies indicated that NRP1 formed a complex with LHBs (large hepatitis B surface proteins) and NTCP. The NRP1 b domain mediated its interaction with conserved arginine residues at positions 88 and 92 in the preS1 domain of LHBs. This NRP1-preS1 interaction subsequently promoted the binding of preS1 to NTCP, facilitating viral infection. Moreover, disruption of the NRP1-preS1 interaction by the NRP1 antagonist EG00229 significantly attenuated the binding affinity between NTCP and preS1, thereby inhibiting HBV infection both in vitro and in vivo.

Our findings indicate that NRP1 is a novel host factor for HBV infection, which interacts with preS1 and NTCP to modulate HBV entry into hepatocytes.

HBV infection is a global public health problem, but the understanding of the early infection process of HBV remains limited. Through single-cell sequencing, we identified a novel host factor, NRP1, which modulates HBV entry by interacting with HBV preS1 and NTCP. Moreover, antagonists targeting NRP1 can inhibit HBV infection both in vitro and in vivo. This study could further advance our comprehension of the early infection process of HBV.

Membrane proteins, especially extracellular domains, are key therapeutic targets due to their role in cell communication and associations. Yet, their functions and interactions often remain unclear. This study presents a general method to discover interactions of membrane proteins with immune cells and subsequently to deorphanize their respective receptors. We developed a comprehensive recombinant protein library of extracellular domains of human transmembrane proteins and proteins found in the ER-Golgi-lysosomal systems. Using this library, we conducted a flow-cytometric screen that identified several cell surface binding events, including an interaction between carbonic anhydrase 9 (CAH9/CA9/CAIX) and CD14high cells. Further analysis revealed this interaction was indirect and mediated via platelets bound to the monocytes. CA9, best known for its diverse roles in cancer, is a promising therapeutic target. We utilized our library to develop an AlphaLISA high-throughput screening assay, identifying CLEC2 as one robust CA9 binding partner. A five-amino-acid sequence (EDLPT) in CA9, identical to a CLEC2 binding domain in Podoplanin (PDPN), was found to be essential for this interaction. Like PDPN, CA9-induced CLEC2 signaling is mediated via Syk. A Hodgkin's lymphoma cell line (HDLM-2) endogenously expressing CA9 can activate Syk-dependent CLEC2 signaling, providing enticing evidence for a novel function of CA9 in hematological cancers. In conclusion, we identified numerous interactions with monocytes and platelets and validated one, CA9, as an endogenous CLEC2 ligand. We provide a new list of other putative CA9 interaction partners and uncovered CA9-induced CLEC2 activation, providing new insights for CA9-based therapeutic strategies.

Despite all the progress in treating SARS-CoV-2, escape mutants to current therapies remain a constant concern. Promising alternative treatments for current and future coronaviruses are those that limit escape mutants by inhibiting multiple pathogenic targets, analogous to the current strategies for treating HCV and HIV. With increasing popularity and ease of manufacturing of RNA technologies for vaccines and drugs, therapeutic microRNAs represent a promising option. In the present work, miR-24-3p was identified to inhibit SARS-CoV-2 entry, replication, and production; furthermore, this inhibition was retained against common mutations improving SARS-CoV-2 fitness. To determine the mechanism of action, bioinformatic tools were employed, identifying numerous potential effectors promoting infection targeted by miR-24-3p. Of these targets, several key host proteins for priming and facilitating SARS-CoV-2 entry were identified: furin, NRP1, NRP2, and SREBP2. With further experimental analysis, we show that miR-24-3p directly downregulates these viral entry factors to impede infection when producing virions and when infecting the target cell. Furthermore, we compare the findings with coronavirus, HCoV-229E, which relies on different factors strengthening the miR-24-3p mechanism. Taken together, the following work suggests that miR-24-3p could be an avenue to treat current coronaviruses and those likely to emerge.

Human cytomegalovirus (HCMV) infection remains a significant global health challenge, particularly for immunocompromised individuals and newborns. This comprehensive review synthesizes current knowledge on HCMV pathogenesis, prevention, and treatment strategies. We examine the molecular mechanisms of HCMV entry, focusing on the structure and function of key envelope glycoproteins (gB, gH/gL/gO, gH/gL/pUL128-131) and their interactions with cellular receptors such as PDGFRα, NRP2, and THBD. The review explores HCMV's sophisticated immune evasion strategies, including interference with pattern recognition receptor signaling, modulation of antigen presentation, and regulation of NK and T cell responses. We highlight recent advancements in developing neutralizing antibodies, various vaccine strategies (live-attenuated, subunit, vector-based, DNA, and mRNA), antiviral compounds (both virus-targeted and host-targeted), and emerging cellular therapies such as TCR-T cell approaches. By integrating insights from structural biology, immunology, and clinical research, we identify critical knowledge gaps and propose future research directions. This analysis aims to stimulate cross-disciplinary collaborations and accelerate the development of more effective prevention and treatment strategies for HCMV infections, addressing a significant unmet medical need.

Intercellular protein-protein interactions (PPIs) have pivotal roles in biological functions and diseases. Membrane proteins are therefore a major class of drug targets. However, studying such intercellular PPIs is challenging because of the properties of membrane proteins. Current methods commonly use purified or modified proteins that are not physiologically relevant and hence might mischaracterize interactions occurring in vivo. Here, we describe Cell-Int: a cell interaction assay for studying plasma membrane PPIs. The interaction signal is measured through conjugate formation between two populations of cells each expressing either a ligand or a receptor. In these settings, membrane proteins are in their native environment thus being physiologically relevant. Cell-Int has been applied to the study of diverse protein partners, and enables to investigate the inhibitory potential of blocking antibodies, as well as the retargeting of fusion proteins for therapeutic development. The assay was also validated for screening applications and could serve as a platform for identifying new protein interactors.

Human cytomegalovirus (HCMV) causes severe birth defects, lifelong health complications, and $4 billion in annual costs in the United States alone. A major challenge in vaccine design is the incomplete understanding of the diverse protein complexes the virus uses to infect cells. In Herpesviridae, the gH/gL glycoprotein heterodimer is expected to be a basal element of virion cell entry machinery. For HCMV, gH/gL forms a "trimer" with gO and a "pentamer" with UL128, UL130, and UL131A, with each complex binding distinct receptors to enter varied cell types. Here, we reveal a third glycoprotein complex, abundant in HCMV virions, which significantly enhances infection of endothelial cells. In this "3-mer" complex, gH, without gL, associates with UL116 and UL141, an immunoevasin previously known to function in an intracellular role. Cryo-EM reveals the virion-surface 3-mer is structurally unique among Herpesviridae gH complexes, with gH-only scaffolding, UL141-mediated dimerization and a heavily glycosylated UL116 cap. Given that antibodies directed at gH and UL141 each can restrict HCMV replication, our work highlights this virion surface complex as a new target for vaccines and antiviral therapies.

Human cytomegalovirus (CMV) causes serious developmental disabilities in newborns infected in utero following oral acquisition by the mother. Thus, neutralizing antibodies in maternal saliva have potential to prevent maternal infection and, consequently, fetal transmission and disease. Based on standard cell culture models, CMV entry mediators (and hence neutralizing targets) are cell type-dependent: entry into fibroblasts requires glycoprotein B (gB) and a trimeric complex (TC) of glycoproteins H, L, and O, whereas endothelial and epithelial cell entry additionally requires a pentameric complex (PC) of glycoproteins H and L with UL128, UL130, and UL131A. However, as the mediators of mucosal cell entry and the potential impact of cellular differentiation remained unclear, the present studies utilized mutant viruses, neutralizing antibodies, and soluble TC-receptor to determine the entry mediators required for infection of mucocutaneus cell lines and primary tonsil epithelial cells. Entry into undifferentiated cells was largely PC-dependent, but PC-independent entry could be induced by differentiation. TC-independent entry was also observed and varied by cell line and differentiation. Infection of primary tonsil cells from some donors was entirely TC-independent. In contrast, an antibody to gB or disruption of virion attachment using heparin blocked entry into all cells. These findings indicate that CMV entry into the spectrum of cell types encountered in vivo is likely to be more complex than has been suggested by standard cell culture models and may be influenced by the relative abundance of virion envelope glycoprotein complexes as well as by cell type, tissue of origin, and state of differentiation.

The Pentamer complex of Human Cytomegalovirus (HCMV) consists of the viral glycoproteins gH, gL, UL128, UL130, and UL131 and is incorporated into infectious virions. HCMV strains propagated extensively in vitro in fibroblasts carry UL128, UL130, or UL131 alleles that do not make a functional complex and thus lack Pentamer function. Adding functional Pentamer to such strains decreases virus growth in fibroblasts. Here, we show that the Pentamer inhibits productive HCMV replication in fibroblasts by repressing viral Immediate Early (IE) transcription. We show that ectopic expression of the viral IE1 protein, a target of Pentamer-mediated transcriptional repression, complements the growth defect of a Pentamer-positive virus. Furthermore, we show that the Pentamer also represses viral IE transcription in cell types where HCMV in vitro latency is studied. Finally, we identify UL130 as a functional subunit of the Pentamer for IE transcriptional repression and demonstrate that cyclic AMP Response Element (CRE) and NFkB sites within the Major Immediate Early Promoter that drives IE1 transcription contribute to this repression. We conclude that the HCMV Pentamer represses viral IE transcription.

This phase 1 trial evaluated the safety, reactogenicity, and immunogenicity of mRNA-1647, a messenger RNA (mRNA)-based cytomegalovirus (CMV) vaccine, in CMV-seronegative and -seropositive adults.

Participants were randomly assigned to receive 30, 90, 180, or 300 µg of mRNA-1647 or placebo on a 0-, 2-, and 6-month schedule and followed for 12 months after the last dose.

A total of 154 (80 CMV-seronegative and 74 CMV-seropositive) participants were enrolled; 118 participants were randomized to mRNA-1647 and 36 to placebo. Mean (standard deviation) age was 32.5 (8.6) and 35.1 (8.9) years in the placebo and mRNA-1647 groups, respectively, in phase B (63% and 64% female) and 42.5 (6.2) and 33.3 (8.7) years, respectively, in phase C (2% and 16% female). No deaths, related serious adverse events, or adverse events of special interest were reported. Most adverse reactions were grade ≤2 severity. Increased neutralizing antibody, binding antibody, and antigen-specific cell-mediated responses were observed across mRNA-1647 treatment groups, regardless of CMV serostatus.

This phase 1, first-in-human trial demonstrated that mRNA-1647 has an acceptable safety profile in adults and elicits humoral and cellular immune responses. Clinical Trials Registration.  NCT03382405.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus found in human breast milk that is frequently transmitted from HCMV-seropositive mothers to their infants during the postnatal period. Despite extensive research, the mechanisms underlying HCMV transmission from breast milk and the anatomical location at which virus transfer takes place remain unclear. Breast milk contains many uniquely differentiated macrophages that undergo specific morphological and functional modifications in the mammary gland during lactation. Although the existence of permissive HCMV infection in differentiated macrophages has been well-described, the role of breast milk in this process remains unknown. Herein, we report that exposure of isolated peripheral blood monocytes to breast milk induces their differentiation into macrophages that exhibit an M2 phenotype (CD14highCD163highCD68highCD206high) and promotes a productive and sustained HCMV infection. We also found that breast milk triggers macrophage proliferation and thus sustains a unique population of proliferating, long-lived, and HCMV-susceptible macrophages that are capable of ongoing production of infectious virions. These results suggest a mechanism that explains chronic HCMV shedding into the breast milk of postpartum seropositive mothers. We also found that HCMV virions released from breast milk-induced macrophages generate a productive infection in primary infant tonsil epithelial cells. Collectively, our results suggest that breast milk may facilitate HCMV transmission from mother to infant via the oropharyngeal mucosa.

While human cytomegalovirus (HCMV) is frequently detected in the breast milk of HCMV-seropositive women and is often transmitted to infants via breastfeeding, the mechanisms by which this transmission occurs remain unclear. In this study, we modeled HCMV transmission at the oropharyngeal mucosa. We treated human monocytes with breast milk to mimic the lactating mammary gland microenvironment. We found that monocytes differentiated into macrophages with an M2 phenotype, which were highly permissive for HCMV. We also discovered that breast milk induces macrophage proliferation. Thus, exposure to breast milk increased the number of HCMV-susceptible macrophages and supported high levels of infectious HCMV. We found that HCMV virions released from breast milk-induced macrophages could infect primary infant tonsil epithelial cells. Collectively, these findings reveal the dual role of breast milk that induces the differentiation and proliferation of macrophages in the mammary gland and thus facilitates mother-to-child HCMV transmission at the oropharyngeal mucosa.

Cytomegalovirus (CMV), a representative member of the Betaherpesvirinae subfamily of herpesviruses, is common in the human population, but immunocompetent individuals are generally asymptomatic when infected with this virus. However, in immunocompromised individuals and immunologically immature fetuses and newborns, CMV can cause a wide range of often long-lasting morbidities and even death. CMV is not only widespread throughout the population but it is also widespread in its hosts, infecting and establishing latency in nearly all tissues and organs. Thus, understanding the pathogenesis of and immune responses to this virus is a prerequisite for developing effective prevention and treatment strategies. Multiple arms of the immune system are engaged to contain the infection, and general concepts of immune control of CMV are now reasonably well understood. Nonetheless, in recent years, tissue-specific immune responses have emerged as an essential factor for resolving CMV infection. As tissues differ in biology and function, so do immune responses to CMV and pathological processes during infection. This review discusses state-of-the-art knowledge of the immune response to CMV infection in tissues, with particular emphasis on several well-studied and most commonly affected organs.

Plasma membrane proteins (PMPs) play critical roles in a myriad of physiological and disease conditions. A unique subset of PMPs functions through interacting with each other in trans at the interface between two contacting cells. These trans-interacting PMPs (tiPMPs) include adhesion molecules and ligands/receptors that facilitate cell-cell contact and direct communication between cells. Among the tiPMPs, a significant number have apparent extracellular binding domains but remain orphans with no known binding partners. Identification of their potential binding partners is therefore important for the understanding of processes such as organismal development and immune cell activation. While a number of methods have been developed for the identification of protein binding partners in general, very few are applicable to tiPMPs, which interact in a two-dimensional fashion with low intrinsic binding affinities. In this review, we present the significance of tiPMP interactions, the challenges of identifying binding partners for tiPMPs, and the landscape of method development. We describe current avidity-based screening approaches for identifying novel tiPMP binding partners and discuss their advantages and limitations. We conclude by highlighting the importance of developing novel methods of identifying new tiPMP interactions for deciphering the complex protein interactome and developing targeted therapeutics for diseases.

The secretome is composed of cell surface membrane proteins and extracellular secreted proteins that are synthesized via secretory machinery, accounting for approximately one-third of human protein-encoding genes and playing central roles in cellular communication with the external environment. Secretome protein-protein interactions (SPPIs) mediate cell proliferation, apoptosis, and differentiation, as well as stimulus- or cell-specific responses that regulate a diverse range of biological processes. Aberrant SPPIs are associated with diseases including cancer, immune disorders, and illness caused by infectious pathogens. Identifying the receptor/ligand for a secretome protein or pathogen can be a challenging task, and many SPPIs remain obscure, with a large number of orphan receptors and ligands, as well as viruses with unknown host receptors, populating the SPPI network. In addition, proteins with known receptors/ligands may also interact with alternative uncharacterized partners and exert context-dependent effects. In the past few decades, multiple varied approaches have been developed to identify SPPIs, and these methods have broad applications in both basic and translational research. Here, we review and discuss the technologies for SPPI profiling and the application of these technologies in identifying novel targets for immunotherapy and anti-infectious agents.

Human cytomegalovirus (HCMV) displays a broad cell tropism, and the infection of biologically relevant cells such as epithelial, endothelial, and hematopoietic cells supports viral transmission, systemic spread, and pathogenesis in the human host. HCMV strains differ in their ability to infect and replicate in these cell types, but the genetic basis of these differences has remained incompletely understood. In this study, we investigated HCMV strain VR1814, which is highly infectious for epithelial cells and macrophages and induces cell-cell fusion in both cell types. A VR1814-derived bacterial artificial chromosome (BAC) clone, FIX-BAC, was generated many years ago but has fallen out of favor because of its modest infectivity. By sequence comparison and genetic engineering of FIX, we demonstrate that the high infectivity of VR1814 and its ability to induce syncytium formation in epithelial cells and macrophages depends on VR1814-specific variants of the envelope glycoproteins gB, UL128, and UL130. We also show that UL130-neutralizing antibodies inhibit syncytium formation, and a FIX-specific mutation in UL130 is responsible for its low infectivity by reducing the amount of the pentameric glycoprotein complex in viral particles. Moreover, we found that a VR1814-specific mutation in US28 further increases viral infectivity in macrophages, possibly by promoting lytic rather than latent infection of these cells. Our findings show that variants of gB and the pentameric complex are major determinants of infectivity and syncytium formation in epithelial cells and macrophages. Furthermore, the VR1814-adjusted FIX strains can serve as valuable tools to study HCMV infection of myeloid cells.IMPORTANCEHuman cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and the leading cause of congenital infections. HCMV infects various cell types, including epithelial cells and macrophages, and some strains induce the fusion of neighboring cells, leading to the formation of large multinucleated cells called syncytia. This process may limit the exposure of the virus to host immune factors and affect pathogenicity. However, the reason why some HCMV strains exhibit a broader cell tropism and why some induce cell fusion more than others is not well understood. We compared two closely related HCMV strains and provided evidence that small differences in viral envelope glycoproteins can massively increase or decrease the virus infectivity and its ability to induce syncytium formation. The results of the study suggest that natural strain variations may influence HCMV infection and pathogenesis in humans.

Human cytomegalovirus (HCMV) is a β-herpesvirus that poses severe disease risk for immunocompromised patients who experience primary infection or reactivation. Development and optimization of safe and effective anti-HCMV therapeutics is of urgent necessity for the prevention and treatment of HCMV-associated diseases in diverse populations. The use of neutralizing monoclonal antibodies (mAbs) to limit HCMV infection poses a promising therapeutic strategy, as anti-HCMV mAbs largely inhibit infection by targeting virion glycoprotein complexes. In contrast, the small-molecule compounds currently approved for patients (e.g., ganciclovir, letermovir, and maribavir) target later stages of the HCMV life cycle. Here, we present a broadly neutralizing human mAb, designated 1C10, elicited from a VelocImmune mouse immunized with infectious HCMV particles. Clone 1C10 neutralizes infection after virion binding to cells by targeting gH/gL envelope complexes and potently reduces infection of diverse HCMV strains in fibroblast, trophoblast, and epithelial cells. Antibody competition assays found that 1C10 recognizes a region of gH associated with broad neutralization and binds to soluble pentamer in the low nanomolar range. Importantly, 1C10 treatment significantly reduced virus proliferation in both fibroblast and epithelial cells. Further, the combination treatment of mAb 1C10 with ganciclovir reduced HCMV infection and proliferation in a synergistic manner. This work characterizes a neutralizing human mAb for potential use as a HCMV treatment, as well as a possible therapeutic strategy utilizing combination-based treatments targeting disparate steps of the viral life cycle. Collectively, the findings support an antibody-based therapy to effectively treat patients at risk for HCMV-associated diseases.

Human cytomegalovirus is a herpesvirus that infects a large proportion of the population and can cause significant disease in diverse patient populations whose immune systems are suppressed or compromised. The development and optimization of safe anti-HCMV therapeutics, especially those that have viral targets and inhibition mechanisms different from current HCMV treatments, are of urgent necessity to better public health. Human monoclonal antibodies (mAbs) that prevent HCMV entry of cells were identified by immunizing transgenic mice and screened for broad and effective neutralization capability. Here, we describe one such mAb, which was found to target gH/gL envelope complexes and effectively limit HCMV infection and dissemination. Further, administration of the antibody in combination with the antiviral drug ganciclovir inhibited HCMV in a synergistic manner, highlighting this approach and the use of anti-HCMV mAbs more broadly, as a potential therapeutic strategy for the treatment of diverse patient populations.

The complement system is an evolutionarily ancient component of innate immunity that serves as an important first line of defense against pathogens, including viruses. In response to infection, the complement system can be activated by three distinct yet converging pathways (classical, lectin, and alternative) capable of engaging multiple antiviral host responses to confront acute, chronic, and recurrent viral infections. Complement can exert profound antiviral effects via multiple mechanisms including the induction of inflammation and chemotaxis to sites of infection, neutralization/opsonization of viruses and virally infected cells, and it can even shape adaptive immune responses. With millions of years of co-evolution and the ability to establish life-long infections, herpesviruses have evolved unique mechanisms to counter complement-mediated antiviral defenses, thus enabling their survival and replication within humans. This review aims to comprehensively summarize how human herpesviruses engage with the complement system and highlight our understanding of the role of complement in human cytomegalovirus (HCMV) infection, immunity, and viral replication. Herein we describe the novel and unorthodox roles of complement proteins beyond their roles in innate immunity and discuss gaps in knowledge and future directions of complement and HCMV research.

Cytomegalovirus (CMV) infection is a significant clinical concern in newborns, immunocompromised patients with acquired immunodeficiency syndrome (AIDS), and patients undergoing immunosuppressive therapy or chemotherapy. CMV infection affects many organs, such as the lungs, digestive organs, the central nerve system, and eyes. In addition, CMV infection sometimes occurs in immunocompetent individuals. CMV ocular diseases includes retinitis, corneal endotheliitis, and iridocyclitis. CMV retinitis often develops in infected newborns and immunocompromised patients. CMV corneal endotheliitis and iridocyclitis sometimes develop in immunocompetent individuals. Systemic infections and CMV ocular diseases often require systemic treatment in addition to topical treatment.

Mammalian orthoreovirus (reovirus) is a nonenveloped virus that establishes primary infection in the intestine and disseminates to sites of secondary infection, including the CNS. Reovirus entry involves multiple engagement factors, but how the virus disseminates systemically and targets neurons remains unclear. In this study, we identified murine neuropilin 1 (mNRP1) as a receptor for reovirus. mNRP1 binds reovirus with nanomolar affinity using a unique mechanism of virus-receptor interaction, which is coordinated by multiple interactions between distinct reovirus capsid subunits and multiple NRP1 extracellular domains. By exchanging essential capsid protein-encoding gene segments, we determined that the multivalent interaction is mediated by outer-capsid protein σ3 and capsid turret protein λ2. Using capsid mutants incapable of binding NRP1, we found that NRP1 contributes to reovirus dissemination and neurovirulence in mice. Collectively, our results demonstrate that NRP1 is an entry receptor for reovirus and uncover mechanisms by which NRPs promote viral entry and pathogenesis.

In this issue of Cell Host & Microbe, Shang et al. identify murine neuropilin 1 as a host factor that binds reovirus particles, directing cell entry and contributing to viral dissemination and neurovirulence. This study highlights the reovirus model system to investigate host receptors and their significance in viral pathogenesis.

A high human cytomegalovirus (HCMV) infection rate accompanied by an increased level of bile duct damage is observed in the perinatal period. The possible mechanism was investigated.

A total of 1,120 HCMV-positive and 9,297 HCMV-negative children were recruited, and depending on age, their liver biochemistry profile was compared. Fetal and infant biliary epithelial cells (F-BECs and I-BECs, respectively) were infected with HCMV, and the differences in cells were revealed by proteomic analysis. Protein-protein interactions were examined by coimmunoprecipitation and mass spectrometry analyses. A murine cytomegalovirus (MCMV) infection model was established to assess treatment effects.

Perinatal HCMV infection significantly increased the level of bile duct damage. Neonatal BALB/c mice inoculated with MCMV showed obvious inflammation in the portal area with an abnormal bile duct structure. Proteomics analysis showed higher CD14 expression in F-BECs than in I-BECs. CD14 siRNA administration hindered HCMV infection, and CD14-knockout mice showed lower MCMV-induced bile duct damage. HCMV infection upregulated CD55 and poly ADP-ribose polymerase-1 (PARP-1) expression in F-BECs. Coimmunoprecipitation and mass spectrometry analyses revealed formation of the CD14-CD55 complex. siRNA-mediated inhibition of CD55 expression reduced sCD14-promoted HCMV replication in F-BECs. In MCMV-infected mice, anti-mouse CD14 antibody and PARP-1 inhibitor treatment diminished cell death, ameliorated bile duct damage, and reduced mortality.

CD14 facilitates perinatal HCMV infection in BECs via CD55, and PARP-1-mediated cell death was detected in perinatal cytomegalovirus-infected BECs. These results provide new insight into the treatment of perinatal HCMV infection with bile duct damage.

Perinatal human cytomegalovirus (HCMV) infection is associated with bile duct damage, but the underlying mechanism is still unknown. We discovered that CD14 expression is increased in biliary epithelial cells during perinatal HCMV infection and facilitates viral entry through CD55. We also detected PARP-1-mediated cell death in perinatal HCMV-infected biliary epithelial cells. We showed that blocking CD14 or inhibiting PARP-1 reduced bile duct damage and mortality in a mouse model of murine cytomegalovirus infection. Our findings provide a new insight into therapeutic strategies for perinatal HCMV infection.

mRNA-1647 is an investigational mRNA-based vaccine against cytomegalovirus (CMV) that contains sequences encoding the CMV proteins glycoprotein B and pentamer. Humoral and cellular immune responses were evaluated in blood samples collected from healthy CMV-seropositive and CMV-seronegative adults who participated in a phase 1 trial of a three-dose series of mRNA-1647 (NCT03382405). Neutralizing antibody (nAb) titers against fibroblast and epithelial cell infection in sera from CMV-seronegative mRNA-1647 recipients were higher than those in sera from control CMV-seropositive samples and remained elevated up to 12 months after dose 3. nAb responses elicited by mRNA-1647 were comparable across 14 human CMV (HCMV) strains. Frequencies of antigen-specific memory B cells increased in CMV-seropositive and CMV-seronegative participants after each mRNA-1647 dose and remained elevated for up to 6 months after dose 3. mRNA-1647 elicited robust increases in frequencies and polyfunctionality of CD4+ T helper type 1 and effector CD8+ T cells in samples from CMV-seronegative and CMV-seropositive participants after stimulation with HCMV-specific peptides. The administration of three doses of mRNA-1647 to healthy adults elicited high nAb titers with wide-breadth, long-lasting memory B cells, and strong polyfunctional T-cell responses. These findings support further clinical development of the mRNA-1647 vaccine against CMV.IMPORTANCECytomegalovirus (CMV), a common virus that can infect people of all ages, may lead to serious health problems in unborn babies and those with a weakened immune system. Currently, there is no approved vaccine available to prevent CMV infection; however, the investigational messenger RNA (mRNA)-based CMV vaccine, mRNA-1647, is undergoing evaluation in clinical trials. The current analysis examined samples from a phase 1 trial of mRNA-1647 in healthy adults to better understand how the immune system reacts to vaccination. Three doses of mRNA-1647 produced a long-lasting immune response, thus supporting further investigation of the vaccine in the prevention of CMV infection.CLINICAL TRIALSRegistered at ClinicalTrials.gov (NCT03382405).

In mammals, a subset of follicle-associated epithelial (FAE) cells, known as M cells, conduct the transcytosis of antigens across the epithelium into the underlying lymphoid tissues. We previously revealed that M cells in the FAE of the chicken lung, bursa of Fabricius (bursa), and caecum based on the expression of CSF1R. Here, we applied RNA-seq analysis on highly enriched CSF1R-expressing bursal M cells to investigate their transcriptome and identify novel chicken M cell-associated genes. Our data show that, like mammalian M cells, those in the FAE of the chicken bursa also express SOX8, MARCKSL1, TNFAIP2 and PRNP. Immunohistochemical analysis also confirmed the expression of SOX8 in CSF1R-expressing cells in the lung, bursa, and caecum. However, we found that many other mammalian M cell-associated genes such as SPIB and GP2 were not expressed by chicken M cells or represented in the chicken genome. Instead, we show bursal M cells express high levels of related genes such as SPI1. Whereas our data show that bursal M cells expressed CSF1R-highly, the M cells in the small intestine lacked CSF1R and both expressed SOX8. This study offers insights into the transcriptome of chicken M cells, revealing the expression of CSF1R in M cells is tissue-specific.

Human cytomegalovirus (HCMV) infection of monocytes is essential for viral dissemination and persistence. We previously identified that HCMV entry/internalization and subsequent productive infection of this clinically relevant cell type is distinct when compared to other infected cells. We showed that internalization and productive infection required activation of epidermal growth factor receptor (EGFR) and integrin/c-Src, via binding of viral glycoprotein B to EGFR, and the pentamer complex to β1/β3 integrins. To understand how virus attachment drives entry, we compared infection of monocytes with viruses containing the pentamer vs. those without the pentamer and then used a phosphoproteomic screen to identify potential phosphorylated proteins that influence HCMV entry and trafficking. The screen revealed that the most prominent pentamer-biased phosphorylated protein was the lipid- and protein-phosphatase phosphatase and tensin homolog (PTEN). PTEN knockdown with siRNA or PTEN inhibition with a PTEN inhibitor decreased pentamer-mediated HCMV entry, without affecting trimer-mediated entry. Inhibition of PTEN activity affected lipid metabolism and interfered with the onset of the endocytic processes required for HCMV entry. PTEN inactivation was sufficient to rescue pentamer-null HCMV from lysosomal degradation. We next examined dephosphorylation of a PTEN substrate Rab7, a regulator of endosomal maturation. Inhibition of PTEN activity prevented dephosphorylation of Rab7. Phosphorylated Rab7, in turn, blocked early endosome to late endosome maturation and promoted nuclear localization of the virus and productive infection.

Immunity has evolved to balance the destructive nature of inflammation with wound healing to overcome trauma, infection, environmental insults, and rogue malignant cells. The inflammatory response is marked by overlapping phases of initiation, resolution, and post-resolution remodeling. However, the disruption of these events can lead to prolonged tissue damage and organ dysfunction, resulting long-term disease states. Macrophages are the archetypic phagocytes present within all tissues and are important contributors to these processes. Pleiotropic and highly plastic in their responses, macrophages support tissue homeostasis, repair, and regeneration, all while balancing immunologic self-tolerance with the clearance of noxious stimuli, pathogens, and malignant threats. Neuropilin-2 (Nrp2), a promiscuous co-receptor for growth factors, semaphorins, and integrins, has increasingly been recognized for its unique role in tissue homeostasis and immune regulation. Notably, recent studies have begun to elucidate the role of Nrp2 in both non-hematopoietic cells and macrophages with cardiothoracic disease. Herein, we describe the unique role of Nrp2 in diseases of the heart and lung, with an emphasis on Nrp2 in macrophages, and explore the potential to target Nrp2 as a therapeutic intervention.

The demand for human cytomegalovirus (HCMV) vaccines was first raised by a committee convened during the 1990s. A comprehensive investigation into the mechanism of viral infection supports the prioritization of developing drugs or vaccines that specifically target receptors and ligands involved in the infection process. As primary targets for neutralizing antibodies to combat HCMV, viral ligands (trimer, pentamer, and glycoprotein B) have crucial roles and exhibit substantial antiviral potential, which could be exploited for breakthroughs in antiviral research.