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Herpesvirus gH/gL envelope glycoprotein complexes are key players in virus entry as ligands for host cell receptors and by promoting fusion of viral envelopes with cellular membranes. Human cytomegalovirus (HCMV) has two alternative gH/gL complexes, gH/gL/gO and gH/gL/UL128,130,131A which both shape the HCMV tropism. By studying binding of HCMV particles to fibroblasts, we could for the first time show that virion gH/gL/gO binds to platelet-derived growth factor-α (PDGFR-α) on the surface of fibroblasts and that gH/gL/gO either directly or indirectly recruits gB to this complex. PDGFR-α functions as an entry receptor for HCMV expressing gH/gL/gO, but not for HCMV mutants lacking the gH/gL/gO complex. PDGFR-α-dependent entry is not dependent on activation of PDGFR-α. We could also show that the gH/gL/gO-PDGFR-α interaction starts the predominant entry pathway for infection of fibroblasts with free virus. Cell-associated virus spread is either driven by gH/gL/gO interacting with PDGFR-α or by the gH/gL/UL128,130,131A complex. PDGFR-α-positive cells may thus be preferred first target cells for infections with free virus which might have implications for the design of future HCMV vaccines or anti-HCMV drugs.

Human cytomegalovirus (HCMV) is an important, ubiquitous pathogen that causes severe clinical disease in immunocompromised individuals, such as organ transplant recipients and infants infected in utero. Antiviral chemotherapy remains problematic due to toxicity of the available compounds and the emergence of viruses resistant to available antiviral therapies. Antiviral antibodies could represent a valuable alternative strategy to limit the clinical consequences of viral disease in patients. The envelope glycoprotein B (gB) of HCMV is a major antigen for the induction of virus neutralizing antibodies. However, the role of anti-gB antibodies in the course of the infection in-vivo remains unknown. We have used a murine CMV (MCMV) model to generate and study a number of anti-gB monoclonal antibodies (mAbs) with differing virus-neutralizing capacities. The mAbs were found to bind to similar antigenic structures on MCMV gB that are represented in HCMV gB. When mAbs were used in immunodeficient RAG-/- hosts to limit an ongoing infection we observed a reduction in viral load both with mAbs having potent neutralizing capacity in-vitro as well as mAbs classified as non-neutralizing. In a therapeutic setting, neutralizing mAbs showed a greater capacity to reduce the viral burden compared to non-neutralizing antibodies. Efficacy was correlated with sustained concentration of virus neutralizing mAbs in-vivo rather than their in-vitro neutralizing capacity. Combinations of neutralizing mAbs further augmented the antiviral effect and were found to be as potent in protection as polyvalent serum from immune animals. Prophylactic administration of mAbs before infection was also protective and both neutralizing and non-neutralizing mAbs were equally effective in preventing lethal infection of immunodeficient mice. In summary, our data argue that therapeutic application of potently neutralizing mAbs against gB represent a strategy to modify the outcome of CMV infection in immunodeficient hosts. When present before infection, both neutralizing and non-neutralizing anti-gB exhibited protective capacity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which was responsible for the COVID-19 pandemic, efficiently spreads cell-to-cell through mechanisms facilitated by its membrane glycoprotein spike. We established a dual split protein (DSP) assay based on the complementation of GFP and luciferase to quantify the fusogenic activity of the SARS-CoV-2 spike protein. We provide several lines of evidence that the spike protein of SARS-CoV-2, but not SARS-CoV-1, induced cell–cell fusion even in the absence of its receptor, angiotensin-converting enzyme 2 (ACE2). This poorly described ACE2-independent cell fusion activity of the spike protein was strictly dependent on the proteasomal cleavage of the spike by furin while TMPRSS2 was dispensable. Previous and current variants of concern (VOCs) differed significantly in their fusogenicity. The Delta spike was extremely potent compared to Alpha, Beta, Gamma and Kappa, while the Omicron spike was almost devoid of receptor-independent fusion activity. Nonetheless, for all analyzed variants, cell fusion was dependent on furin cleavage and could be pharmacologically inhibited with CMK. Mapping studies revealed that amino acids 652-1273 conferred the ACE2-independent fusion activity of the spike. Unexpectedly, residues proximal to the furin cleavage site were not of major relevance, whereas residue 655 critically regulated fusion. Finally, we found that the spike’s fusion activity in the absence of ACE2 could be inhibited by antibodies directed against its N-terminal domain (NTD) but not by antibodies targeting its receptor-binding domain (RBD). In conclusion, our BSL-1-compatible DSP assay allowed us to screen for inhibitors or antibodies that interfere with the spike’s fusogenic activity and may therefore contribute to both rational vaccine design and development of novel treatment options against SARS-CoV-2.

Human cytomegalovirus (HCMV) can cause severe clinical disease in immunocompromised individuals, such as allograft recipients and infants infected in utero. Neutralizing activity of antibodies, measured as the ability to prevent the entry of cell-free virus, has been correlated with the reduction in HCMV transmission and the severity of HCMV-associated disease. However, in vivo HCMV amplification may occur mainly via cell-to-cell spread. Thus, quantifying the inhibition of cell-to-cell transmission could be important in the evaluation of therapeutic antibodies and/or humoral responses to infection or immunization. Here, we established a quantitative plaque reduction assay, which allowed for the measurement of the capacity of antibodies to limit HCMV spread in vitro. Using an automated fluorescence spot reader, infection progression was assayed by the expansion of viral plaques during the course of infection with various GFP-expressing viruses. We found that in contrast to non-neutralizing monoclonal antibodies (mAbs), neutralizing mAbs against both glycoprotein B and H (gB and gH) could significantly inhibit viral plaque expansion of different HCMV strains and was equally efficient in fibroblasts as in epithelial cells. In contrast, an anti-pentamer mAb was active only in epithelial cells. Taken together, our data demonstrate that specific anti-HCMV mAbs can significantly limit cell-associated virus spread in vitro.

Infections with cytomegalovirus (CMV) can cause severe disease in immunosuppressed patients and infected newborns. Innate as well as cellular and humoral adaptive immune effector functions contribute to the control of CMV in immunocompetent individuals. None of the innate or adaptive immune functions are essential for virus control, however. Expansion of γδ T cells has been observed during human CMV (HCMV) infection in the fetus and in transplant patients with HCMV reactivation but the protective function of γδ T cells under these conditions remains unclear. Here we show for murine CMV (MCMV) infections that mice that lack CD8 and CD4 αβ-T cells as well as B lymphocytes can control a MCMV infection that is lethal in RAG-1(-/-) mice lacking any T- and B-cells. γδ T cells, isolated from infected mice can kill MCMV infected target cells in vitro and, importantly, provide long-term protection in infected RAG-1(-/-) mice after adoptive transfer. γδ T cells in MCMV infected hosts undergo a prominent and long-lasting phenotypic change most compatible with the view that the majority of the γδ T cell population persists in an effector/memory state even after resolution of the acute phase of the infection. A clonotypically focused Vγ1 and Vγ2 repertoire was observed at later stages of the infection in the organs where MCMV persists. These findings add γδ T cells as yet another protective component to the anti-CMV immune response. Our data provide clear evidence that γδ T cells can provide an effective control mechanism of acute CMV infections, particularly when conventional adaptive immune mechanisms are insufficient or absent, like in transplant patient or in the developing immune system in utero. The findings have implications in the stem cell transplant setting, as antigen recognition by γδ T cells is not MHC-restricted and dual reactivity against CMV and tumors has been described.

The human cytomegalovirus (HCMV) glycoprotein B (gB) is the viral fusogen required for entry into cells and for direct cell-to-cell spread of the virus. We have previously demonstrated that the exchange of the carboxy-terminal domain (CTD) of gB for the CTD of the structurally related fusion protein G of the vesicular stomatitis virus (VSV-G) resulted in an intrinsically fusion-active gB variant (gB/VSV-G). In this present study, we employed a dual split protein (DSP)-based cell fusion assay to further characterize the determinants of fusion activity in the CTD of gB. We generated a comprehensive library of gB CTD truncation mutants and identified two mutants, gB-787 and gB-807, which were fusion-competent and induced the formation of multinucleated cell syncytia in the absence of other HCMV proteins. Structural modeling coupled with site-directed mutagenesis revealed that gB fusion activity is primarily mediated by the CTD helix 2, and secondarily by the recruitment of cellular SH2/WW-domain-containing proteins. The fusion activity of gB-807 was inhibited by gB-specific monoclonal antibodies (MAbs) targeting the antigenic domains AD-1 to AD-5 within the ectodomain and not restricted to MAbs directed against AD-4 and AD-5 as observed for gB/VSV-G. This finding suggested a differential regulation of the fusion-active conformational state of both gB variants. Collectively, our findings underscore a pivotal role of the CTD in regulating the fusogenicity of HCMV gB, with important implications for understanding the conformations of gB that facilitate membrane fusion, including antigenic structures that could be targeted by antibodies to block this essential step in HCMV infection.

Human cytomegalovirus (HCMV) is the most common viral infection acquired in utero and a leading cause of neurodevelopmental abnormalities, including sensorineural hearing loss (SNHL). In previous studies using a murine model of HCMV induced SNHL, hearing loss was correlated with virus-induced cochlear inflammation but not cochlear viral load. However, these previous findings were determined at the time of auditory testing, a time poiont well past critical periods of auditory development. In the current study, cochlear virus load early in auditory development could be correlated with the magnitude of virus-induced cochlear inflammation, cochlear histopathology and the development of hearing loss. Transcriptional profiling at early times after infection revealed dysregulation of multiple well described deafness-related genes (DRG). Treatment with antiviral antibodies early after infection decreased cochlear virus load, cochlear inflammation, cochlear histopathology, and normalized DRG expression arguing that virus-induced cochlear inflammation can result in pleiotropic effects on the developing auditory system. Finally, this model also demonstrated that sterilizing immunity was unnecessary for prevention of SNHL, thus providing a rationale for inteventions that could limit, but not completely prevent HCMV infection of the developing auditory system.

Following infection of permissive cells, human cytomegalovirus (HCMV) induces the reorganization of intracellular membranes resulting in the formation of a distinctive membranous compartment in the cytoplasm of infected cells. This compartment has been designated the viral assembly compartment (AC) and is thought to be a site for cytoplasmic virion assembly and envelopment. In this study, we have demonstrated that a single virion envelope glycoprotein is essential for AC formation in infected cells, and in its absence, there is a significant decrease in the production of infectious virions. These findings are consistent with those from other studies that have demonstrated the importance of host cell proteins in the formation of the AC and demonstrate a critical role of a single virion protein in AC formation and the efficient assembly of infectious virus. The human cytomegalovirus (HCMV) UL132 open reading frame encodes a 270-amino-acid type I envelope glycoprotein, gpUL132. The deletion of UL132 (ΔUL132) from the HCMV genome results in a pronounced deficit in virus yield, with an approximately 2-log decrease in the production of infectious virus compared to the wild-type (WT) virus. Characterization of the ΔUL132 mutant virus indicated that it was less infectious with a high particle-to-infectious unit ratio and an altered composition of virion proteins compared to the WT virus. In addition, the viral assembly compartment (AC) failed to form in cells infected with the ΔUL132 mutant virus. The expression of gpUL132 in trans rescued the defects in the morphogenesis of the AC in cells infected with the ΔUL132 mutant virus and in infectious virus production. Furthermore, using cell lines expressing chimeric proteins, we demonstrated that the cytosolic domain of gpUL132 was sufficient to rescue AC formation and WT levels of virus production. Progeny virions from ΔUL132-infected cells expressing the cytosolic domain of gpUL132 exhibited particle-to-infectious unit ratios similar to those of the WT virus. Together, our findings argue that gpUL132 is essential for HCMV AC formation and the efficient production of infectious particles, thus highlighting the importance of this envelope protein for the virus-induced reorganization of intracellular membranes and AC formation in the assembly of infectious virus. IMPORTANCE Following infection of permissive cells, human cytomegalovirus (HCMV) induces the reorganization of intracellular membranes resulting in the formation of a distinctive membranous compartment in the cytoplasm of infected cells. This compartment has been designated the viral assembly compartment (AC) and is thought to be a site for cytoplasmic virion assembly and envelopment. In this study, we have demonstrated that a single virion envelope glycoprotein is essential for AC formation in infected cells, and in its absence, there is a significant decrease in the production of infectious virions. These findings are consistent with those from other studies that have demonstrated the importance of host cell proteins in the formation of the AC and demonstrate a critical role of a single virion protein in AC formation and the efficient assembly of infectious virus.

Human cytomegalovirus (HCMV), a herpesvirus, is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and infected newborns. Efforts are underway to prepare effective subunit vaccines and therapies including antiviral antibodies. However, current vaccine efforts are hampered by the lack of information on protective immune responses against HCMV. Characterizing the B-cell response in healthy infected individuals could aid in the design of optimal vaccines and therapeutic antibodies. To address this problem, we determined, for the first time, the B-cell repertoire against glycoprotein B (gB) of HCMV in different healthy HCMV seropositive individuals in an unbiased fashion. HCMV gB represents a dominant viral antigenic determinant for induction of neutralizing antibodies during infection and is also a component in several experimental HCMV vaccines currently being tested in humans. Our findings have revealed that the vast majority (>90%) of gB-specific antibodies secreted from B-cell clones do not have virus neutralizing activity. Most neutralizing antibodies were found to bind to epitopes not located within the previously characterized antigenic domains (AD) of gB. To map the target structures of these neutralizing antibodies, we generated a 3D model of HCMV gB and used it to identify surface exposed protein domains. Two protein domains were found to be targeted by the majority of neutralizing antibodies. Domain I, located between amino acids (aa) 133-343 of gB and domain II, a discontinuous domain, built from residues 121-132 and 344-438. Analysis of a larger panel of human sera from HCMV seropositive individuals revealed positivity rates of >50% against domain I and >90% against domain II, respectively. In accordance with previous nomenclature the domains were designated AD-4 (Dom II) and AD-5 (Dom I), respectively. Collectively, these data will contribute to optimal vaccine design and development of antibodies effective in passive immunization.

Viruses transmit via the environmental and social interactions of their hosts. Herpesviruses have colonized mammals since their earliest origins, suggesting that they exploit ancient, common pathways. Cytomegaloviruses (CMVs) are assumed to enter new hosts orally, but no site has been identified. We show by live imaging that murine CMV (MCMV) infects nasally rather than orally, both after experimental virus uptake and during natural transmission. Replication-deficient virions revealed the primary target as olfactory neurons. Local, nasal replication by wild-type MCMV was not extensive, but there was rapid systemic spread, associated with macrophage infection. A long-term, transmissible infection was then maintained in the salivary glands. The viral m131/m129 chemokine homolog, which influences tropism, promoted salivary gland colonization after nasal entry but was not required for entry per se . The capacity of MCMV to transmit via olfaction, together with previous demonstrations of experimental olfactory infection by murid herpesvirus 4 (MuHV-4) and herpes simplex virus 1 (HSV-1), suggest that this is a common, conserved route of mammalian herpesvirus entry. IMPORTANCE Cytomegaloviruses (CMVs) infect most mammals. Human CMV (HCMV) harms people with poor immune function and can damage the unborn fetus. It infects approximately 1% of live births. We lack a good vaccine. One problem is that how CMVs first enter new hosts remains unclear. Oral entry is often assumed, but the evidence is indirect, and no infection site is known. The difficulty of analyzing HCMV makes related animal viruses an important source of insights. Murine CMV (MCMV) infected not orally but nasally. Specifically, it targeted olfactory neurons. Viral transmission was also a nasal infection. Like HCMV, MCMV infected cells by binding to heparan, and olfactory surfaces display heparan to incoming viruses, whereas most other mucosal surfaces do not. These data establish a new understanding of CMV infections and a basis for infection control. Cytomegaloviruses (CMVs) infect most mammals. Human CMV (HCMV) harms people with poor immune function and can damage the unborn fetus. It infects approximately 1% of live births. We lack a good vaccine. One problem is that how CMVs first enter new hosts remains unclear. Oral entry is often assumed, but the evidence is indirect, and no infection site is known. The difficulty of analyzing HCMV makes related animal viruses an important source of insights. Murine CMV (MCMV) infected not orally but nasally. Specifically, it targeted olfactory neurons. Viral transmission was also a nasal infection. Like HCMV, MCMV infected cells by binding to heparan, and olfactory surfaces display heparan to incoming viruses, whereas most other mucosal surfaces do not. These data establish a new understanding of CMV infections and a basis for infection control.