SARS-CoV-2 Spike Protein Is Capable of Inducing Cell-Cell Fusions Independent from Its Receptor ACE2 and This Activity Can Be Impaired by Furin Inhibitors or a Subset of Monoclonal Antibodies.

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.

Neutralizing Antibodies Limit Cell-Associated Spread of Human Cytomegalovirus in Epithelial Cells and Fibroblasts.

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.

A Novel Strain-Specific Neutralizing Epitope on Glycoprotein H of Human Cytomegalovirus.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that causes severe clinical disease in immunosuppressed patients and congenitally infected newborn infants. Viral envelope glycoproteins represent attractive targets for vaccination or passive immunotherapy. To extend the knowledge of mechanisms of virus neutralization, monoclonal antibodies (MAbs) were generated following immunization of mice with HCMV virions. Hybridoma supernatants were screened for in vitro neutralization activity, yielding three potent MAbs, 6E3, 3C11, and 2B10. MAbs 6E3 and 3C11 blocked infection of all viral strains that were tested, while MAb 2B10 neutralized only 50% of the HCMV strains analyzed. Characterization of the MAbs using indirect immunofluorescence analyses demonstrated their reactivity with recombinantly derived gH. While MAbs 6E3 and 3C11 reacted with gH when expressed alone, 2B10 detected gH only when it was coexpressed with gB and gL. Recognition of gH by 3C11 was dependent on the expression of the entire ectodomain of gH, whereas 6E3 required residues 1 to 629 of gH. The strain-specific determinant for neutralization by Mab 2B10 was identified as a single Met→Ile amino acid polymorphism within gH, located within the central part of the protein. The polymorphism is evenly distributed among described HCMV strains. The 2B10 epitope thus represents a novel strain-specific antibody target site on gH of HCMV. The dependence of the reactivity of 2B10 on the simultaneous presence of gB/gH/gL will be of value in the structural definition of this tripartite complex. The 2B10 epitope may also represent a valuable tool for diagnostics to monitor infections/reinfections with different HCMV strains during pregnancy or after transplantation. IMPORTANCE HCMV infections are life threatening to people with compromised or immature immune systems. Understanding the antiviral antibody repertoire induced during HCMV infection is a necessary prerequisite to define protective antibody responses. Here, we report three novel anti-gH MAbs that potently neutralized HCMV infectivity. One of these MAbs (2B10) targets a novel strain-specific conformational epitope on gH that only becomes accessible upon coexpression of the minimal fusion machinery gB/gH/gL. Strain specificity is dependent on a single amino acid polymorphism within gH. Our data highlight the importance of strain-specific neutralizing antibody responses against HCMV. The 2B10 epitope may also represent a valuable tool for diagnostics to monitor infections/reinfections with different HCMV strains during pregnancy or after transplantation. In addition, the dependence of the reactivity of 2B10 on the simultaneous presence of gB/gH/gL will be of value in the structural definition of this tripartite complex.

Phenotypical Characterization of the Nuclear Egress of Recombinant Cytomegaloviruses Reveals Defective Replication upon ORF-UL50 Deletion but Not pUL50 Phosphosite Mutation.

Nuclear egress is a common herpesviral process regulating nucleocytoplasmic capsid release. For human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the pUL50-pUL53 core that regulates multicomponent assembly with NEC-associated proteins and capsids. Recently, NEC crystal structures were resolved for α-, ß- and γ-herpesviruses, revealing profound structural conservation, which was not mirrored, however, by primary sequence and binding properties. The NEC binding principle is based on hook-into-groove interaction through an N-terminal hook-like pUL53 protrusion that embraces an α-helical pUL50 binding groove. So far, pUL50 has been considered as the major kinase-interacting determinant and massive phosphorylation of pUL50-pUL53 was assigned to NEC formation and functionality. Here, we addressed the question of phenotypical changes of ORF-UL50-mutated HCMVs. Surprisingly, our analyses did not detect a predominant replication defect for most of these viral mutants, concerning parameters of replication kinetics (qPCR), viral protein production (Western blot/CoIP) and capsid egress (confocal imaging/EM). Specifically, only the ORF-UL50 deletion rescue virus showed a block of genome synthesis during late stages of infection, whereas all phosphosite mutants exhibited marginal differences compared to wild-type or revertants. These results (i) emphasize a rate-limiting function of pUL50 for nuclear egress, and (ii) demonstrate that mutations in all mapped pUL50 phosphosites may be largely compensated. A refined mechanistic concept points to a multifaceted nuclear egress regulation, for which the dependence on the expression and phosphorylation of pUL50 is discussed.

Diazadispiroalkane Derivatives Are New Viral Entry Inhibitors.

Herpesviruses are widespread and can cause serious illness. Many currently available antiviral drugs have limited effects, result in rapid development of resistance, and often exhibit dose-dependent toxicity. Especially for human cytomegalovirus (HCMV), new well-tolerated compounds with novel mechanisms of action are urgently needed. In this study, we characterized the antiviral activity of two new diazadispiroalkane derivatives, 11826091 and 11826236. These two small molecules exhibited strong activity against low-passage-number HCMV. Pretreatment of cell-free virus with these compounds greatly reduced infection. Time-of-addition assays where 11826091 or 11826236 was added to cells before infection, before and during infection, or during or after infection demonstrated an inhibitory effect on early steps of infection. Interestingly, 11826236 had an effect by addition to cells after infection. Results from entry assays showed the major effect to be on attachment. Only 11826236 had a minimal effect on penetration comparable to heparin. Further, no effect on virus infection was found for cell lines with a defect in heparan sulfate expression or lacking all surface glycosaminoglycans, indicating that these small molecules bind to heparan sulfate on the cell surface. To test this further, we extended our analyses to pseudorabies virus (PrV), a member of the Alphaherpesvirinae, which is known to use cell surface heparan sulfate for initial attachment via nonessential glycoprotein C (gC). While infection with PrV wild type was strongly impaired by 11826091 or 11826236, as with heparin, a mutant lacking gC was unaffected by either treatment, demonstrating that primary attachment to heparan sulfate via gC is targeted by these small molecules.

Human Cytomegalovirus Envelope Protein gpUL132 Regulates Infectious Virus Production through Formation of the Viral Assembly Compartment.

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.

Cell Fusion Induced by a Fusion-Active Form of Human Cytomegalovirus Glycoprotein B (gB) Is Inhibited by Antibodies Directed at Antigenic Domain 5 in the Ectodomain of gB.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen that can cause severe clinical disease in allograft recipients and infants infected in utero Virus-neutralizing antibodies defined in vitro have been proposed to confer protection against HCMV infection, and the virion envelope glycoprotein B (gB) serves as a major target of neutralizing antibodies. The viral fusion protein gB is nonfusogenic on its own and requires glycoproteins H (gH) and L (gL) for membrane fusion, which is in contrast to requirements of related class III fusion proteins, including vesicular stomatitis virus glycoprotein G (VSV-G) or baculovirus gp64. To explore requirements for gB's fusion activity, we generated a set of chimeras composed of gB and VSV-G or gp64, respectively. These gB chimeras were intrinsically fusion active and led to the formation of multinucleated cell syncytia when expressed in the absence of other viral proteins. Utilizing a panel of virus-neutralizing gB-specific monoclonal antibodies (MAbs), we could demonstrate that syncytium formation of the fusogenic gB/VSV-G chimera can be significantly inhibited by only a subset of neutralizing MAbs which target antigenic domain 5 (AD-5) of gB. This observation argues for differential modes of action of neutralizing anti-gB MAbs and suggests that blocking the membrane fusion function of gB could be one mechanism of antibody-mediated virus neutralization. In addition, our data have important implications for the further understanding of the conformation of gB that promotes membrane fusion as well as the identification of structures in AD-5 that could be targeted by antibodies to block this early step in HCMV infection.IMPORTANCE HCMV is a major global health concern, and antiviral chemotherapy remains problematic due to toxicity of available compounds and the emergence of drug-resistant viruses. Thus, an HCMV vaccine represents a priority for both governmental and pharmaceutical research programs. A major obstacle for the development of a vaccine is a lack of knowledge of the nature and specificities of protective immune responses that should be induced by such a vaccine. Glycoprotein B of HCMV is an important target for neutralizing antibodies and, hence, is often included as a component of intervention strategies. By generation of fusion-active gB chimeras, we were able to identify target structures of neutralizing antibodies that potently block gB-induced membrane fusion. This experimental system provides an approach to screen for antibodies that interfere with gB's fusogenic activity. In summary, our data will likely contribute to both rational vaccine design and the development of antibody-based therapies against HCMV.

Influence of Human Cytomegalovirus Glycoprotein O Polymorphism on the Inhibitory Effect of Soluble Forms of Trimer- and Pentamer-Specific Entry Receptors.

Human cytomegalovirus (HCMV) envelope glycoprotein complexes, gH/gL/gO trimer and gH/gL/UL128-131 pentamer, are important for cell-free HCMV entry. While soluble NRP2-Fc (sNRP2-Fc) interferes with epithelial/endothelial cell entry through UL128, soluble platelet-derived growth factor receptor α-Fc (sPDGFRα-Fc) interacts with gO, thereby inhibiting infection of all cell types. Since gO is the most variable subunit, we investigated the influence of gO polymorphism on the inhibitory capacities of sPDGFRα-Fc and sNRP2-Fc. Accordingly, gO genotype 1c (GT1c) sequence was fully or partially replaced by gO GT2b, GT3, and GT5 sequences in the bacterial artificial chromosome (BAC) TB40-BAC4-luc background (where luc is luciferase). All mutants were tested for fibroblast and epithelial cell infectivity, for virion content of gB, gH, and gO, and for infection inhibition by sPDGFRα-Fc and sNRP2-Fc. Full-length and partial gO GT swapping may increase epithelial-to-fibroblast ratios due to subtle alterations in fibroblast and/or epithelial infectivity but without substantial changes in gB and gH levels in mutant virions. All gO GT mutants except recombinant gO GT1c/3 displayed a nearly complete inhibition at 1.25 µg/ml sPDGFRα-Fc on epithelial cells (98% versus 91%), and all experienced complete inhibition on fibroblasts (≥99%). While gO GT replacement did not influence sNRP2-Fc inhibition at 1.25 µg/ml on epithelial cells (97% to 99%), it rendered recombinant mutant GT1c/3 moderately accessible to fibroblast inhibition (40%). In contrast to the steep sPDGFRα-Fc inhibition curves (slope of >1.0), sNRP2-Fc dose-response curves on epithelial cells displayed slopes of ∼1.0, suggesting functional differences between these entry inhibitors. Our findings demonstrate that artificially generated gO recombinants rather than the major gO genotypic forms may affect the inhibitory capacities of sPDGFRα and sNRP2 in a cell type-dependent manner.IMPORTANCE Human cytomegalovirus (HCMV) is known for its broad cell tropism, as reflected by the different organs and tissues affected by HCMV infection. Hence, inhibition of HCMV entry into distinct cell types could be considered a promising therapeutic option to limit cell-free HCMV infection. Soluble forms of cellular entry receptor PDGFRα rather than those of entry receptor neuropilin-2 inhibit infection of multiple cell types. sPDGFRα specifically interacts with gO of the trimeric gH/gL/gO envelope glycoprotein complex. HCMV strains may differ with respect to the amounts of trimer in virions and the highly polymorphic gO sequence. In this study, we show that the major gO genotypes of HCMV that are also found in vivo are similarly well inhibited by sPDGFRα. Novel gO genotypic forms potentially emerging through recombination, however, may evade sPDGFRα inhibition on epithelial cells. These findings provide useful additional information for the future development of anti-HCMV therapeutic compounds based on sPDGFRα.

Detection of antibody-secreting cells specific for the cytomegalovirus and herpes simplex virus surface antigens.

Infections with the herpes simplex virus (HSV) and the human cytomegalovirus (HCMV) can lead to life-threatening diseases, particularly in immunosuppressed patients. Furthermore, HSV infections at birth (herpes neonatorum) can result in a disseminated disease associated with a fatal multiorgan failure. Congenital HCMV infections can result in miscarriage, serious birth defects or developmental disabilities. Antibody-based interventions with hyperimmunoglobulins showed encouraging results in clinical studies, but clearly need to be improved. The isolation of highly neutralizing monoclonal antibodies is a promising strategy to establish potent therapy options against HSV and HCMV infections. Monoclonal antibodies are commonly isolated from hybridomas or EBV-immortalized B-cell clones. The screening procedure to identify virus-specific cells from a cell mixture is a challenging step, since most of the highly neutralizing antibodies target complex conformational epitopes on the virus surface. Conventional assays such as ELISA are based on purified viral proteins and inappropriate to display complex epitopes. To overcome this obstacle, we have established two full-virus based methods that allow screening for cells and antibodies targeting complex conformational epitopes on viral surface antigens. The methods are suitable to detect surface antigen-specific cells from a cell mixture and may facilitate the isolation of highly neutralizing antibodies against HSV and HCMV.

Protection from cytomegalovirus viremia following glycoprotein B vaccination is not dependent on neutralizing antibodies.

Human cytomegalovirus (HCMV) is an important pathogen in transplant patients and in congenital infection. Previously, we demonstrated that vaccination with a recombinant viral glycoprotein B (gB)/MF59 adjuvant formulation before solid organ transplant reduced viral load parameters post transplant. Reduced posttransplant viremia was directly correlated with antibody titers against gB consistent with a humoral response against gB being important. Here we show that sera from the vaccinated seronegative patients displayed little evidence of a neutralizing antibody response against cell-free HCMV in vitro. Additionally, sera from seronegative vaccine recipients had minimal effect on the replication of a strain of HCMV engineered to be cell-associated in a viral spread assay. Furthermore, although natural infection can induce antibody-dependent cellular cytotoxicity (ADCC) responses, serological analysis of seronegative vaccinees again presented no evidence of a substantial ADCC-promoting antibody response being generated de novo. Finally, analyses for responses against major antigenic domains of gB following vaccination were variable, and their pattern was distinct compared with natural infection. Taken together, these data argue that the protective effect elicited by the gB vaccine is via a mechanism of action in seronegative vaccinees that cannot be explained by neutralization or the induction of ADCC. More generally, these data, which are derived from a human challenge model that demonstrated that the gB vaccine is protective, highlight the need for more sophisticated analyses of new HCMV vaccines over and above the quantification of an ability to induce potent neutralizing antibody responses in vitro.

Epitope-Specific Humoral Responses to Human Cytomegalovirus Glycoprotein-B Vaccine With MF59: Anti-AD2 Levels Correlate With Protection From Viremia.

The human cytomegalovirus (HCMV) virion envelope protein glycoprotein B (gB) is essential for viral entry and represents a major target for humoral responses following infection. Previously, a phase 2 placebo-controlled clinical trial conducted in solid organ transplant candidates demonstrated that vaccination with gB plus MF59 adjuvant significantly increased gB enzyme-linked immunosorbent assay (ELISA) antibody levels whose titer correlated directly with protection against posttransplant viremia. The aim of the current study was to investigate in more detail this protective humoral response in vaccinated seropositive transplant recipients. We focused on 4 key antigenic domains (AD) of gB (AD1, AD2, AD4, and AD5), measuring antibody levels in patient sera and correlating these with posttransplant HCMV viremia. Vaccination of seropositive patients significantly boosted preexisting antibody levels against the immunodominant region AD1 as well as against AD2, AD4, and AD5. A decreased incidence of viremia correlated with higher antibody levels against AD2 but not with antibody levels against the other 3 ADs. Overall, these data support the hypothesis that antibodies against AD2 are a major component of the immune protection of seropositives seen following vaccination with gB/MF59 vaccine and identify a correlate of protective immunity in allograft patients.

Differences in Growth Properties among Two Human Cytomegalovirus Glycoprotein O Genotypes.

Glycoprotein O (gO) of the human cytomegalovirus (HCMV) is the critical subunit of the envelope trimer gH/gL/gO as it interacts with platelet-derived growth factor alpha receptor upon fibroblast entry, and triggers gB-mediated fusion for fibroblast and epithelial cell infection. Eight genotypes (GT) of the highly polymorphic gO gene are described, yet it is unclear whether the distinct GTs differ in their function. Thus, we aimed to elucidate potential functional differences between two highly diverse gO GTs in an otherwise genomically identical HCMV strain. Therefore, resident gO GT1c sequence of strain TB40-BAC4-luc was entirely replaced by gO GT4 of strain Towne and both, GT1c and GT4 viruses, were investigated for their growth properties in fibroblasts and epithelial cells. In addition, two conserved gO cysteines involved in gH/gL/gO stabilization were mutated to serine either in GT1c (C218S and C343S) or GT4 (C216S and C336S) and their effects on cell-free infectivity were assessed. GT4 viruses displayed a significantly enhanced epithelial cell tropism and this resulted in higher virus release upon replication in epithelial cells when compared to GT1c viruses. Further, when the two cysteines were individually mutated in gO GT1c no impairment in cell-free infectivity was observed. This, however, was in sharp contrast to gO GT4, in which both of the corresponding cysteine mutations led to a substantial reduction in cell-free infectivity which was even more pronounced upon mutation of GT4-C336 than of GT4-C216. In conclusion, these findings provide evidence that the two highly diverse gO genotypes, GT1c and GT4, differ in their functional properties as revealed by their different infection capacities for epithelial cells and by their different responsiveness to mutation of strictly conserved cysteine residues. Thus, it is likely that the gO heterogeneity influences cell-free infectivity of HCMV also in vivo which may have important implications for virus host transmission.

Protective capacity of neutralizing and non-neutralizing antibodies against glycoprotein B of cytomegalovirus.

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.

Generation of a Gaussia luciferase-expressing endotheliotropic cytomegalovirus for screening approaches and mutant analyses.

For many questions in human cytomegalovirus (HCMV) research, assays are desired that allow robust and fast quantification of infection efficiencies under high-throughput conditions. The secreted Gaussia luciferase has been demonstrated as a suitable reporter in the context of a fibroblast-adapted HCMV strain, which however is greatly restricted in the number of cell types to which it can be applied. We inserted the Gaussia luciferase expression cassette into the BAC-cloned virus strain TB40-BAC4, which displays the natural broad cell tropism of HCMV and hence allows application to screening approaches in a variety of cell types including fibroblasts, epithelial, and endothelial cells. Here, we applied the reporter virus TB40-BAC4-IE-GLuc to identify mouse hybridoma clones that preferentially neutralize infection of endothelial cells. In addition, as the Gaussia luciferase is secreted into culture supernatants from infected cells it allows kinetic analyses in living cultures. This can speed up and facilitate phenotypic characterization of BAC-cloned mutants. For example, we analyzed a UL74 stop-mutant of TB40-BAC4-IE-GLuc immediately after reconstitution in transfected cultures and found the increase of luciferase delayed and reduced as compared to wild type. Phenotypic monitoring directly in transfected cultures can minimize the risk of compensating mutations that might occur with extended passaging.

Glycoprotein N of human cytomegalovirus protects the virus from neutralizing antibodies.

Herpes viruses persist in the infected host and are transmitted between hosts in the presence of a fully functional humoral immune response, suggesting that they can evade neutralization by antiviral antibodies. Human cytomegalovirus (HCMV) encodes a number of polymorphic highly glycosylated virion glycoproteins (g), including the essential envelope glycoprotein, gN. We have tested the hypothesis that glycosylation of gN contributes to resistance of the virus to neutralizing antibodies. Recombinant viruses carrying deletions in serine/threonine rich sequences within the glycosylated surface domain of gN were constructed in the genetic background of HCMV strain AD169. The deletions had no influence on the formation of the gM/gN complex and in vitro replication of the respective viruses compared to the parent virus. The gN-truncated viruses were significantly more susceptible to neutralization by a gN-specific monoclonal antibody and in addition by a number of gB- and gH-specific monoclonal antibodies. Sera from individuals previously infected with HCMV also more efficiently neutralized gN-truncated viruses. Immunization of mice with viruses that expressed the truncated forms of gN resulted in significantly higher serum neutralizing antibody titers against the homologous strain that was accompanied by increased antibody titers against known neutralizing epitopes on gB and gH. Importantly, neutralization activity of sera from animals immunized with gN-truncated virus did not exhibit enhanced neutralizing activity against the parental wild type virus carrying the fully glycosylated wild type gN. Our results indicate that the extensive glycosylation of gN could represent a potentially important mechanism by which HCMV neutralization by a number of different antibody reactivities can be inhibited.

UL74 of human cytomegalovirus reduces the inhibitory effect of gH-specific and gB-specific antibodies.

The human cytomegalovirus (HCMV) glycoproteins gH (UL75) and gL (UL115) can form complexes with gO (UL74) or with proteins of the UL128-UL131A locus. Deletion of gO abolishes cell-free virus transmission and renders cell-associated virus transmission in fibroblasts more sensitive to inhibition by human anti-HCMV serum. To test whether the latter effect is specific for gO, we compared mutants with deletions in UL74, UL99 and the UL128-131A locus regarding their sensitivity to anti-HCMV antibodies. UL74 deletion mutants were more sensitive to a further restriction by polyspecific or gH-specific antibodies than control mutants, showing that gO specifically protects focal growth against inhibitory antibodies. This effect was not confined to gH-specific antibodies, as UL74 deletion mutants were also inhibited by an anti-gB antibody. In conclusion, gO specifically promotes focal spread in the presence of gH and gB antibodies, thus contributing to the ability of HCMV to resist the host's immune response.

Optimal replication of human cytomegalovirus correlates with endocytosis of glycoprotein gpUL132.

Envelopment of a herpesvirus particle is a complex process of which much is still to be learned. We previously identified the glycoprotein gpUL132 of human cytomegalovirus (HCMV) as an envelope component of the virion. In its carboxy-terminal portion, gpUL132 contains at least four motifs for sorting of transmembrane proteins to endosomes; among them are one dileucine-based signal and three tyrosine-based signals of the YXXØ and NPXY (where X stands for any amino acid, and Ø stands for any bulky hydrophobic amino acid) types. To investigate the role of each of these trafficking signals in intracellular localization and viral replication, we constructed a panel of expression plasmids and recombinant viruses in which the signals were rendered nonfunctional by mutagenesis. In transfected cells wild-type gpUL132 was mainly associated with the trans-Golgi network. Consecutive mutation of the trafficking signals resulted in increasing fractions of the protein localized at the cell surface, with gpUL132 mutated in all four trafficking motifs predominantly associated with the plasma membrane. Concomitant with increased surface expression, endocytosis of mutant gpUL132 was reduced, with a gpUL132 expressing all four motifs in mutated form being almost completely impaired in endocytosis. The replication of recombinant viruses harboring mutations in single trafficking motifs was comparable to replication of wild-type virus. In contrast, viruses containing mutations in three or four of the trafficking signals showed pronounced deficits in replication with a reduction of approximately 100-fold. Moreover, recombinant viruses expressing gpUL132 with three or four trafficking motifs mutated failed to incorporate the mutant protein into the virus particle. These results demonstrate a role of endocytosis of an HCMV envelope glycoprotein for incorporation into the virion and optimal virus replication.

The carboxy-terminal domain of glycoprotein N of human cytomegalovirus is required for virion morphogenesis.

Glycoproteins M and N (gM and gN, respectively) are among the few proteins that are conserved across the herpesvirus family. The function of the complex is largely unknown. Whereas deletion from most alphaherpesviruses has marginal effects on the replication of the respective viruses, both proteins are essential for replication of human cytomegalovirus (HCMV). We have constructed a series of mutants in gN to study the function of this protein. gN of HCMV is a type I glycoprotein containing a short carboxy-terminal domain of 14 amino acids, including two cysteine residues directly adjacent to the predicted transmembrane anchor at positions 125 and 126. Deletion of the entire carboxy-terminal domain as well as substitution with the corresponding region from alpha herpesviruses or mutations of both cysteine residues resulted in a replication-incompetent virus. Recombinant viruses containing point mutations of either cysteine residue could be generated. These viruses were profoundly defective for replication. Complex formation of the mutant gNs with gM and transport of the complex to the viral assembly compartment appeared unaltered compared to the wild type. However, in infected cells, large numbers of capsids accumulated in the cytoplasm that failed to acquire an envelope. Transiently expressed gN was shown to be modified by palmitic acid at both cysteine residues. In summary, our data suggest that the carboxy-terminal domain of gN plays a critical role in secondary envelopment of HCMV and that palmitoylation of gN appears to be essential for function in secondary envelopment of HCMV and virus replication.

Deletion of gpUL132, a structural component of human cytomegalovirus, results in impaired virus replication in fibroblasts.

The coding capacity of human cytomegalovirus (HCMV) for glycoproteins by far exceeds that of other herpesviruses. Few of these proteins have been characterized so far. We have investigated the gene product of reading frame UL132. The putative protein product of UL132 is a glycoprotein with a theoretical mass of 29.8 kDa. Transcription analysis revealed that the gene is transcribed with a true late kinetics from the laboratory-adapted strain AD169 and the low-passage isolate TB40E. Two proteins of 22 to 28 kDa and 45 to 60 kDa were detected in virus-infected cells as well as in extracellular virions. The larger protein carried N-linked carbohydrates. Both protein forms were present in laboratory-adapted strains as well as in low-passage isolates of HCMV. Recombinant viruses with the UL132 gene deleted were constructed in the low-passage HCMV isolate PAN as well as the high-passage isolate AD169. Deletion of UL132 from either genome resulted in a pronounced replication deficit with a reduction of approximately 100-fold for HCMV strain AD169. Thus, the protein product of the UL132 reading frame represents a structural viral glycoprotein of HCMV that has an important function for viral replication in tissue culture.

Complex formation by glycoproteins M and N of human cytomegalovirus: structural and functional aspects.

The genomes of herpesviruses contain a number of genes which are conserved throughout the family of Herpesviridae, indicating that the proteins may serve important functions in the replication of these viruses. Among these are several envelope glycoproteins, including glycoprotein M (gM) and gN, which form a complex that is covalently linked via disulfide bonds in some herpesviruses. However, deletion of gM and/or gN from most alphaherpesviruses has limited effects on replication of the respective viruses in vitro. In contrast, insertional inactivation of the gM gene of the betaherpesvirus human cytomegalovirus (HCMV) results in a replication-incompetent virus. We have started to analyze the structural and functional aspects of the interaction between gM and gN of HCMV. We show that large parts of gM are dispensable for the formation of a gM/gN complex that is transported to distal parts of the cellular secretory pathway. In addition, we demonstrate that the disulfide bond is between the cysteine at position 44 in gM and cysteine 90 in gN. However, disulfide linkage is not a prerequisite for modification and transport of the gM/gN complex. Moreover, mutant viruses that lack a disulfide bridge between gM and gN replicate with efficiencies similar to that of wild-type viruses.