Single-cell analysis of memory B cells from top neutralizers reveals multiple sites of vulnerability within HCMV Trimer and Pentamer.

Human cytomegalovirus (HCMV) can cause severe diseases in fetuses, newborns, and immunocompromised individuals. Currently, no vaccines are approved, and treatment options are limited. Here, we analyzed the human B cell response of four HCMV top neutralizers from a cohort of 9,000 individuals. By single-cell analyses of memory B cells targeting the pentameric and trimeric HCMV surface complexes, we identified vulnerable sites on the shared gH/gL subunits as well as complex-specific subunits UL128/130/131A and gO. Using high-resolution cryogenic electron microscopy, we revealed the structural basis of the neutralization mechanisms of antibodies targeting various binding sites. Moreover, we identified highly potent antibodies that neutralized a broad spectrum of HCMV strains, including primary clinical isolates, that outperform known antibodies used in clinical trials. Our study provides a deep understanding of the mechanisms of HCMV neutralization and identifies promising antibody candidates to prevent and treat HCMV infection.

Generation of UL128-shRNA transduced fibroblasts for the release of cell-free virus from clinical human cytomegalovirus isolates.

Working with recent isolates of human cytomegalovirus (HCMV) is complicated by their strictly cell-associated growth with lack of infectivity in the supernatant. Adaptation to cell-free growth is associated with disruption of the viral UL128 gene locus. The authors transduced fibroblasts with a lentiviral vector encoding UL128-specific-shRNA to allow the release of cell-free infectivity without genetic alteration. Transduced cells were cocultured with fibroblasts containing cell-associated isolates, and knockdown of the UL128 protein was validated by immunoblotting. Cell-free infectivity increased 1000-fold in isolate cocultures with UL128-shRNA compared with controls, and virions could be purified by density gradients. Transduced fibroblasts also allowed direct isolation of HCMV from a clinical specimen and cell-free transfer to other cell types. In conclusion, UL128-shRNA-transduced fibroblasts allow applications previously unsuitable for recent isolates.

Characterization of Plasma Immunoglobulin G Responses in Elite Neutralizers of Human Cytomegalovirus.

BACKGROUND: Human cytomegalovirus (HCMV) is the most common infectious complication of organ transplantation and cause of birth defects worldwide. There are limited therapeutic options and no licensed vaccine to prevent HCMV infection or disease. To inform development of HCMV antibody-based interventions, a previous study identified individuals with potent and broad plasma HCMV-neutralizing activity, termed elite neutralizers (ENs), from a cohort of HCMV-seropositive (SP) blood donors. However, the specificities and functions of plasma antibodies associated with EN status remained undefined. METHODS: We sought to determine the plasma antibody specificities, breadth, and Fc-mediated antibody effector functions associated with the most potent HCMV-neutralizing responses in plasma from ENs (n = 25) relative to that from SP donors (n = 19). We measured antibody binding against various HCMV strains and glycoprotein targets and evaluated Fc-mediated effector functions, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). RESULTS: We demonstrate that ENs have elevated immunoglobulin G binding responses against multiple viral glycoproteins, relative to SP donors. Our study also revealed potent HCMV-specific antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis activity of plasma from ENs. CONCLUSIONS: We conclude that antibody responses against multiple glycoprotein specificities may be needed to achieve potent plasma neutralization and that potently HCMV elite-neutralizing plasma antibodies can also mediate polyfunctional responses.

Viral and Cellular Factors Contributing to the Hematogenous Dissemination of Human Cytomegalovirus via Polymorphonuclear Leukocytes.

Polymorphonuclear leukocytes (PMNs) presumably transmit human cytomegalovirus (HCMV) between endothelial cells in blood vessels and thereby facilitate spread to peripheral organs. We aimed to identify viral components that contribute to PMN-mediated transmission and test the hypothesis that cellular adhesion molecules shield transmission sites from entry inhibitors. Stop codons were introduced into the genome of HCMV strain Merlin to delete pUL74 of the trimeric and pUL128 of the pentameric glycoprotein complex and the tegument proteins pp65 and pp71. Mutants were analyzed regarding virus uptake by PMNs and transfer of infection to endothelial cells. Cellular adhesion molecules were evaluated for their contribution to virus transmission using function-blocking antibodies, and hits were further analyzed regarding shielding against inhibitors of virus entry. The viral proteins pUL128, pp65, and pp71 were required for efficient PMN-mediated transmission, whereas pUL74 was dispensable. On the cellular side, the blocking of the αLß2-integrin LFA-1 reduced virus transfer by 50% and allowed entry inhibitors to reduce it further by 30%. In conclusion, these data show that PMN-mediated transmission depends on the pentameric complex and an intact tegument and supports the idea of a virological synapse that promotes this dissemination mode both directly and via immune evasion.

Combined knockdown of RL13 and UL128 for release of cell-free infectivity from recent HCMV isolates.

Due to strictly cell-associated growth, experiments requiring cell-free virus are not applicable to recent clinical HCMV isolates to date. On the other hand, adaptation to cell-free growth is associated with undesirable changes in the viral gene regions RL13 and UL128. We had previously found that siRNA-mediated reduction of UL128 expression allowed transient release of cell-free virus by clinical isolates, and now hypothesized that virus yield could be further increased by additional knockdown of RL13. Despite the extensive polymorphism of RL13, effective RL13-specific siRNAs could be designed for three recent isolates and the Merlin strain. Knockdown efficiency was demonstrated at the protein level with a Merlin variant expressing V5-tagged pRL13. Knockdown of RL13 alone did not result in measurable release of cell-free virus, but combined knockdown of RL13 and UL128 increased infectivity in cell-free supernatants by a factor of 10-2000 compared to knockdown of UL128 alone. These supernatants could be used in dose-response assays to compare the effect of a neutralizing antibody on the various HCMV isolates. In summary, combined knockdown of RL13 and UL128 by specific siRNAs allows reliable release of cell-free infectivity from otherwise strictly cell-associated HCMV isolates without the need to modify the viral genome.

Peptide Derivatives of Platelet-Derived Growth Factor Receptor Alpha Inhibit Cell-Associated Spread of Human Cytomegalovirus.

Cell-free human cytomegalovirus (HCMV) can be inhibited by a soluble form of the cellular HCMV-receptor PDGFRα, resembling neutralization by antibodies. The cell-associated growth of recent HCMV isolates, however, is resistant against antibodies. We investigated whether PDGFRα-derivatives can inhibit this transmission mode. A protein containing the extracellular PDGFRα-domain and 40-mer peptides derived therefrom were tested regarding the inhibition of the cell-associated HCMV strain Merlin-pAL1502, hits were validated with recent isolates, and the most effective peptide was modified to increase its potency. The modified peptide was further analyzed regarding its mode of action on the virion level. While full-length PDGFRα failed to inhibit HCMV isolates, three peptides significantly reduced virus growth. A 30-mer version of the lead peptide (GD30) proved even more effective against the cell-free virus, and this effect was HCMV-specific and depended on the viral glycoprotein O. In cell-associated spread, GD30 reduced both the number of transferred particles and their penetration. This effect was reversible after peptide removal, which allowed the synchronized analysis of particle transfer, showing that two virions per hour were transferred to neighboring cells and one virion was sufficient for infection. In conclusion, PDGFRα-derived peptides are novel inhibitors of the cell-associated spread of HCMV and facilitate the investigation of this transmission mode.

Selection of Human Cytomegalovirus Mutants with Resistance against PDGFRα-Derived Entry Inhibitors.

The human cytomegalovirus (HCMV) infects fibroblasts via an interaction of its envelope glycoprotein gO with the cellular platelet-derived growth factor receptor alpha (PDGFRα), and soluble derivatives of this receptor can inhibit viral entry. We aimed to select mutants with resistance against PDGFRα-Fc and the PDGFRα-derived peptides GT40 and IK40 to gain insight into the underlying mechanisms and determine the genetic barrier to resistance. An error-prone variant of strain AD169 was propagated in the presence of inhibitors, cell cultures were monitored weekly for signs of increased viral growth, and selected viruses were tested regarding their sensitivity to the inhibitor. Resistant virus was analyzed by DNA sequencing, candidate mutations were transferred into AD169 clone pHB5 by seamless mutagenesis, and reconstituted virus was again tested for loss of sensitivity by dose-response analyses. An S48Y mutation in gO was identified that conferred a three-fold loss of sensitivity against PDGFRα-Fc, a combination of mutations in gO, gH, gB and gN reduced sensitivity to GT40 by factor 4, and no loss of sensitivity occurred with IK40. The resistance-conferring mutations support the notion that PDGFRα-Fc and GT40 perturb the interaction of gO with its receptor, but the relatively weak effect indicates a high genetic barrier to resistance.

Transmission of cell-associated human cytomegalovirus isolates between various cell types using polymorphonuclear leukocytes as a vehicle.

Polymorphonuclear leukocytes (PMNs) are regarded as vehicles for the hematogenous dissemination of human cytomegalovirus (HCMV). In cell culture, this concept has been validated with cell-free laboratory strains but not yet with clinical HCMV isolates that grow strictly cell-associated. We, therefore, aimed to evaluate whether PMNs can also transmit such isolates from initially infected fibroblasts to other cell types, which might further clarify the role of PMNs in HCMV dissemination and provide a model to search for potential inhibitors. PMNs, which have been isolated from HCMV-seronegative individuals, were added for 3 h to fibroblasts infected with recent cell-associated HCMV isolates, then removed and transferred to various recipient cell cultures. The transfer efficiency in the recipient cultures was evaluated by immunofluorescence staining of viral immediate early antigens. Soluble derivatives of the cellular HCMV entry receptor PDGFRα were analyzed for their potential to interfere with this transfer. All of five tested HCMV isolates could be transferred to fibroblasts, endothelial and epithelial cells with transfer rates ranging from 2 to 9%, and the transferred viruses could spread focally in these recipient cells within 1 week. The PDGFRα-derived peptides IK40 and GT40 reduced transfer by 40 and 70% when added during the uptake step. However, when added during the transfer step, only IK40 was effective, inhibiting transmission by 20% on endothelial cells and 50-60% on epithelial cells and fibroblasts. These findings further corroborate the assumption of cell-associated HCMV dissemination by PMNs and demonstrate that it is possible to inhibit this transmission mode.

Role of Envelope Glycoprotein Complexes in Cell-Associated Spread of Human Cytomegalovirus.

The role of viral envelope glycoproteins, particularly the accessory proteins of trimeric and pentameric gH/gL-complexes, in cell-associated spread of human cytomegalovirus (HCMV) is unclear. We aimed to investigate their contribution in the context of HCMV variants that grow in a strictly cell-associated manner. In the genome of Merlin pAL1502, the glycoproteins gB, gH, gL, gM, and gN were deleted by introducing stop codons, and the mutants were analyzed for viral growth. Merlin and recent HCMV isolates were compared by quantitative immunoblotting for expression of accessory proteins of the trimeric and pentameric gH/gL-complexes, gO and pUL128. Isolates were treated with siRNAs against gO and pUL128 and analyzed regarding focal growth and release of infectious virus. All five tested glycoproteins were essential for growth of Merlin pAL1502. Compared with this model virus, higher gO levels were measured in recent isolates of HCMV, and its knockdown decreased viral growth. Knockdown of pUL128 abrogated the strict cell-association and led to release of infectivity, which allowed cell-free transfer to epithelial cells where the virus grew again strictly cell-associated. We conclude that both trimer and pentamer contribute to cell-associated spread of recent clinical HCMV isolates and downregulation of pentamer can release infectious virus into the supernatant.

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.

In vivo Downregulation of MHC Class I Molecules by HCMV Occurs During All Phases of Viral Replication but Is Not Always Complete.

Based on cell culture data, MHC class I downregulation by HCMV on infected cells has been suggested as a means of immune evasion by this virus. In order to address this issue in vivo, an immunohistochemical analysis of tissue sections from biopsy and autopsy materials of HCMV infected organs was performed. HCMV antigens from the immediate early, early, and late phase of viral replication, and cellular MHC class I molecules were detected simultaneously or in serial sections by immuno-peroxidase and immuno-alkaline phosphatase techniques. Investigated organs included lung, gastrointestinal tract, and placenta. Colocalization of MHC molecules with sites of viral replication as well as MHC expression in individual infected cells were analyzed. To detect immune effector cells at sites of viral replication, leukocytes, CD8+ lymphocytes, and HCMV antigens were stained in serial sections. While strong MHC class I expression was detected in the cells surrounding infected cells, it appeared downregulated in the majority of infected cells themselves, particularly in the late replication phase. Despite significantly reduced MHC class I signals on infected cells, sites of infection were infiltrated by inflammatory cells that consisted predominantly of CD8+ lymphocytes. The extent of inflammatory infiltrates was negatively correlated with the extent of HCMV infected cells. Taken together, our findings indicate that HCMV can downmodulate MHC class I expression in vivo, whereas cytokines originating from infiltrating immune effector cells probably up regulates MHC class I expression in noninfected bystander cells. The presence of cytotoxic lymphocytes in close contact to infected cells may reflect control of viral spread by these cells despite MHC class I downmodulation.

Dense Bodies of a gH/gL/UL128/UL130/UL131 Pentamer-Repaired Towne Strain of Human Cytomegalovirus Induce an Enhanced Neutralizing Antibody Response.

The development of a vaccine against human cytomegalovirus infection (HCMV) is a high-priority medical goal. The viral pentameric protein complex consisting of glycoprotein H (gH)/gL/UL128-131A (PC) is considered to be an important vaccine component. Its relevance to the induction of a protective antibody response is, however, still a matter of debate. We addressed this issue by using subviral dense bodies (DBs) of HCMV. DBs are exceptionally immunogenic. Laboratory HCMV strain DBs harbor important neutralizing antibody targets, like the glycoproteins B, H, L, M, and N, but they are devoid of the PC. To be able to directly compare the impact of the PC on the levels of neutralizing antibody (NT-abs) responses, a PC-positive variant of the HCMV laboratory strain Towne was established by bacterial artificial chromosome (BAC) mutagenesis (Towne-UL130rep). This strain synthesized PC-positive DBs upon infection of fibroblasts. These DBs were used in side-by-side immunizations with PC-negative Towne DBs. Mouse and rabbit sera were tested to address the impact of the PC on DB immunogenicity. The neutralizing antibody response to PC-positive DBs was superior to that of PC-negative DBs, as tested on fibroblasts, epithelial cells, and endothelial cells and for both animal species used. The experiments revealed the potential of the PC to enhance the antibody response against HCMV. Of particular interest was the finding that PC-positive DBs induced an antibody response that blocked the infection of fibroblasts by a PC-positive viral strain more efficiently than sera following immunizations with PC-negative particles.IMPORTANCE Infections with the human cytomegalovirus (HCMV) may cause severe and even life-threatening disease manifestations in newborns and immunosuppressed individuals. Several strategies for the development of a vaccine against this virus are currently pursued. A critical question in this respect refers to the antigenic composition of a successful vaccine. Using a subviral particle vaccine candidate, we show here that one protein complex of HCMV, termed the pentameric complex (PC), enhances the neutralizing antibody response against viral infection of different cell types. We further show for the first time that this not only relates to the infection of epithelial or endothelial cells; the presence of the PC in the particles also enhanced the neutralizing antibody response against the infection of fibroblasts by HCMV. Together, these findings argue in favor of including the PC in strategies for HCMV vaccine development.

The N Terminus of Human Cytomegalovirus Glycoprotein O Is Important for Binding to the Cellular Receptor PDGFRα.

The human cytomegalovirus (HCMV) glycoprotein complex gH/gL/gO is required for the infection of cells by cell-free virions. It was recently shown that entry into fibroblasts depends on the interaction of gO with the platelet-derived growth factor receptor alpha (PDGFRα). This interaction can be blocked with soluble PDGFRα-Fc, which binds to HCMV virions and inhibits entry. The aim of this study was to identify parts of gO that contribute to PDGFRα binding. In a systematic mutational approach, we targeted potential interaction sites by exchanging conserved clusters of charged amino acids of gO with alanines. To screen for impaired interaction with PDGFRα, virus mutants were tested for sensitivity to inhibition by soluble PDGFRα-Fc. Two mutants with mutations within the N terminus of gO (amino acids 56 to 61 and 117 to 121) were partially resistant to neutralization. To validate whether these mutations impair interaction with PDGFRα-Fc, we compared binding of PDGFRα-Fc to mutant and wild-type virions via quantitative immunofluorescence analysis. PDGFRα-Fc staining intensities were reduced by 30% to 60% with mutant virus particles compared to wild-type particles. In concordance with the reduced binding to the soluble receptor, virus penetration into fibroblasts, which relies on binding to the cellular PDGFRα, was also reduced. In contrast, PDGFRα-independent penetration into endothelial cells was unaltered, demonstrating that the phenotypes of the gO mutant viruses were specific for the interaction with PDGFRα. In conclusion, the mutational screening of gO revealed that the N terminus of gO contributes to efficient spread in fibroblasts by promoting the interaction of virions with its cellular receptor.IMPORTANCE The human cytomegalovirus is a highly prevalent pathogen that can cause severe disease in immunocompromised hosts. Currently used drugs successfully target the viral replication within the host cell, but their use is restricted due to side effects and the development of resistance. An alternative approach is the inhibition of virus entry, for which understanding the details of the initial virus-cell interaction is desirable. As binding of the viral gH/gL/gO complex to the cellular PDGFRα drives infection of fibroblasts, this is a potential target for inhibition of infection. Our mutational mapping approach suggests the N terminus as the receptor binding portion of the protein. The respective mutants were partially resistant to inhibition by PDGFRα-Fc but also attenuated for infection of fibroblasts, indicating that such mutations have little if any benefit for the virus. These findings highlight the potential of targeting the interaction of gH/gL/gO with PDGFRα for therapeutic inhibition of HCMV.

Signatures of T and B Cell Development, Functional Responses and PD-1 Upregulation After HCMV Latent Infections and Reactivations in Nod.Rag.Gamma Mice Humanized With Cord Blood CD34+ Cells.

Human cytomegalovirus (HCMV) latency is typically harmless but reactivation can be largely detrimental to immune compromised hosts. We modeled latency and reactivation using a traceable HCMV laboratory strain expressing the Gaussia luciferase reporter gene (HCMV/GLuc) in order to interrogate the viral modulatory effects on the human adaptive immunity. Humanized mice with long-term (more than 17 weeks) steady human T and B cell immune reconstitutions were infected with HCMV/GLuc and 7 weeks later were further treated with granulocyte-colony stimulating factor (G-CSF) to induce viral reactivations. Whole body bio-luminescence imaging analyses clearly differentiated mice with latent viral infections vs. reactivations. Foci of vigorous viral reactivations were detectable in liver, lymph nodes and salivary glands. The number of viral genome copies in various tissues increased upon reactivations and were detectable in sorted human CD14+, CD169+, and CD34+ cells. Compared with non-infected controls, mice after infections and reactivations showed higher thymopoiesis, systemic expansion of Th, CTL, Treg, and Tfh cells and functional antiviral T cell responses. Latent infections promoted vast development of memory CD4+ T cells while reactivations triggered a shift toward effector T cells expressing PD-1. Further, reactivations prompted a marked development of B cells, maturation of IgG+ plasma cells, and HCMV-specific antibody responses. Multivariate statistical methods were employed using T and B cell immune phenotypic profiles obtained with cells from several tissues of individual mice. The data was used to identify combinations of markers that could predict an HCMV infection vs. reactivation status. In spleen, but not in lymph nodes, higher frequencies of effector CD4+ T cells expressing PD-1 were among the factors most suited to distinguish HCMV reactivations from infections. These results suggest a shift from a T cell dominated immune response during latent infections toward an exhausted T cell phenotype and active humoral immune response upon reactivations. In sum, this novel in vivo humanized model combined with advanced analyses highlights a dynamic system clearly specifying the immunological spatial signatures of HCMV latency and reactivations. These signatures can be merged as predictive biomarker clusters that can be applied in the clinical translation of new therapies for the control of HCMV reactivation.

Inhibition of Tetraspanin Functions Impairs Human Papillomavirus and Cytomegalovirus Infections.

Tetraspanins are suggested to regulate the composition of cell membrane components and control intracellular transport, which leaves them vulnerable to utilization by pathogens such as human papillomaviruses (HPV) and cytomegaloviruses (HCMV) to facilitate host cell entry and subsequent infection. In this study, by means of cellular depletion, the cluster of differentiation (CD) tetraspanins CD9, CD63, and CD151 were found to reduce HPV16 infection in HeLa cells by 50 to 80%. Moreover, we tested recombinant proteins or peptides of specific tetraspanin domains on their effect on the most oncogenic HPV type, HPV16, and HCMV. We found that the C-terminal tails of CD63 and CD151 significantly inhibited infections of both HPV16 and HCMV. Although CD9 was newly identified as a key cellular factor for HPV16 infection, the recombinant CD9 C-terminal peptide had no effect on infection. Based on the determined half-maximal inhibitory concentration (IC50), we classified CD63 and CD151 C-terminal peptides as moderate to potent inhibitors of HPV16 infection in HeLa and HaCaT cells, and in EA.hy926, HFF (human foreskin fibroblast) cells, and HEC-LTT (human endothelial cell-large T antigen and telomerase) cells for HCMV, respectively. These results indicate that HPV16 and HCMV share similar cellular requirements for their entry into host cells and reveal the necessity of the cytoplasmic CD151 and CD63 C-termini in virus infections. Furthermore, this highlights the suitability of these peptides for functional investigation of tetraspanin domains and as inhibitors of pathogen infections.

A TB40/E-derived human cytomegalovirus genome with an intact US-gene region and a self-excisable BAC cassette for immunological research.

For immunological research on the human cytomegalovirus (HCMV), a virus that combines the broad cell tropism of clinical isolates, efficient replication in cell culture, the complete set of MHC-I modulator genes, and suitability for genetic engineering is desired. Here, we aimed to generate a genetically complete derivative of HCMV strain TB40/E as a bacterial artificial chromosome (BAC) with a self-excisable BAC cassette. The BAC cassette was inserted into the US2-US6 gene region (yielding TB40-BACKL7), relocated into the UL73/UL74 region with modifications that favor excision of the BAC cassette during replication in fibroblasts, and finally the US2-US6 region was restored, resulting in BAC clone TB40-BACKL7-SE When this BAC clone was transfected into fibroblasts at efficiencies >0.1%, replicating virus that had lost the BAC cassette appeared within 2 weeks after transfection, grew to high titers, and displayed the broad tropism of the parental virus. The degree of MHC-I down-regulation by this virus was consistent with functional restoration of US2-US6. To enable detection of infected cells by flow cytometry, an enhanced green fluorescent protein (EGFP)-expression cassette was inserted downstream of US34A, yielding the fluorescent virus RV-TB40-BACKL7-SE-EGFP.

A derivative of platelet-derived growth factor receptor alpha binds to the trimer of human cytomegalovirus and inhibits entry into fibroblasts and endothelial cells.

Human cytomegalovirus (HCMV) is a widely distributed herpesvirus that causes significant morbidity in immunocompromised hosts. Inhibitors of viral DNA replication are available, but adverse effects limit their use. Alternative antiviral strategies may include inhibition of entry. We show that soluble derivatives of the platelet-derived growth factor receptor alpha (PDGFR-alpha), a putative receptor of HCMV, can inhibit HCMV infection of various cell types. A PDGFR-alpha-Fc fusion protein binds to and neutralizes cell-free virus particles at an EC50 of 10-30 ng/ml. Treatment of particles reduced both attachment to and fusion with cells. In line with the latter, PDGFR-alpha-Fc was also effective when applied postattachment. A peptide scan of the extracellular domain of PDGFR-alpha identified a 40mer peptide that inhibits infection at an EC50 of 1-2 nmol/ml. Both, peptide and fusion protein, were effective against various HCMV strains and are hence promising candidates for the development of novel anti-HCMV therapies.

Importance of Highly Conserved Peptide Sites of Human Cytomegalovirus gO for Formation of the gH/gL/gO Complex.

The glycoprotein O (gO) is betaherpesvirus specific. Together with the viral glycoproteins H and L, gO forms a covalent trimeric complex that is part of the viral envelope. This trimer is crucial for cell-free infectivity of human cytomegalovirus (HCMV) but dispensable for cell-associated spread. We hypothesized that the amino acids that are conserved among gOs of different cytomegaloviruses are important for the formation of the trimeric complex and hence for efficient virus spread. In a mutational approach, nine peptide sites, containing all 13 highly conserved amino acids, were analyzed in the context of HCMV strain TB40-BAC4 with regard to infection efficiency and formation of the gH/gL/gO complex. Mutation of amino acids (aa) 181 to 186 or aa 193 to 198 resulted in the loss of the trimer and a complete small-plaque phenotype, whereas mutation of aa 108 or aa 249 to 254 caused an intermediate phenotype. While individual mutations of the five conserved cysteines had little impact, their relevance was revealed in a combined mutation, which abrogated both complex formation and cell-free infectivity. C343 was unique, as it was sufficient and necessary for covalent binding of gO to gH/gL. Remarkably, however, C218 together with C167 rescued infectivity in the absence of detectable covalent complex formation. We conclude that all highly conserved amino acids contribute to the function of gO to some extent but that aa 181 to 198 and cysteines 343, 218, and 167 are particularly relevant. Surprisingly, covalent binding of gO to gH/gL is required neither for its incorporation into virions nor for proper function in cell-free infection. IMPORTANCE: Like all herpesviruses, the widespread human pathogen HCMV depends on glycoproteins gB, gH, and gL for entry into target cells. Additionally, gH and gL have to bind gO in a trimeric complex for efficient cell-free infection. Homologs of gO are shared by all cytomegaloviruses, with 13 amino acids being highly conserved. In a mutational approach we analyzed these amino acids to elucidate their role in the function of gO. All conserved amino acids contributed either to formation of the trimeric complex or to cell-free infection. Notably, these two phenotypes were not inevitably linked as the mutation of a charged cluster in the center of gO abrogated cell-free infection while trimeric complexes were still being formed. Cysteine 343 was essential for covalent binding of gO to gH/gL; however, noncovalent complex formation in the absence of cysteine 343 also allowed for cell-free infectivity.

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.

Tetraspanin CD151 Promotes Initial Events in Human Cytomegalovirus Infection.

UNLABELLED: Human cytomegalovirus (HCMV), a betaherpesvirus, can cause life-threatening disease in immunocompromised individuals. Viral envelope glycoproteins that mediate binding to and penetration into target cells have been identified previously. In contrast, cellular proteins supporting HCMV during entry are largely unknown. In order to systematically identify host genes affecting initial steps of HCMV infection, a targeted RNA interference screen of 96 cellular genes was performed in endothelial cells by use of a virus strain expressing the full set of known glycoprotein H and L (gH/gL) complexes. The approach yielded five proviral host factors from different protein families and eight antiviral host factors, mostly growth factor receptors. The tetraspanin CD151 was uncovered as a novel proviral host factor and was analyzed further. Like endothelial cells, fibroblasts were also less susceptible to HCMV infection after CD151 depletion. Virus strains with different sets of gH/gL complexes conferring either broad or narrow cell tropism were equally impaired. Infection of CD151-depleted cells by a fluorescent virus with differentially labeled capsid and envelope proteins revealed a role of CD151 in viral penetration but not in adsorption to the cell. In conclusion, the tetraspanin CD151 has emerged as a novel host factor in HCMV entry and as a putative antiviral target. IMPORTANCE: At present, the events at the virus-cell interface and the cellular proteins involved during the HCMV entry steps are scarcely understood. In this study, several host factors with putative roles in this process were identified. The tetraspanin CD151 was discovered as a previously unrecognized proviral host factor for HCMV and was found to support viral penetration into the target cells. The findings of this study shed light on the cellular contribution during the initial steps of HCMV infection and open a new direction in HCMV research.