Human cytomegalovirus (HCMV) poses a significant threat to immunocompromised individuals and neonates infected in utero. Glycoprotein B (gB), the herpesvirus fusion protein, is a target for neutralizing antibodies and a vaccine candidate due to its indispensable role in infection. Here we show the crystal structure of the HCMV gB ectodomain bound to the Fab fragment of 1G2, a neutralizing human monoclonal antibody isolated from a seropositive subject. The gB/1G2 interaction is dominated by aromatic residues in the 1G2 heavy chain CDR3 protruding into a hydrophobic cleft in the gB antigenic domain 5 (AD-5). Structural analysis and comparison with HSV gB suggest the location of additional neutralizing antibody binding sites on HCMV gB. Finally, immunoprecipitation experiments reveal that 1G2 can bind to HCMV virion gB suggesting that its epitope is exposed and accessible on the virus surface. Our data will support the development of vaccines and therapeutic antibodies against HCMV infection.

The human cytomegalovirus (HCMV) protein RL13 has recently been described to be present in all primary isolates but rapidly mutated in culture adapted viruses. Although these data suggest a crucial role for this gene product in HCMV primary infection, no function has so far been assigned to this protein. Working with RL13 expressed in isolation in transfected human epithelial cells, we demonstrated that recombinant RL13 from the clinical HCMV isolates TR and Merlin have selective human immunoglobulin (Ig)-binding properties towards IgG1 and IgG2 subtypes. An additional Fc binding protein, RL12, was also identified as an IgG1 and IgG2 binding protein but not further characterized. The glycoprotein RL13 trafficked to the plasma membrane where it bound and internalized exogenous IgG or its constant fragment (Fcγ). Analysis of RL13 ectodomain mutants suggested that the RL13 Ig-like domain is responsible for the Fc binding activity. Ligand-dependent internalization relied on a YxxL endocytic motif located in the C-terminal tail of RL13. Additionally, we showed that the tyrosine residue could be replaced by phenylalanine but not by alanine, indicating that the internalization signal was independent from phosphorylation events. In sum, RL13 binds human IgG and may contribute to HCMV immune evasion in the infected host, but this function does not readily explain the instability of the RL13 gene during viral propagation in cultured cells.

Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in transplant patients and is the leading viral cause of birth defects after congenital infection. HCMV infection relies on the recognition of cell-specific receptors by one of the viral envelope glycoprotein complexes. Either the gH/gL/gO or the gH/gL/UL128/UL130/UL131A (Pentamer) complex has been found to fulfill this role, accounting for HCMV entry into almost all cell types. We have studied the UL116 gene product, a putative open reading frame identified by in silico analysis and predicted to code for a secreted protein. Virus infection experiments in mammalian cells demonstrated that UL116 is expressed late in the HCMV replication cycle and is a heavily glycosylated protein that first localizes to the cellular site of virus assembly and then inserts into the virion envelope. Transient-transfection studies revealed that UL116 is efficiently transported to the plasma membrane when coexpressed with gH and that gL competes with UL116 for gH binding. Further evidence for gH/UL116 complex formation was obtained by coimmunoprecipitation experiments on both transfected and infected cells and biochemical characterization of the purified complex. In summary, our results show that the product of the UL116 gene is an HCMV envelope glycoprotein that forms a novel gH-based complex alternative to gH/gL. Remarkably, the gH/UL116 complex is the first herpesvirus gH-based gL-less complex. IMPORTANCE HCMV infection can cause severe disease in immunocompromised adults and infants infected in utero.The dissection of the HCMV entry machinery is important to understand the mechanism of viral infection and to identify new vaccine antigens. The gH/gL/gO and gH/gL/UL128/UL130/UL131 (Pentamer) complexes play a key role in HCMV cell entry and tropism. Both complexes are formed by an invariant gH/gL scaffold on which the other subunits assemble. Here, we show that the UL116 gene product is expressed in infected cells and forms a heterodimer with gH. The gH/UL116 complex is carried on the infectious virions, although in smaller amounts than gH/gL complexes. No gH/UL116/gL ternary complex formed in transfected cells, suggesting that the gH/UL116 complex is independent from gL. This new gH-based gL-free complex represents a potential target for a protective HCMV vaccine and opens new perspectives on the comprehension of the HCMV cell entry mechanism and tropism. H uman cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus infecting 40 to 60% of the human population (1).HCMV infection usually is mild or asymptomatic in immunocompetent individuals, while it can cause severe disease in immunocompromised adults. Infants infected in utero can suffer from disseminated HCMV disease and impaired neurological development (2). Transplant patients do not adequately control HCMV infection or reactivation, often resulting in graft rejection and death (3). Preventing primary HCMV infection could significantly decrease the frequency of organ rejection in seronegative recip...

Human cytomegalovirus (HCMV) is known to exert suppressive effects on the host immune system through expression of various viral genes, thus directly and indirectly affecting antiviral immunity of the infected individuals. We report here that HCMV UL10 encodes a protein (pUL10) with immunosuppressive properties. UL10 has been classified as a member of the HCMV RL11 gene family. Although pUL10 is known to be dispensable for viral replication in cultured cells, its amino-acid sequence is well conserved among different HCMV isolates, suggesting that the protein has a crucial role in viral survival in the host environment. We show that pUL10 is cleaved from the cell surface of fibroblasts as well as epithelial cells and interacts with a cellular receptor ubiquitously expressed on the surface of human leukocytes, demonstrated by ex vivo cell-based assays and flow cytometric analyses on both lymphoid cell lines and primary blood cells. Furthermore, preincubation of peripheral blood mononuclear cells with purified pUL10 ectodomain results in significantly impaired proliferation and substantially reduced pro-inflammatory cytokine production, in particular in CD4 T cells upon in vitro T-cell stimulation. The inhibitory effect of pUL10 is also observed on antigen receptor-mediated intracellular tyrosine phosphorylation in a T-cell line. Based on these observations, we suggest that pUL10 is a newly identified immunomodulatory protein encoded by HCMV. Further elucidation of interactions between pUL10 and the host immune system during HCMV may contribute to finding ways towards new therapies for HCMV infection.