Human Cytomegalovirus Glycoprotein B Is Required for Virus Entry and Cell-to-Cell Spread but Not for Virion Attachment, Assembly, or Egress

Human cytomegalovirus (HCMV), a ubiquitous, opportunistic pathogen, is a member of the Herpesviridae family of viruses (1, 44). Infection with HCMV is generally asymptomatic, but naive or immunosuppressed individuals, such as neonates, AIDS patients, and transplant recipients, often manifest serious disease (1, 44). HCMV is a promiscuous pathogen, able to enter and initiate infection in almost every vertebrate cell type tested. Similarly, disease can occur in nearly every organ system and cell type in the human body (1, 44). The ability of HCMV to enter such a wide variety of cell types requires the coordinated interaction of several virus-encoded envelope glycoproteins with multiple cell surface receptors (15). Herpesvirus entry, in general, is very complex and not well understood. For HCMV, the initial attachment step involves glycoprotein M (gM) and gB, both of which bind to heparan sulfate proteoglycans (HSPGs) on the cell surface (17, 33). The virus then quickly moves to a more stable docking interaction with one or more cellular receptors (15). It has recently been discovered that gB is able to interact with specific integrin heterodimers, a step that greatly enhances HCMV entry (21). It has also been reported that gB interacts with the epidermal growth factor receptor to mediate virus entry (62, 63), although our laboratory (28) and others (13) have not been able to confirm these findings. During the final phase of virus entry, the viral and cellular membranes fuse, releasing the tegument proteins and capsid into the cytoplasm. This fusion event is thought to require gB, as well as the gH/gL complex (6, 35, 46, 58, 59). It is possible that gB interaction with β1 integrins activates its fusogenic activity by some as yet unknown mechanism (21; A. L. Feire and T. Compton, unpublished data). Additionally, αVβ3 integrins were also recently identified as cellular receptors for gH (62) and are possibly required to activate its fusogenic activity, as well. gB, an abundant glycoprotein on the virus envelope (61) and the most highly conserved glycoprotein of the human herpesviruses, plays a critical role in the HCMV entry process, as it is believed to be involved in the initial virion-tethering step and the second, more stable attachment step, as well as the fusion event itself (reviewed in reference 15). Evidence supporting an attachment role for gB includes the ability of soluble gB (gBs) to bind heparin, a soluble mimic of HSPGs (17, 34). Additionally, gBs manifests two-step binding kinetics to cells, in which the protein is initially dissociable with soluble heparin but quickly becomes resistant to removal by heparin washes. This suggests that gB moves from one receptor, presumably HSPGs, to another, likely cellular integrins, during the attachment process (7). Evidence supporting a role of cellular integrins in the HCMV entry process includes the disintegrin-like domain (DLD) of gB binding specifically to β1 integrins (21; Feire et al., unpublished). Antibodies to HCMV gB, including those to the DLD and to specific integrin heterodimers, are able to efficiently neutralize virus entry at a postattachment stage, suggesting that gB and integrins are involved in the fusion event (23, 48; Feire et al., unpublished). Additionally, by comparison with other gB homologs, such as herpes simplex virus type 1 (HSV-1) gB, which has a well-defined role in virus entry, it is likely that HMCV gB also acts as a fusion mediator for the virus. Finally, gB contains a heptad repeat region, which is predicted to form an alpha-helical coiled coil, commonly found in many class I viral fusion proteins (41). Synthetic peptides that mimic the heptad repeat region of HCMV gB efficiently inhibit virus entry without interfering with virus attachment, presumably at the level of virus fusion (19, 41). Aside from its role in virus entry and fusion, gB is also believed to be required for cell-to-cell spread of the virus (23, 46, 48). During plaque formation, virions are thought to spread directly through cellular contacts or across cellular junctions (reviewed in reference 30). Evidence for the role of gB in HCMV cell-to-cell spread is indirect and includes the ability of gB-neutralizing antibodies to prevent plaque formation in infected cells (23, 46, 48). However, gB proteins from other herpesviruses, including HSV-1 and HSV-2, pseudorabies virus (PRV), Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus (KSHV), are also required for cell-to-cell spread (25, 45, 50, 52, 60). gB proteins from a variety of herpesviruses have also been proposed to be necessary to mediate virion assembly and egress from infected cells. Herpesvirus egress is thought to involve an initial envelopment step at the inner nuclear membrane, followed by a de-envelopment step at the outer nuclear membrane and, last, a reenvelopment step at a Golgi apparatus-derived membrane before the virion-containing vesicle fuses with the plasma membrane to release the virion outside the cell (43). Presumably, the fusogenic activity of viral glycoproteins is required for the initially enveloped particles to fuse with the outer nuclear membrane to be released into the cytoplasm (reviewed in reference 43). PRV, EBV, and KSHV have all been reported to require gB for virion egress (37, 39, 49). However, reports on HSV-1, as well as another, conflicting report on PRV, suggest that gB is not absolutely required for egress from infected cells, although slight defects are seen with HSV-1 (11, 20, 52, 53). For HSV-1, it appears that either gB or gH on the virion envelope is sufficient to mediate virion egress; however, mutants lacking both glycoproteins are unable to be released into the cytoplasm (20). Other than the fusogenic activity of HCMV gB, the protein also initiates multiple signaling events, even in the absence of other virion components (reviewed in reference 4). HCMV attachment and entry induce activation of innate immune responses, including the interferon response and inflammatory cytokine induction (reviewed in reference 4). gB has been demonstrated to bind Toll-like receptor 2, likely leading to the induction of inflammatory cytokines (5); however, the mechanism of activation of the interferon response is currently unknown. Additionally, the induction of cytoskeletal rearrangements and activation of focal adhesion kinase likely occur through gB binding to β1 integrins through its DLD and/or gH binding to β3 integrins during the entry process (21, 62; Feire, et al. unpublished). Genetic analysis is a powerful tool to determine functional roles in the context of viral infections. The recent development of the bacterial artificial chromosome (BAC) system has proven to be a great asset for reverse genetics studies; however, like all mutagenesis systems, it is limited. Essential gene mutants cannot be isolated and propagated unless the mutation is able to be efficiently complemented. Until now, all attempts to complement a gB deletion virus, or any essential HCMV glycoprotein knockout, have been unsuccessful. To further define the role of gB in the HCMV life cycle, we constructed a BAC plasmid containing a replacement of the UL55 gene, which encodes gB, with a galactose operon gene, galK (pAD/CreΔUL55). This ΔUL55 BAC was transfected into gB-expressing normal human dermal fibroblasts (NHDF-gB), which compensated for the loss of the UL55 gene, generating infectious virus with a deletion of the UL55 gene in the viral genome but pseudotyped with wild-type gB on the virion surface (ΔUL55-gB HCMV). When noncomplementing NHDF were infected with ΔUL55-gB HCMV, all stages of gene expression were detected and significant quantities of DNase-resistant viral genomes were released into the supernatant, indicating that viral egress occurs in the absence of gB. These ΔUL55 gB-null virions were gradient purified and demonstrated to contain other virion structural proteins, including gH, and tegument proteins pp150, pp71, and pp28. Using gB-null virions, we found HCMV could attach to cells in the absence of gB, as well as wild-type gB-containing virions, but the virus was completely noninfectious, confirming that gB is required for virus-cell fusion. These gB-null virions could, however, be chemically forced to fuse with NHDF by treatment with polyethylene glycol (PEG), which artificially induces the fusion of nearby membranes, leading to immediate-early (IE) gene expression. Additionally, the ΔUL55-gB virus was not able to spread from cell to cell in an NHDF plaque overlay assay, indicating that gB is also required for cell-to-cell spread of the virus. These genetic data complement prior approaches and demonstrates that gB mediates entry, but not egress. Additionally, this is the first report of a successful complementation of an HCMV glycoprotein mutant and establishes the ability to examine essential glycoprotein mutants in the context of a viral infection using a complementation system.