CD8(+) T cells from mice transnuclear for a TCR that recognizes a single H-2K(b)-restricted MHV68 epitope derived from gB-ORF8 help control infection.

To study the CD8(+) T cell response against a mouse γ-herpes virus, we generated K(b)-MHV-68-ORF8(604-612)RAG(-/-) CD8(+) T cell receptor transnuclear (TN) mice as a source of virus-specific CD8(+) T cells. K(b)-ORF8-Tet(+) CD8(+) T cells, expanded in the course of a resolving MHV-68 infection, served as a source of nucleus donors. Various in vivo and ex vivo assay criteria demonstrated the fine specificity and functionality of TN cells. TN cells proliferated extensively in response to viral infection, helped control viral burden, and exhibited a phenotype similar to that of endogenous K(b)-ORF8-Tet(+) cells. When compared to OT-1 cells, TN cells displayed distinct properties in response to lymphopenia and cognate antigen stimulation, which may be attributable to the affinity of the TCR expressed by the TN cells. The availability of MHV-68-specific CD8(+) TCR TN mice provides a new tool for investigating aspects of host-pathogen interactions unique to γ-herpes viruses.

A gammaherpesvirus ubiquitin-specific protease is involved in the establishment of murine gammaherpesvirus 68 infection.

Murine gammaherpesvirus 68 (MHV-68) contains a ubiquitin (Ub)-specific cysteine protease (USP) domain embedded within the large tegument protein ORF64, as do all other herpesviruses. The biological role of this protease is still unclear, but for the alphaherpesvirus Marek's disease virus, its USP is involved in T-cell lymphoma formation. We here study the role of the MHV-68 USP, encoded by ORF64. By constructing a mutant virus with a single cysteine-to-alanine replacement in the active site of ORF64, we demonstrate that the USP activity of ORF64 is abolished. The mutant virus replicates less efficiently in vitro, and plaque size is reduced compared to that of a revertant virus. Electron microscopy of infected cells did not reveal any obvious differences in virion morphogenesis or differences in egress for the mutant and revertant viruses. Intraperitoneal infection of C57/BL6 mice demonstrates that the mutant virus is generally cleared by day 7, indicating a role for the USP in the persistence of MHV-68 infection or efficient replication. However, the USP activity in MHV-68 is unlikely to be involved in the establishment of latency or reactivation, since we observed no significant difference in viral DNA genome copy number in the spleen or in the number of cells that reactivate MHV-68 from latency. Our results for MHV-68 ORF64 are consistent with an enzymatic function of the tegument protein that is beneficial to the virus during acute infection, particularly in vivo.

Human cytomegalovirus glycoprotein B is required for virus entry and cell-to-cell spread but not for virion attachment, assembly, or egress.

Glycoprotein B (gB) homologs are conserved throughout the family Herpesviridae and appear to serve essential, universal functions, as well as specific functions unique to a particular herpesvirus. Genetic analysis is a powerful tool to analyze protein function, and while it has been possible to generate virus mutants, complementation of essential virus knockouts has been problematic. Human cytomegalovirus (HCMV) gB (UL55) plays an essential role in the replication cycle of the virus. To define the function(s) of gB in HCMV infection, the BAC system was used to generate a recombinant virus in which the UL55 gene was replaced with galK (pAD/CreDeltaUL55). UL55 deletions in the viral genome have been made before, demonstrating that UL55 is an essential gene. However, without being able to successfully complement the genetic defect, a phenotypic analysis of the mutant virus was impossible. We generated fibroblasts expressing HCMV gB that complement pAD/CreDeltaUL55 and produce infectious virions lacking the UL55 gene but containing wild-type gB on the virion surface (DeltaUL55-gB HCMV). This is the first successful complementation of an HCMV mutant with a glycoprotein deleted. To characterize DeltaUL55 infection in the absence of gB, noncomplementing cells were infected with DeltaUL55-gB virus. All stages of gene expression were detected, and significant amounts of DNase-resistant viral DNA genomes, representing whole intact virions, were released into the infected cell supernatant. Gradient purification of these virions revealed they lacked gB but contained other viral structural proteins. The gB-null virions were able to attach to the cell surface similarly to wild-type gB-containing virions but were defective in virus entry and cell-to-cell spread. Glycoprotein B-null virions do, however, contain infectious DNA, as IE gene expression can be detected in fibroblasts following treatment of attached gB-null virions with a membrane fusion agent, polyethylene glycol. Taken together, our results indicate that gB is required for virus entry and cell-to-cell spread of the virus. However, HCMV gB is not absolutely required for virus attachment or assembly and egress from infected cells.

Epidermal growth factor receptor is not required for human cytomegalovirus entry or signaling.

Human cytomegalovirus (HCMV) can bind, fuse, and initiate gene expression in a diverse range of vertebrate cell types. This broad cellular tropism suggests that multiple receptors and/or universally distributed receptors mediate HCMV entry. Our laboratory has recently discovered that certain beta1 and beta3 integrin heterodimers are critical mediators of HCMV entry into permissive fibroblasts (A. L. Feire, H. Koss, and T. Compton, Proc. Natl. Acad. Sci. USA 101:15470-15475, 2004). It has also been reported that epidermal growth factor receptor (EGFR) is necessary for HCMV-mediated signaling and entry (X. Wang, S. M. Huong, M. L. Chiu, N. Raab-Traub, and E. E. Huang, Nature 424:456-461, 2003). Integrins are known to signal synergistically with growth factor receptors, and this coordination was recently reported for EGFR and beta3 integrins in the context of HCMV entry (X. Wang, D. Y. Huang, S. M. Huong, and E. S. Huang, Nat. Med. 11:515-521, 2005). However, EGFR-negative cell lines, such as hematopoietic cells, are known to be infected by HCMV. Therefore, we wished to confirm a role for EGFR in HCMV entry and then examine any interaction between beta1 integrins and EGFR during the entry process. Surprisingly, we were unable to detect any role for EGFR in the process of HCMV entry into fibroblast, epithelial, or endothelial cell lines. Additionally, HCMV did not activate the EGFR kinase in fibroblast cell lines. We first examined HCMV entry into two EGFR-positive or -negative cell lines but observed no increase in entry when EGFR was expressed to high levels. Physically blocking EGFR with a neutralizing antibody in fibroblast, epithelial, or endothelial cell lines or blocking EGFR kinase signaling with a chemical inhibitor in fibroblast cells did not inhibit virus entry. Lastly, we were unable to detect phosphorylation of EGFR in fibroblasts cells in response to HCMV stimulation. Our findings demonstrate that EGFR does not play a significant role in HCMV entry or signaling. These results suggest that specific integrin heterodimers either act alone as the primary entry receptors or interact in conjunction with an additional receptor(s), other than EGFR, to facilitate virus entry.