Eviction of linker histone H1 by NAP-family histone chaperones enhances activated transcription.

BACKGROUND: In the Metazoan nucleus, core histones assemble the genomic DNA to form nucleosome arrays, which are further compacted into dense chromatin structures by the linker histone H1. The extraordinary density of chromatin creates an obstacle for accessing the genetic information. Regulation of chromatin dynamics is therefore critical to cellular homeostasis, and histone chaperones serve as prominent players in these processes. In the current study, we examined the role of specific histone chaperones in negotiating the inherently repressive chromatin structure during transcriptional activation. RESULTS: Using a model promoter, we demonstrate that the human nucleosome assembly protein family members hNap1 and SET/Taf1ß stimulate transcription in vitro during pre-initiation complex formation, prior to elongation. This stimulatory effect is dependent upon the presence of activators, p300, and Acetyl-CoA. We show that transcription from our chromatin template is strongly repressed by H1, and that both histone chaperones enhance RNA synthesis by overcoming H1-induced repression. Importantly, both hNap1 and SET/Taf1ß directly bind H1, and function to enhance transcription by evicting the linker histone from chromatin reconstituted with H1. In vivo studies demonstrate that SET/Taf1ß, but not hNap1, strongly stimulates activated transcription from the chromosomally-integrated model promoter, consistent with the observation that SET/Taf1ß is nuclear, whereas hNap1 is primarily cytoplasmic. Together, these observations indicate that SET/Taf1ß may serve as a critical regulator of H1 dynamics and gene activation in vivo. CONCLUSIONS: These studies uncover a novel function for SET that mechanistically couples transcriptional derepression with H1 dynamics. Furthermore, they underscore the significance of chaperone-dependent H1 displacement as an essential early step in the transition of a promoter from a dense chromatin state into one that is permissive to transcription factor binding and robust activation.

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.

Ubiquitination, ubiquitin-like modifiers, and deubiquitination in viral infection.

Ubiquitin is important for nearly every aspect of cellular physiology. All viruses rely extensively on host machinery for replication; therefore, it is not surprising that viruses connect to the ubiquitin pathway at many levels. Viral involvement with ubiquitin occurs either adventitiously because of the unavoidable usurpation of cellular processes, or for some specific purpose selected for by the virus to enhance viral replication. Here, we review current knowledge of how the ubiquitin pathway alters viral replication and how viruses influence the ubiquitin pathway to enhance their own replication.

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.

The CD8 T-cell response against murine gammaherpesvirus 68 is directed toward a broad repertoire of epitopes from both early and late antigens.

Infection of mice with murine gammaherpesvirus 68 (MHV-68) robustly activates CD8 T cells, but only six class I major histocompatibility complex (MHC)-restricted epitopes have been described to date for the widely used H-2(b) haplotype mice. To explore the specificity and kinetics of the cytotoxic T-lymphocyte response in MHV-68-infected C57BL/6 mice, we screened for H-2K(b)- and H-2D(b)-restricted epitopes using a set of 384 candidate epitopes in an MHC tetramer-based approach and identified 19 new epitopes in 16 different open reading frames. Of the six known H-2K(b)- and H-2D(b)-restricted epitopes, we confirmed a response against three and did not detect CD8 T-cell-specific responses for the remaining three. The peak of the CD8 T-cell response to most peptides occurs between 6 and 10 days postinfection. The respective MHC tetramer-positive CD8 T cells display an activated/effector phenotype (CD62L(lo) and CD44(hi)) and produce gamma interferon upon peptide stimulation ex vivo. MHV-68 infection in vivo elicits a response to multiple viral epitopes, derived from both early and late viral antigens, illustrating a far broader T-cell repertoire and more-rapid activation than those previously recorded.

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.