Viral gene expression patterns in human herpesvirus 6B-infected T cells

Herpesvirus gene expression is divided into immediate-early (IE) or alpha genes, early (E) or beta genes, and late (L) or gamma genes on the basis of temporal expression and dependency on other gene products. By using real-time PCR, we have investigated the expression of 35 human herpesvirus 6B (HHV-6B) genes in T cells infected by strain PL-1. Kinetic analysis and dependency on de novo protein synthesis and viral DNA polymerase activity suggest that the HHV-6B genes segregate into six separate kinetic groups. The genes expressed early (groups I and II) and late (groups V and VI) corresponded well with IE and L genes, whereas the intermediate groups III and IV contained E and L genes. Although HHV-6B has characteristics similar to those of other roseoloviruses in its overall gene regulation, we detected three B-variant-specific IE genes. Moreover, genes that were independent of de novo protein synthesis clustered in an area of the viral genome that has the lowest identity to the HHV-6A variant. The organization of IE genes in an area of the genome that differs from that of HHV-6A underscores the distinct differences between HHV-6B and HHV-6A and may provide a basis for further molecular and immunological analyses to elucidate their different biological behaviors.     

Two gamma interferon-activated site-like elements in the human cytomegalovirus major immediate-early promoter/enhancer are important for viral replication

Human cytomegalovirus (HCMV) infection directly initiates a signal transduction pathway that leads to activation of a large number of cellular interferon-stimulated genes (ISGs). Our previous studies demonstrated that two interferon response elements, the interferon-stimulated response element and gamma interferon-activated site (GAS), in the ISG promoters serve as HCMV response sites (VRS). Interestingly, two GAS-like VRS elements (VRS1) were also present in the HCMV major immediate-early promoter-enhancer (MIEP/E). In this study, the importance of these VRS elements in viral replication was investigated. We demonstrate that the expression of the major IE genes, IE1 and IE2, is interferon inducible. To understand the biological significance of this signal transduction pathway in HCMV major IE expression, the two VRS1 in the MIEP/E were mutated. Mutant HCMVs in which the VRS elements were deleted or that contained point mutations grew dramatically more slowly than wild-type virus at a low multiplicity of infection (MOI). Insertion of wild-type VRS1 into the mutant viral genome rescued the slow growth phenotype. Furthermore, the expression levels of major IE RNAs and proteins were greatly reduced during infection with the VRS mutants at a low MOI. HCMV microarray analysis indicated that infection of host cells with the VRS mutant virus resulted in a global reduction in the expression of viral genes. Collectively, these data demonstrate that the two VRS elements in the MIEP/E are necessary for efficient viral gene expression and replication. This study suggests that although the HCMV-initiated signal transduction pathway results in induction of cellular antiviral genes, it also functions to stimulate viral major IE gene expression. This might be a new viral strategy in which the pathway is used to regulate gene expression and play a role in reactivation.     

Human cytomegalovirus UL84 insertion mutant defective for viral DNA synthesis and growth

Human cytomegalovirus (HCMV) UL84 is required for oriLyt-dependent DNA replication, and evidence from transient transfection assays suggests that UL84 directly participates in DNA synthesis. In addition, because of its apparent interaction with IE2, UL84 is implicated as a possible regulatory protein. To address the role of UL84 in the context of the viral genome, we generated a recombinant HCMV bacterial artificial chromosome (BAC) construct that did not express the UL84 gene product. This construct, BAC-IN84/Ep, displayed a null phenotype in that it failed to produce infectious virus after transfection into human fibroblast cells, whereas a revertant virus readily produced viral plaques and, subsequently, infectious virus. Real-time quantitative PCR showed that BAC-IN84/Ep was defective for DNA synthesis in that no increase in the accumulation of viral DNA was observed in transfected cells. We were unable to complement BAC-IN84/Ep in trans; however, oriLyt-dependent DNA replication was observed by the cotransfection of UL84 and BAC-IN84/Ep. An analysis of viral mRNA by real-time PCR indicated that, even in the absence of DNA synthesis, all representative kinetic classes of genes were expressed in cells transfected with BAC-IN84/Ep. The detection of UL44 and IE2 by immunofluorescence in BAC-IN84/Ep-transfected cells showed that these proteins failed to partition into replication compartments, indicating that UL84 expression is essential for the formation of these proteins into replication centers within the context of the viral genome. These results show that UL84 provides an essential DNA replication function and influences the subcellular localization of other viral proteins.     

CCAAT box-dependent activation of the TATA-less human DNA polymerase alpha promoter by the human cytomegalovirus 72-kilodalton major immediate-early protein.

Human cytomegalovirus (HCMV) immediate-early (IE) proteins are known potent transregulators of viral and cellular gene expression upon HCMV infection. HCMV is known to activate a number of cellular genes intimately associated with the cell cycle and DNA replication by mechanisms involving the viral major IE 86-kDa protein (IE2). We have recently shown that IE2 mediates this activation in a TATA-dependent manner and interacts directly with the TATA-binding protein. However, a number of TATA-less cellular promoters, e.g., DNA polymerase alpha and dihydrofolate reductase, are also activated by HCMV infection. Consequently, we have asked how HCMV mediates this activation. We show that, consistent with its known TATA dependency, IE2 does not activate the DNA polymerase alpha promoter. In contrast, this promoter is strongly activated by the major IE 72-kDa protein (IE1). Whilst deletion of ATF or E2F sites within the DNA polymerase alpha promoter had little effect on IE1-mediated activation, removal of the CCAAT box appeared to abolish high levels of activation by IE1. Consistent with this observation, we also find that IE1 interacts directly with the CCAAT box binding factor CTF1 in vitro and massively augments CTF1-mediated activation of the DNA polymerase alpha promoter in transient transfection assays.     

DNA microarrays of the complex human cytomegalovirus genome: profiling kinetic class with drug sensitivity of viral gene expression.

We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the largest member of the herpesvirus family, human cytomegalovirus (HCMV). In this study, an HCMV chip was fabricated and used to characterize the temporal class of viral gene expression. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of oligonucleotides on glass for ORFs in the HCMV genome. Viral gene expression was monitored by hybridization to the oligonucleotide microarrays with fluorescently labelled cDNAs prepared from mock-infected or infected human foreskin fibroblast cells. By using cycloheximide and ganciclovir to block de novo viral protein synthesis and viral DNA replication, respectively, the kinetic classes of array elements were classified. The expression profiles of known ORFs and many previously uncharacterized ORFs provided a temporal map of immediate-early (alpha), early (beta), early-late (gamma1), and late (gamma2) genes in the entire genome of HCMV. Sequence compositional analysis of the 5' noncoding DNA sequences of the temporal classes, performed by using algorithms that automatically search for defined and recurring motifs in unaligned sequences, indicated the presence of potential regulatory motifs for beta, gamma1, and gamma2 genes. In summary, these fabricated microarrays of viral DNA allow rapid and parallel analysis of gene expression at the whole viral genome level. The viral chip approach coupled with global biochemical and genetic strategies should greatly speed the functional analysis of established as well as newly discovered large viral genomes.     

A Novel DDB2-ATM Feedback Loop Regulates Human Cytomegalovirus Replication

Human cytomegalovirus (HCMV) genome replication requires host DNA damage responses (DDRs) and raises the possibility that DNA repair pathways may influence viral replication. We report here that a nucleotide excision repair (NER)-associated-factor is required for efficient HCMV DNA replication. Mutations in genes encoding NER factors are associated with xeroderma pigmentosum (XP). One of the XP complementation groups, XPE, involves mutation in ddb2, which encodes DNA damage binding protein 2 (DDB2). Infectious progeny virus production was reduced by >2 logs in XPE fibroblasts compared to levels in normal fibroblasts. The levels of immediate early (IE) (IE2), early (E) (pp65), and early/late (E/L) (gB55) proteins were decreased in XPE cells. These replication defects were rescued by infection with a retrovirus expressing DDB2 cDNA. Similar patterns of reduced viral gene expression and progeny virus production were also observed in normal fibroblasts that were depleted for DDB2 by RNA interference (RNAi). Mature replication compartments (RCs) were nearly absent in XPE cells, and there were 1.5- to 2.0-log reductions in viral DNA loads in infected XPE cells relative to those in normal fibroblasts. The expression of viral genes (UL122, UL44, UL54, UL55, and UL84) affected by DDB2 status was also sensitive to a viral DNA replication inhibitor, phosphonoacetic acid (PAA), suggesting that DDB2 affects gene expression upstream of or events associated with the initiation of DNA replication. Finally, a novel, infection-associated feedback loop between DDB2 and ataxia telangiectasia mutated (ATM) was observed in infected cells. Together, these results demonstrate that DDB2 and a DDB2-ATM feedback loop influence HCMV replication.