The nonclassical major histocompatibility complex (MHC) class I molecule HLA-E inhibits natural killer (NK) cell-mediated lysis by interacting with CD94/NKG2A receptors. Surface expression of HLA-E depends on binding of conserved peptides derived from MHC class I molecules. The same peptide is present in the leader sequence of the human cytomegalovirus (HCMV) glycoprotein UL40 (gpUL40). It is shown that, independently of the transporter associated with antigen processing, gpUL40 can up-regulate expression of HLA-E, which protects targets from NK cell lysis. While classical MHC class I molecules are down-regulated, HLA-E is up-regulated by HCMV. Induction of HLA-E surface expression by gpUL40 may represent an escape route for HCMV.

Human cytomegalovirus (HCMV) in clinical material cannot replicate efficiently in vitro until it has adapted by mutation. Consequently, wild-type HCMV differ fundamentally from the passaged strains used for research. To generate a genetically intact source of HCMV, we cloned strain Merlin into a self-excising BAC. The Merlin BAC clone had mutations in the RL13 gene and UL128 locus that were acquired during limited replication in vitro prior to cloning. The complete wild-type HCMV gene complement was reconstructed by reference to the original clinical sample. Characterization of viruses generated from repaired BACs revealed that RL13 efficiently repressed HCMV replication in multiple cell types; moreover, RL13 mutants rapidly and reproducibly emerged in transfectants. Virus also acquired mutations in genes UL128, UL130, or UL131A, which inhibited virus growth specifically in fibroblast cells in wild-type form. We further report that RL13 encodes a highly glycosylated virion envelope protein and thus has the potential to modulate tropism. To overcome rapid emergence of mutations in genetically intact HCMV, we developed a system in which RL13 and UL131A were conditionally repressed during virus propagation. This technological advance now permits studies to be undertaken with a clonal, characterized HCMV strain containing the complete wild-type gene complement and promises to enhance the clinical relevance of fundamental research on HCMV.

SummaryHuman cytomegalovirus (HCMV) is an important pathogen with multiple immune evasion strategies, including virally facilitated degradation of host antiviral restriction factors. Here, we describe a multiplexed approach to discover proteins with innate immune function on the basis of active degradation by the proteasome or lysosome during early-phase HCMV infection. Using three orthogonal proteomic/transcriptomic screens to quantify protein degradation, with high confidence we identified 35 proteins enriched in antiviral restriction factors. A final screen employed a comprehensive panel of viral mutants to predict viral genes that target >250 human proteins. This approach revealed that helicase-like transcription factor (HLTF), a DNA helicase important in DNA repair, potently inhibits early viral gene expression but is rapidly degraded during infection. The functionally unknown HCMV protein UL145 facilitates HLTF degradation by recruiting the Cullin4 E3 ligase complex. Our approach and data will enable further identifications of innate pathways targeted by HCMV and other viruses.

Upon herpesvirus infection, viral DNA becomes associated with nuclear structures known as nuclear domain 10 (ND10). The role of ND10 during herpesvirus infection has long been contentious; data arguing for a role for ND10 in repression of infection have been countered by other data showing little effect of ND10 on virus infection. Here we show that knockdown of human Daxx (hDaxx) expression, an important component of ND10, prior to infection with human cytomegalovirus resulted in increased levels of viral immediate early RNA and protein expression and that this correlated with an increased association of the major immediate early promoter with markers of transcriptionally active chromatin. Conversely, we also show that stable overexpression of hDaxx renders cells refractory to cytomegalovirus immediate early gene expression. Intriguingly, this hDaxxmediated repression appears to be restricted to cells stably overexpressing hDaxx and is not recapitulated in transient transfection assays. Finally, hDaxx-mediated repression of cytomegalovirus major immediate early gene expression was overcome by infecting at higher virus titers, suggesting that an incoming viral structural protein or viral DNA is responsible for overcoming the repression of viral gene expression in hDaxx superexpressing cells. These data suggest that hDaxx in ND10 functions at the site of cytomegalovirus genome deposition to repress transcription of incoming viral genomes and that this repression is mediated by a direct and immediate effect of hDaxx on chromatin modification around the viral major immediate early promoter. Human cytomegalovirus (HCMV)3 is an extremely widespread, opportunistic pathogen of considerable clinical significance. Primary infection of healthy, immunocompetent individuals with HCMV is normally asymptomatic. However, as is the case with all herpesviruses, after the initial control of primary infection by the immune system, HCMV establishes lifelong latency in the host. During latency and throughout the lifetime of the infected individual, the herpesviruses can reactivate. Although generally asymptomatic, such reactivation can result in severe disease. As such, the disease burden of herpesviruses can be life-long and particularly severe when associated with immunosuppression, such as in transplant patients and in human immunodeficiency virus-infected patients who have developed immunodeficiency (1, 2).After infection with herpes simplex virus-1, the viral genomes appear to co-localize with cellular nuclear structures known as nuclear domain 10 (ND10) and give rise to replication centers in close proximity to these sites (3, 4). ND10 are discrete interchromosomal accumulations of a number of proteins, many of which are transcriptional repressors. Intriguingly, many of the human herpesviruses encode an immediate early protein, which functions to destroy or redistribute components of ND10 (5-17), and a number of studies have attempted to address the role of ND10 during infection. One of the first observations was that ICP0 (int...

The genomic characteristics of human cytomegalovirus (HCMV) strains sequenced directly from clinical pathology samples were investigated, focusing on variation, multiple-strain infection, recombination, and gene loss. A total of 207 datasets generated in this and previous studies using target enrichment and high-throughput sequencing were analyzed, in the process enabling the determination of genome sequences for 91 strains. Key findings were that (i) it is important to monitor the quality of sequencing libraries in investigating variation; (ii) many recombinant strains have been transmitted during HCMV evolution, and some have apparently survived for thousands of years without further recombination; (iii) mutants with nonfunctional genes (pseudogenes) have been circulating and recombining for long periods and can cause congenital infection and resulting clinical sequelae; and (iv) intrahost variation in single-strain infections is much less than that in multiple-strain infections. Future population-based studies are likely to continue illuminating the evolution, epidemiology, and pathogenesis of HCMV.