Human cytomegalovirus (CMV), one of the eight herpesviruses that commonly infect humans, is best known for its propensity to cause disease in immunocompromised patients, especially transplant recipients, patients with advanced AIDS, and congenitally infected newborns. Advances in molecular virology coupled with improvements in diagnostic methods and treatment options have vastly improved our understanding of and ability to manage CMV, but many uncertainties remain, including the mechanisms of persistence and pathogenesis and its hypothesized roles in a variety of human illnesses. Here we review recent advances that are reshaping our view and approach to this fascinating virus.

During infection with human cytomegalovirus (HCMV), cellular protein synthesis continues even as viral proteins are being synthesized in abundance. Thus, HCMV may have a mechanism for counteracting host cell antiviral pathways that act by shutting off translation. Consistent with this view, HCMV infection of human fibroblasts rescues the replication of a vaccinia virus mutant lacking the double-stranded RNA-binding protein gene E3L (VVΔE3L). HCMV also prevents the phosphorylation of the eukaryotic translation initiation factor eIF-2α, the activation of RNase L, and the shutoff of viral and cellular protein synthesis that otherwise result from VVΔE3L infection. To identify the HCMV gene(s) responsible for these effects, we prepared a library of VVΔE3L recombinants containing HCMV genomic fragments. By infecting nonpermissive cells with this library and screening for VV gene expression and replication, we isolated a virus containing a 2.8-kb HCMV fragment that rescues replication of VVΔE3L. The fragment comprises the 3′ end of the J1S open reading frame through the entire TRS1 gene. Analyses of additional VVΔE3L recombinants revealed that the protein encoded by TRS1 , pTRS1, as well as the closely related IRS1 gene, rescues VVΔE3L replication and prevent the shutoff of protein synthesis, the phosphorylation of eIF-2α, and activation of RNase L. These results demonstrate that TRS1 and IRS1 are able to counteract critical host cell antiviral response pathways.

The human cytomegalovirus (HCMV) TRS1 and IRS1 genes block the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α) and the consequent shutoff of cellular protein synthesis that occur during infection with vaccinia virus (VV) deleted of the double-stranded RNA binding protein gene E3L (VVΔE3L). To further define the underlying mechanism, we first evaluated the effect of pTRS1 on protein kinase R (PKR), the double-stranded RNA (dsRNA)-dependent eIF2α kinase. Immunoblot analyses revealed that pTRS1 expression in the context of a VVΔE3L recombinant decreased levels of PKR in the cytoplasm and increased its levels in the nucleus of infected cells, an effect not seen with wild-type VV or a VVΔE3L recombinant virus expressing E3L. This effect of pTRS1 was confirmed by visualizing the nuclear relocalization of PKR-EGFP expressed by transient transfection. PKR present in both the nuclear and cytoplasmic fractions was nonphosphorylated, indicating that it was unactivated when TRS1 was present. PKR also accumulated in the nucleus during HCMV infection as determined by indirect immunofluorescence and immunoblot analysis. Binding assays revealed that pTRS1 interacted with PKR in mammalian cells and in vitro. This interaction required the same carboxy-terminal region of pTRS1 that is necessary to rescue VVΔE3L replication in HeLa cells. The carboxy terminus of pIRS1 was also required for rescue of VVΔE3L and for mediating an interaction of pIRS1 with PKR. These results suggest that these HCMV genes directly interact with PKR and inhibit its activation by sequestering it in the nucleus, away from both its activator, cytoplasmic dsRNA, and its substrate, eIF2α.

Overexpression of the HER-2 (neu,erbB-2) receptor results in cellular transformation and is associated with a variety of human cancers. Multiple mechanisms, including gene amplification and transcriptional, post-transcriptional, and translational controls contribute to the regulation of HER-2 expression. One of the components of these regulatory mechanisms is a short upstream open reading frame (uORF) in the HER-2 mRNA that represses downstream translation in a variety of cell types. Here we explore the mechanism by which this uORF exerts its inhibitory effect. As judged by comparisons of protein and mRNA abundance and by polysomal distribution analyses, the uORF represses translation of the HER-2 cistron or of a heterologous reporter gene. Despite its conservation among mammalian species, the peptide sequence of the uORF is not required for this inhibitory effect. Rather, the majority of ribosomes that load on the HER-2 mRNA most likely translate the uORF and are then unable to reinitiate at the downstream AUG codon, in part due to the short intercistronic spacing. A minority of ribosomes gain access to the HER-2 initiation codon either by leaky scanning past the upstream AUG codon or by reinitiating after having translated the uORF despite the short intercistronic region. These results suggest that the HER-2 uORF controls synthesis of this oncoprotein by limiting ribosomal access to downstream initiation sites.

Viral infections often produce double-stranded RNA (dsRNA), which in turn triggers potent antiviral responses, including the global repression of protein synthesis mediated by protein kinase R (PKR) and 2′-5′ oligoadenylate synthetase (OAS). As a consequence, many viruses have evolved genes, such as those encoding dsRNA-binding proteins, which counteract these pathways. Human cytomegalovirus (HCMV) encodes two related proteins, pTRS1 and pIRS1, which bind dsRNA and can prevent activation of the PKR and OAS pathways. HCMV mutants lacking either IRS1 or TRS1 replicate at least moderately well in cell culture. However, as we demonstrate in the present study, an HCMV mutant lacking both IRS1 and TRS1 (HCMV[ΔI/ΔT]) has a severe replication defect. Infection with HCMV[ΔI/ΔT] results in a profound inhibition of overall and viral protein synthesis, as well as increased phosphorylation of eukaryotic initiation factor 2α (eIF2α). The vaccinia virus E3L gene can substitute for IRS1 or TRS1 , enabling HCMV replication. Despite the accumulation of dsRNA in HCMV-infected cells, the OAS pathway remains inactive, even in HCMV[ΔI/ΔT]-infected cells. These results suggest that PKR-mediated phosphorylation of eIF2α is the dominant dsRNA-activated pathway responsible for inhibition of protein synthesis and HCMV replication in the absence of both IRS1 and TRS1 and that the requirement for evasion of the PKR pathway likely explains the necessity for IRS1 or TRS1 for productive infection.

Human cytomegalovirus (HCMV), which infects the majority of the population worldwide, causes few, if any, symptoms in otherwise healthy people but is responsible for considerable morbidity and mortality in immunocompromised patients and in congenitally infected newborns. The evolutionary success of HCMV depends in part on its ability to evade host defense systems. Here we review recent progress in elucidating the remarkable assortment of mechanisms employed by HCMV and the related β-herpesviruses, murine cytomegaloviruses (MCMV) and rhesus cytomegaloviruses (RhCMV), for counteracting the host interferon (IFN) response. Very early after infection, cellular membrane sensors such as the lymphotoxin β receptor initiate the production of antiviral cytokines including type I IFNs. However, virion factors, such as pp65 (ppUL83) and viral proteins made soon after infection including the immediate early gene 2 protein (pUL122), repress this response by interfering with steps in the activation of IFN regulatory factor 3 and NF-κB. CMVs then exert a multi-pronged attack on downstream IFN signaling. HCMV infection results in decreased accumulation and phosphorylation of the IFN signaling kinases Jak1 and Stat2, and the MCMV protein pM27 mediates Stat2 down-regulation, blocking both type I and type II IFN signaling. The HCMV immediate early gene 1 protein (pUL123) interacts with Stat2 and inhibits transcriptional activation of IFN-regulated genes. Infection also causes reduction in the abundance of p48/IRF9, a component of the ISGF3 transcription factor complex. Furthermore, CMVs have multiple genes involved in blocking the function of IFN-induced effectors. For example, viral double-stranded RNA-binding proteins are required to prevent the shutoff of protein synthesis by protein kinase R, further demonstrating the vital importance of evading the IFN response at multiple levels during infection.