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Latency-associated nuclear antigen (LANA), a multifunctional protein expressed by the Kaposi sarcoma-associated herpesvirus (KSHV) in latently infected cells, is required for stable maintenance of the viral episome. This is mediated by two interactions: LANA binds to specific sequences (LBS1 and LBS2) on viral DNA and also engages host histones, tethering the viral genome to host chromosomes in mitosis. LANA has also been suggested to affect host gene expression, but both the mechanism(s) and role of this dysregulation in KSHV biology remain unclear. Here, we have examined LANA interactions with host chromatin on a genome-wide scale using chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) and show that LANA predominantly targets human genes near their transcriptional start sites (TSSs). These host LANA-binding sites are generally found within transcriptionally active promoters and display striking overrepresentation of a consensus DNA sequence virtually identical to the LANA-binding site 1 (LBS1) motif in KSHV DNA. Comparison of the ChIP-seq profile with whole-transcriptome (high-throughput sequencing of RNA transcripts [RNA-seq]) data reveals that few of the genes that are differentially regulated in latent infection are occupied by LANA at their promoters. This suggests that direct LANA binding to promoters is not the prime determinant of altered host transcription in KSHV-infected cells. Most surprisingly, the association of LANA to both host and viral DNA is strongly disrupted during the lytic cycle of KSHV. This disruption can be prevented by the inhibition of viral DNA synthesis, suggesting the existence of novel and potent regulatory mechanisms linked to either viral DNA replication or late gene expression. [ABSTRACT FROM AUTHOR]

Although picornavirus RNA genomes contain a 3'-terminal poly(A) tract that is critical for their replication, the impact of encephalomyocarditis virus (EMCV) infection on the host poly(A)-binding protein (PABP) remains unknown. Here, we establish that EMCV infection stimulates site-specific PABP proteolysis, resulting in accumulation of a 45-kDa N-terminal PABP fragment in virus-infected cells. Expression of a functional EMCV 3C proteinase was necessary and sufficient to stimulate PABP cleavage in uninfected cells, and bacterially expressed 3C cleaved recombinant PABP in vitro in the absence of any virus-encoded or eukaryotic cellular cofactors. N-terminal sequencing of the resulting C-terminal PABP fragment identified a 3Cpro cleavage site on PABP between amino acids Q437 and G438, severing the C-terminal protein-interacting domain from the N-terminal RNA binding fragment. Single amino acid substitution mutants with changes at Q437 were resistant to 3Cpro cleavage in vitro and in vivo, validating that this is the sole detectable PABP cleavage site. Finally, while ongoing protein synthesis was not detectably altered in EMCV-infected cells expressing a cleavage-resistant PABP variant, viral RNA synthesis and infectious virus production were both reduced. Together, these results establish that the EMCV 3C proteinase mediates site-specific PABP cleavage and demonstrate that PABP cleavage by 3C regulates EMCV replication. [ABSTRACT FROM AUTHOR]

In the efforts of viruses to dominate and control critical cellular pathways, viruses generate considerable intracellular stress within their hosts. In particular, the capacity of resident endoplasmic reticulum (ER) chaperones to properly process the acute increase in client protein load is significantly challenged. Such alterations typically induce the unfolded protein response, one component of which acts through IRE1 to restore ER homeostasis by expanding the folding capabilities, whereas the other arm activates the eIF-2α (αsubunit of eukaryotic initiation factor 2) kinase PERK to transiently arrest production of new polypeptide clientele. Viruses, such as herpes simplex virus type 1 (HSV-1), however, go to great lengths to prevent the inhibition of translation resulting from eIF-2α phosphorylation. Here, we establish that PERK, but not IRE1, resists activation by acute ER stress in HSV-1-infected cells. This requires the ER luminal domain of PERK, which associates with the viral glycoprotein gB. Strikingly, gB regulates viral protein accumulation in a PERK-dependent manner. This is the first description of a virus-encoded PERK-specific effector and defines a new strategy by which viruses are able to maintain ER homeostasis. [ABSTRACT FROM AUTHOR]

ia careful control of multiple kinases that inactivate the critical translation initiation factor eIF2 by phosphorylation of its alpha subunit, the cellular translation machinery can rapidly respond to a spectrum of environmental stresses, including viral infection. Indeed, virus replication produces a battery of stresses, such as endoplasmic reticulum (ER) stress resulting from misfolded proteins accumulating within the lumen of this organelle, which could potentially result in eIF2α phosphorylation and inhibit translation. While cellular translation is exquisitely sensitive to ER stress-inducing agents, protein synthesis in herpes simplex virus type 1 (HSV-1)-infected cells is notably resistant. Sustained translation in HSV-1-infected cells exposed to acute ER stress does not involve the interferon-induced, double-stranded RNA-responsive eIF2α kinase PKR, and it does not require either the PKR inhibitor encoded by the Us11 gene or the eIF2α phosphatase component specified by the γ134.5 gene, the two viral functions known to regulate eIF2α phosphorylation. In addition, although ER stress potently induced the GADD34 cellular eIF2α phosphatase subunit in uninfected cells, it did not accumulate to detectable levels in HSV-1-infected cells under identical exposure conditions. Significantly, resistance of translation to the acute ER stress observed in infected cells requires HSV-1 gene expression. Whereas blocking entry into the true late phase of the viral developmental program does not abrogate ER stress-resistant translation, the presence of viral immediate-early proteins is sufficient to establish a state permissive of continued polypeptide synthesis in the presence of ER stress-inducing agents. Thus, one or more previously uncharacterized viral functions exist to counteract the accumulation of phosphorylated eIF2α in response to ER stress in HSV-1-infected cells. [ABSTRACT FROM AUTHOR]