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Essential role of protein kinase R antagonism by TRS1 in human cytomegalovirus replication.

ABSTRACT: Human cytomegalovirus (HCMV) lacking TRS1 and IRS1 (HCMV[?I/?T]) cannot replicate in cell culture. Although both proteins can block the protein kinase R (PKR) pathway, they have multiple other activities and binding partners. It remains unknown which functions are essential for HCMV replication. To investigate this issue, we first identified a TRS1 mutant that is unable to bind to PKR. Like HCMV[?I/?T], a recombinant HCMV containing this mutant (HCMV[TRS1-Mut 1]) did not replicate in wild-type cells. However, HCMV[?I/?T] did replicate in cells in which PKR expression was reduced by RNA interference. Moreover, HCMV[?I/?T] and HCMV[TRS1-Mut 1] replicated to similar levels as virus containing wild-type TRS1 in cell lines in which PKR expression was knocked out by CRISPR/Cas9-mediated genome editing. These results demonstrate that the sole essential function of TRS1 is to antagonize PKR and that its other activities do not substantially enhance HCMV replication, at least in cultured human fibroblasts.

SUBMITTER: Braggin JE

PROVIDER: S-EPMC4761322 | biostudies-literature | 2016 Feb

REPOSITORIES: biostudies-literature

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Essential role of protein kinase R antagonism by TRS1 in human cytomegalovirus replication.

Braggin Jacquelyn E JE Child Stephanie J SJ Geballe Adam P AP

Virology 20151221

Human cytomegalovirus (HCMV) lacking TRS1 and IRS1 (HCMV[ΔI/ΔT]) cannot replicate in cell culture. Although both proteins can block the protein kinase R (PKR) pathway, they have multiple other activities and binding partners. It remains unknown which functions are essential for HCMV replication. To investigate this issue, we first identified a TRS1 mutant that is unable to bind to PKR. Like HCMV[ΔI/ΔT], a recombinant HCMV containing this mutant (HCMV[TRS1-Mut 1]) did not replicate in wild-type c ...[more]

PMID: 26716879

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Project description:UnlabelledHuman cytomegalovirus (HCMV) counteracts host defenses that otherwise act to limit viral protein synthesis. One such defense is the antiviral kinase protein kinase R (PKR), which inactivates the eukaryotic initiation factor 2 (eIF2) translation initiation factor upon binding to viral double-stranded RNAs. Previously, the viral TRS1 and IRS1 proteins were found to antagonize the antiviral kinase PKR outside the context of HCMV infection, and the expression of either pTRS1 or pIRS1 was shown to be necessary for HCMV replication. In this study, we found that expression of either pTRS1 or pIRS1 is necessary to prevent PKR activation during HCMV infection and that antagonism of PKR is critical for efficient viral replication. Consistent with a previous study, we observed decreased overall levels of protein synthesis, reduced viral protein expression, and diminished virus replication in the absence of both pTRS1 and pIRS1. In addition, both PKR and eIF2α were phosphorylated during infection when pTRS1 and pIRS1 were absent. We also found that expression of pTRS1 was both necessary and sufficient to prevent stress granule formation in response to eIF2α phosphorylation. Depletion of PKR prevented eIF2α phosphorylation, rescued HCMV replication and protein synthesis, and reversed the accumulation of stress granules in infected cells. Infection with an HCMV mutant lacking the pTRS1 PKR binding domain resulted in PKR activation, suggesting that pTRS1 inhibits PKR through a direct interaction. Together our results show that antagonism of PKR by HCMV pTRS1 and pIRS1 is critical for viral protein expression and efficient HCMV replication.ImportanceTo successfully replicate, viruses must counteract host defenses that limit viral protein synthesis. We have identified inhibition of the antiviral kinase PKR by the viral proteins TRS1 and IRS1 and shown that this is a critical step in HCMV replication. Our results suggest that inhibiting pTRS1 and pIRS1 function or restoring PKR activity during infection may be a successful strategy to limit HCMV disease.

Project description:Picornavirus replication is known to cause extensive remodeling of Golgi and endoplasmic reticulum membranes, and a number of the host proteins involved in the viral replication complex have been identified, including oxysterol binding protein (OSBP) and phosphatidylinositol 4-kinase III beta (PI4KB). Since both OSBP and PI4KB are substrates for protein kinase D (PKD) and PKD is known to be involved in the control of Golgi membrane vesicular and lipid transport, we hypothesized that PKD played a role in viral replication. We present multiple lines of evidence in support of this hypothesis. First, infection of HeLa cells with human rhinovirus (HRV) induced the phosphorylation of PKD. Second, PKD inhibitors reduced HRV genome replication, protein expression, and titers in a concentration-dependent fashion and also blocked the replication of poliovirus (PV) and foot-and-mouth disease virus (FMDV) in a variety of cells. Third, HRV replication was significantly reduced in HeLa cells overexpressing wild-type and mutant forms of PKD1. Fourth, HRV genome replication was reduced in HAP1 cells in which the PKD1 gene was knocked out by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9. Although we have not identified the molecular mechanism through which PKD regulates viral replication, our data suggest that this is not due to enhanced interferon signaling or an inhibition of clathrin-mediated endocytosis, and PKD inhibitors do not need to be present during viral uptake. Our data show for the first time that targeting PKD with small molecules can inhibit the replication of HRV, PV, and FMDV, and therefore, PKD may represent a novel antiviral target for drug discovery.IMPORTANCE Picornaviruses remain an important family of human and animal pathogens for which we have a very limited arsenal of antiviral agents. HRV is the causative agent of the common cold, which in itself is a relatively trivial infection; however, in asthma and chronic obstructive pulmonary disease (COPD) patients, this virus is a major cause of exacerbations resulting in an increased use of medication, worsening symptoms, and, frequently, hospital admission. Thus, HRV represents a substantial health care and economic burden for which there are no approved therapies. We sought to identify a novel host target as a potential anti-HRV therapy. HRV infection induces the phosphorylation of PKD, and inhibitors of this kinase effectively block HRV replication at an early stage of the viral life cycle. Moreover, PKD inhibitors also block PV and FMDV replication. This is the first description that PKD may represent a target for antiviral drug discovery.

Dataset Information

Essential role of protein kinase R antagonism by TRS1 in human cytomegalovirus replication.

Publications

Essential role of protein kinase R antagonism by TRS1 in human cytomegalovirus replication.

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