title: Mechanism of Protein Kinase R Inhibition by Human Cytomegalovirus pTRS1.
abstract: Double-stranded RNAs (dsRNA) produced during human cytomegalovirus (HCMV) infection activate the antiviral kinase protein kinase R (PKR), which potently inhibits virus replication. The HCMV pTRS1 and pIRS1 proteins antagonize PKR to promote HCMV protein synthesis and replication; however, the mechanism by which pTRS1 inhibits PKR is unclear. PKR activation occurs in a three-step cascade. First, binding to dsRNA triggers PKR homodimerizaton. PKR dimers then autophosphorylate, leading to a conformational shift that exposes the binding site for the PKR substrate eIF2α. Consistent with previousin vitrostudies, we found that pTRS1 bound and inhibited PKR. pTRS1 binding to PKR was not mediated by an RNA intermediate, and mutations in the pTRS1 RNA binding domain did not affect PKR binding or inhibition. Rather, mutations that disrupted the pTRS1 interaction with PKR ablated the ability of pTRS1 to antagonize PKR activation by dsRNA. pTRS1 did not block PKR dimerization and could bind and inhibit a constitutively dimerized PKR kinase domain. In addition, pTRS1 binding to PKR inhibited PKR kinase activity. Single amino acid point mutations in the conserved eIF2α binding domain of PKR disrupted pTRS1 binding and rendered PKR resistant to inhibition by pTRS1. Consistent with a critical role for the conserved eIF2α contact site in PKR binding, pTRS1 bound an additional eIF2α kinase, heme-regulated inhibitor (HRI), and inhibited eIF2α phosphorylation in response to an HRI agonist. Together our data suggest that pTRS1 inhibits PKR by binding to conserved amino acids in the PKR eIF2α binding site and blocking PKR kinase activity.IMPORTANCEThe antiviral kinase PKR plays a critical role in controlling HCMV replication. This study furthered our understanding of how HCMV evades inhibition by PKR and identified new strategies for how PKR activity might be restored during infection to limit HCMV disease.
authors.affiliation: Department of Microbiology & Immunology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Heather A
authors.initials: HA
authors.lastname: Vincent
authors.affiliation: Department of Microbiology & Immunology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Ziehr
authors.affiliation: Department of Microbiology & Immunology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA nmoorman@med.unc.edu.
authors.forename: Nathaniel J
authors.initials: NJ
authors.lastname: Moorman
mesh_terms: D020816:Amino Acid Motifs; D003587:Cytomegalovirus / Q000502:physiology*; D003586:Cytomegalovirus Infections / Q000201:enzymology* / Q000821:virology; D004789:Enzyme Activation; D057809:HEK293 Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D010766:Phosphorylation; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D012330:RNA, Double-Stranded / Q000502:physiology; D012367:RNA, Viral / Q000502:physiology; D014764:Viral Proteins / Q000502:physiology; D014779:Virus Replication; D019892:eIF-2 Kinase / Q000378:metabolism*
chemical_list: D012330:RNA, Double-Stranded; D012367:RNA, Viral; C072216:TRS1 protein, Human herpesvirus 5; D014764:Viral Proteins; D019892:eIF-2 Kinase
keywords: HCMV; PKR; eIF2α; gene expression; human cytomegalovirus; human herpesvirus; innate immunity; mRNA translation; protein kinase R; protein synthesis