The role of host and viral translation factors during HCMV infection

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DESCRIPTION (provided by applicant): No virus encodes a ribosome. Thus all viruses require the host translation machinery for the synthesis of viral proteins. This fundamental host:pathogen interface creates two critical barriers to human cytomegalovirus (HCMV) mRNA translation. The first is competition for limiting components of the translation machinery. The second is the inactivation of critical translation factors by host antiviral defenses. How HCMV avoids competition and blunts antiviral defenses to ensure efficient viral protein synthesis is poorly understood, highlighting a critical gap in our knowledge of the mechanisms controlling HCMV mRNA translation. Many viruses prevent competition for the translation machinery by inhibiting host translation, however host translation is maintained during HCMV infection. How then do HCMV mRNAs effectively compete with host mRNAs for access to ribosomes? Formation of the eIF4F translation initiation complex is the limiting step in host mRNA translation due to the low abundance of eIF4F subunits. Thus competition between host and viral mRNAs would be most intense for recruiting eIF4F. Our data suggests that HCMV avoids competition for eIF4F by using an unknown complement of alternative initiation factors. We find that the eIF4F complex is necessary for the ongoing translation of host mRNAs during infection. However HCMV mRNAs translate efficiently when the eIF4F complex is disrupted. In Aim1 we define the factors necessary for the eIF4F-independent translation of the IE1 and IE2 mRNAs, two critical regulators of the HCMV lytic cycle, and then determine how these factors contribute to the widespread eIF4F-independent translation of HCMV mRNAs as a whole. The second barrier to HCMV mRNA translation is the inactivation of critical translation factors by host antiviral defenses. Our lab and others have shown that the HCMV TRS1 protein (pTRS1) inhibits the antiviral kinase PKR. Activated PKR inhibits the eIF2 translation factor by phosphorylating the eIF2α subunit. However phosphorylated eIF2α accumulates in infected cells treated with a serine/threonine phosphatase inhibitor, even as PKR remains inactive. This suggests that HCMV has an unknown additional mechanism to ensure eIF2 remains active during infection. Analysis of pTRS1 binding partners revealed that pTRS1 binds the protein phosphatase PP1, which dephosphorylates eIF2α, independent of its ability to bind PKR. Further, pTRS1 binds the host DHX29 RNA helicase, which can promote translation independent of the eIF4F complex. In Aim 2 we determine how HCMV pTRS1 increases translation independent of PKR inhibition by 1) binding to PP1 to limit eIF2α phosphorylation and 2) promoting translation of HCMV mRNAs through its interaction with DHX29. Together these aims will determine how HCMV overcomes these two critical barriers to viral mRNA translation, and identify regulatory events unique to HCMV mRNA translation that could be targeted to limit HCMV disease.