The role of host and viral translation factors during HCMV infection

描述（由申请人提供）：无病毒编码核糖体。因此，所有病毒都需要宿主 病毒蛋白合成的翻译机制。这个基本的宿主：病原体界面创造了两个 人巨细胞病毒（HCMV）mRNA翻译的关键障碍。首先是限制竞争 翻译机器的组成部分。二是宿主对关键翻译因子的失活 抗病毒防御HCMV如何避免竞争并削弱抗病毒防御以确保有效的病毒蛋白 合成是知之甚少，突出了我们的知识的关键差距的机制控制 HCMV mRNA翻译。许多病毒通过抑制宿主细胞来阻止翻译机器的竞争 宿主翻译，然而宿主翻译在HCMV感染期间维持。那么HCMV mRNAs 能有效地与宿主mRNA竞争进入核糖体吗eIF4F翻译起始的形成 由于eIF4F亚基丰度低，因此复合物是宿主mRNA翻译的限制步骤。因此 宿主和病毒mRNA之间的竞争对于募集eIF4F最为激烈。我们的数据显示， HCMV通过使用一种未知的替代起始因子来避免与eIF4F竞争。我们发现 eIF4F复合物对于感染期间宿主mRNA的持续翻译是必需的。然而 当eIF4F复合物被破坏时，HCMV mRNA有效翻译。在目标1中，我们定义了 IE1和IE2 mRNA的eIF4F非依赖性翻译所必需的，IE1和IE2 mRNA是两个关键的调节因子， HCMV裂解周期，然后确定这些因素如何有助于广泛的eIF4F非依赖性 HCMV mRNA的翻译。HCMV mRNA翻译的第二个障碍是 宿主抗病毒防御的关键翻译因子。我们的实验室和其他实验室已经表明，HCMV TRS 1 蛋白（pTRS 1）抑制抗病毒激酶PKR。激活的PKR通过以下途径抑制eIF2翻译因子： 磷酸化eIF2 α亚基。然而，磷酸化的eIF2 α在用抗肿瘤药物处理的感染细胞中积累。 丝氨酸/苏氨酸磷酸酶抑制剂，即使PKR保持无活性。这表明HCMV具有一种 未知的其他机制，以确保eIF2在感染期间保持活性。pTRS 1结合分析 合作伙伴揭示，pTRS 1结合蛋白磷酸酶PP 1，使eIF 2 α去磷酸化， 独立于其结合PKR的能力。此外，pTRS 1结合宿主DHX29 RNA解旋酶，其可以促进 翻译独立于eIF4F复合物。在目标2中，我们确定了HCMV pTRS1如何增加翻译 不依赖于PKR抑制，通过1）与PP1结合以限制eIF2 α磷酸化和2）促进翻译 通过与DHX29的相互作用，这些目标将共同决定HCMV如何 克服了病毒mRNA翻译的这两个关键障碍，并确定了HCMV特有的调控事件， 可以靶向限制HCMV疾病的mRNA翻译。

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.