Ziehr Benjamin, Vincent Heather A, Moorman Nathaniel J
Department of Microbiology & Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
J Virol. 2016 Mar 28;90(8):3839-3848. doi: 10.1128/JVI.02714-15. Print 2016 Apr.
Human 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.
To 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.
人巨细胞病毒（HCMV）可对抗宿主防御机制，否则这些防御机制会限制病毒蛋白合成。其中一种防御机制是抗病毒激酶蛋白激酶R（PKR），它在与病毒双链RNA结合后会使真核起始因子2（eIF2）失活。此前，在HCMV感染背景之外发现病毒TRS1和IRS1蛋白可拮抗抗病毒激酶PKR，并且pTRS1或pIRS1的表达对于HCMV复制是必需的。在本研究中，我们发现pTRS1或pIRS1的表达对于在HCMV感染期间防止PKR激活是必需的，并且PKR的拮抗作用对于有效的病毒复制至关重要。与先前的研究一致，我们观察到在缺乏pTRS1和pIRS1的情况下，蛋白质合成的总体水平降低、病毒蛋白表达减少以及病毒复制减弱。此外，在缺乏pTRS1和pIRS1的感染过程中，PKR和eIF2α均被磷酸化。我们还发现pTRS1的表达对于响应eIF2α磷酸化而防止应激颗粒形成既是必需的也是充分的。PKR的缺失可防止eIF2α磷酸化，挽救HCMV复制和蛋白质合成，并逆转感染细胞中应激颗粒的积累。用缺乏pTRS1 PKR结合域的HCMV突变体感染导致PKR激活，表明pTRS1通过直接相互作用抑制PKR。我们的结果共同表明，HCMV pTRS1和pIRS1对PKR的拮抗作用对于病毒蛋白表达和有效的HCMV复制至关重要。
为了成功复制，病毒必须对抗限制病毒蛋白合成的宿主防御机制。我们已经确定病毒蛋白TRS1和IRS1可抑制抗病毒激酶PKR，并表明这是HCMV复制中的关键步骤。我们的结果表明，在感染期间抑制pTRS1和pIRS1的功能或恢复PKR活性可能是限制HCMV疾病的成功策略。
