Pham Alissa M, Santa Maria Felicia Gilfoy, Lahiri Tanaya, Friedman Eugene, Marié Isabelle J, Levy David E
Departments of Pathology and Microbiology and NYU Cancer Institute, NYU School of Medicine, New York, New York, United States of America.
PLoS Pathog. 2016 Mar 3;12(3):e1005489. doi: 10.1371/journal.ppat.1005489. eCollection 2016 Mar.
Sensing invading pathogens early in infection is critical for establishing host defense. Two cytosolic RIG-like RNA helicases, RIG-I and MDA5, are key to type I interferon (IFN) induction in response to viral infection. Mounting evidence suggests that another viral RNA sensor, protein kinase R (PKR), may also be critical for IFN induction during infection, although its exact contribution and mechanism of action are not completely understood. Using PKR-deficient cells, we found that PKR was required for type I IFN induction in response to infection by vaccinia virus lacking the PKR antagonist E3L (VVΔE3L), but not by Sendai virus or influenza A virus lacking the IFN-antagonist NS1 (FluΔNS1). IFN induction required the catalytic activity of PKR, but not the phosphorylation of its principal substrate, eIF2α, or the resulting inhibition of host translation. In the absence of PKR, IRF3 nuclear translocation was impaired in response to MDA5 activators, VVΔE3L and encephalomyocarditis virus, but not during infection with a RIG-I-activating virus. Interestingly, PKR interacted with both RIG-I and MDA5; however, PKR was only required for MDA5-mediated, but not RIG-I-mediated, IFN production. Using an artificially activated form of PKR, we showed that PKR activity alone was sufficient for IFN induction. This effect required MAVS and correlated with IRF3 activation, but no longer required MDA5. Nonetheless, PKR activation during viral infection was enhanced by MDA5, as virus-stimulated catalytic activity was impaired in MDA5-null cells. Taken together, our data describe a critical and non-redundant role for PKR following MDA5, but not RIG-I, activation to mediate MAVS-dependent induction of type I IFN through a kinase-dependent mechanism.
在感染早期感知入侵病原体对于建立宿主防御至关重要。两种胞质内视黄酸诱导基因（RIG）样RNA解旋酶，即RIG-I和黑色素瘤分化相关基因5（MDA5），是病毒感染后I型干扰素（IFN）诱导的关键。越来越多的证据表明，另一种病毒RNA传感器，即蛋白激酶R（PKR），在感染期间对IFN诱导可能也至关重要，尽管其确切作用和作用机制尚未完全明确。利用PKR缺陷细胞，我们发现PKR是缺乏PKR拮抗剂E3L的痘苗病毒（VVΔE3L）感染后I型IFN诱导所必需的，但不是缺乏IFN拮抗剂NS1的仙台病毒或甲型流感病毒（FluΔNS1）感染后所必需的。IFN诱导需要PKR的催化活性，但不需要其主要底物真核生物翻译起始因子2α（eIF2α）的磷酸化，也不需要由此导致的宿主翻译抑制。在没有PKR的情况下，对MDA5激活剂、VVΔE3L和脑心肌炎病毒的反应中，干扰素调节因子3（IRF3）的核转位受损，但在RIG-I激活病毒感染期间未受损。有趣的是，PKR与RIG-I和MDA5都相互作用；然而，PKR仅对MDA5介导的而非RIG-I介导的IFN产生是必需的。使用人工激活形式的PKR，我们表明仅PKR活性就足以诱导IFN。这种效应需要线粒体抗病毒信号蛋白（MAVS），并与IRF3激活相关，但不再需要MDA5。尽管如此，MDA5可增强病毒感染期间PKR的激活，因为在MDA5缺失的细胞中病毒刺激的催化活性受损。综上所述，我们的数据描述了PKR在MDA5而非RIG-I激活后通过激酶依赖性机制介导MAVS依赖性I型IFN诱导中的关键且非冗余作用。
