Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)FBXW11-NSs E3 Ligase

Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)FBXW11 E3 ligase. NSs binds to the F-box protein, FBXW11, via the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through an alternate binding site to the SCFFBXW11 E3 ligase. We further show that disrupting the assembly of the SCFFBXW11-NSs E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCFFBXW11-NSs E3 ligase is sufficient to inhibit RVFV infection. Furthermore, FBXW11 and BTRC are the two homologues of the βTrCP (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCFFBXW11 complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection.活化的蛋白激酶R（Protein Kinase R, PKR）主要通过抑制蛋白质合成、增强干扰素应答，在抗病毒防御中发挥关键作用。诸多病毒蛋白已演化出独特策略，以拮抗PKR的有害效应。裂谷热病毒（Rift Valley Fever Virus, RVFV）编码的NSs（非结构蛋白s）可通过此前未被阐明的机制，促进PKR的早期蛋白酶体降解。本研究证实，NSs通过组装SCF（SKP1-CUL1-F-box）FBXW11 E3泛素连接酶实现该功能：NSs可通过一段名为降解基序（degron sequence）的六氨基酸序列DDGFVE，与F-box蛋白FBXW11结合，并通过另一结合位点将PKR招募至SCFFBXW11 E3泛素连接酶复合物中。本研究进一步发现，使用MLN4924（一种SCF E3泛素连接酶活性小分子抑制剂）、NSs降解基序病毒突变株，或通过小干扰RNA（siRNA）敲低FBXW11以破坏SCFFBXW11-NSs E3泛素连接酶的组装，均可阻断PKR的降解。令人意外的是，在PKR降解被阻断的上述条件下，NSs足以且必须激活PKR，通过抑制病毒蛋白质合成强效阻断RVFV感染；若PKR表达缺失或使用NSs缺失突变病毒，则上述抗病毒效应会被拮抗。因此，通过解离SCFFBXW11-NSs E3泛素连接酶实现的早期PKR激活，足以抑制RVFV感染。此外，FBXW11与BTRC是βTrCP（β转导蛋白重复序列包含蛋白，Beta-transducin repeat containing protein）基因的两个同源物，此前研究认为二者功能冗余；但在RVFV感染过程中，在βTrCP的两个同源物中，FBXW11在PKR降解中发挥主导作用，且是SCFFBXW11复合物组装的限速因子。因此，FBXW11通过促进PKR降解，成为RVFV感染的核心调控因子。综上，本研究的发现为NSs调控PKR活性提供了新视角，也为RVFV感染的治疗干预提供了潜在方向。