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.

Author Summary Rift Valley fever (RVF) is a severe disease caused by infection with the Rift Valley fever virus (RVFV) that affects humans and livestock and occurs in large epidemics. Currently there are no FDA-approved drugs or vaccines to treat RVF. Many viruses have evolved unique strategies to overcome host immune responses in order to establish infection. One protein of RVFV called NSs is responsible for over-powering cellular antiviral defenses. NSs is known to degrade double-stranded (ds) RNA-dependent protein kinase (PKR), but neither the mechanism nor the functional significance of this activity has been fully understood. In this study we show that NSs promotes PKR degradation by recruiting PKR to the E3 ligase complex called SCF (SKP1-CUL1-F-box)FBXW11. A short stretch of six amino acids called the degron sequence in NSs regulates the NSs- FBXW11 interaction and is required for the assembly of the SCFFBXW11 complex. We further show that disruption of the SCFFBXW11-NSs complex, with either a small molecule or with NSs degron viral mutants, can block PKR degradation. Surprisingly, when NSs mediated PKR degradation was blocked, NSs was essential and sufficient to activate PKR, causing potent inhibition of RVFV infection by suppressing viral protein synthesis. Therefore early PKR activation induced by inactivation of the SCFFBXW11 is sufficient to induce potent inhibition of RVFV infection. These findings may provide new molecular targets for therapeutic intervention of this important disease.