NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and β-TRCP1 To Degrade the Antiviral Protein Kinase PKR.
Rift Valley fever virus (RVFV, family Bunyaviridae, genus Phlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as β-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and β-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and β-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells.



Rift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we demonstrate that NSs requires E3 ubiquitin ligase complexes of the SCF (Skp1, Cul1, F-box protein) type to destroy PKR. SCF-type complexes can engage variant ubiquitination substrate recognition subunits, and we found the F-box proteins FBXW11 and β-TRCP1 to be relevant for the action of NSs against PKR. Thus, we identified the host cell factors that are critically needed by Rift Valley fever virus to uphold its replication against the potent antiviral kinase PKR.
Institute for Virology, Philipps-Universität Marburg, Marburg, Germany.
Markus
M
Kainulainen
Institute for Virology, Philipps-Universität Marburg, Marburg, Germany Institute for Virology, FB10-Veterinary Medicine, Justus-Liebig-Universität Gießen, Giessen, Germany.
Simone
S
Lau
Department of Molecular, Cellular and Developmental Biology, University of California, Santa Barbara, California, USA Biomolecular Sciences and Engineering Program, University of California, Santa Barbara, California, USA.
Charles E
CE
Samuel
Institute of Molecular Medicine, University Hospital, Universität Bonn, Bonn, Germany Gene Center and Department of Biochemistry, Ludwig-Maximilians-Universität München, Munich, Germany.
Veit
V
Hornung
Institute for Virology, Philipps-Universität Marburg, Marburg, Germany Institute for Virology, FB10-Veterinary Medicine, Justus-Liebig-Universität Gießen, Giessen, Germany friedemann.weber@vetmed.uni-giessen.de.
Friedemann
0000-0001-9737-337X
F
Weber
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism; D044783:F-Box Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D034741:RNA, Small Interfering; D012296:Rift Valley fever virus / Q000737:chemistry / Q000378:metabolism / Q000472:pathogenicity*; D044786:S-Phase Kinase-Associated Proteins / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D014709:Vero Cells; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D044844:beta-Transducin Repeat-Containing Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D019892:eIF-2 Kinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
C100318:Cullin 1; D044842:Cullin Proteins; D044783:F-Box Proteins; C471645:FBXW11 protein, human; D034741:RNA, Small Interfering; D044786:S-Phase Kinase-Associated Proteins; C097351:SKP1 protein, human; D017361:Viral Nonstructural Proteins; D037521:Virulence Factors; D044844:beta-Transducin Repeat-Containing Proteins; D044767:Ubiquitin-Protein Ligases; D019892:eIF-2 Kinase