RESUMO
UNLABELLED: The nonstructural protein NSs is the main virulence factor of Rift Valley fever virus (RVFV; family Bunyaviridae, genus Phlebovirus), a serious pathogen of livestock and humans in Africa. RVFV NSs blocks transcriptional upregulation of antiviral type I interferons (IFN) and destroys the general transcription factor TFIIH subunit p62 via the ubiquitin/proteasome pathway. Here, we identified a subunit of E3 ubiquitin ligases, F-box protein FBXO3, as a host cell interactor of NSs. Small interfering RNA (siRNA)-mediated depletion of FBXO3 rescued p62 protein levels in RVFV-infected cells and elevated IFN transcription by 1 order of magnitude. NSs interacts with the full-length FBXO3 protein as well as with a truncated isoform that lacks the C-terminal acidic and poly(R)-rich domains. These isoforms are present in both the nucleus and the cytoplasm. NSs exclusively removes the nuclear pool of full-length FBXO3, likely due to consumption during the degradation process. F-box proteins form the variable substrate recognition subunit of the so-called SCF ubiquitin ligases, which also contain the constant components Skp1, cullin 1 (or cullin 7), and Rbx1. siRNA knockdown of Skp1 also protected p62 from degradation, suggesting involvement in NSs action. However, knockdown of cullin 1, cullin 7, or Rbx1 could not rescue p62 degradation by NSs. Our data show that the enzymatic removal of p62 via the host cell factor FBXO3 is a major mechanism of IFN suppression by RVFV. IMPORTANCE: Rift Valley fever virus is a serious emerging pathogen of animals and humans. Its main virulence factor, NSs, enables unhindered virus replication by suppressing the antiviral innate immune system. We identified the E3 ubiquitin ligase FBXO3 as a novel host cell interactor of NSs. NSs recruits FBXO3 to destroy the general host cell transcription factor TFIIH-p62, resulting in suppression of the transcriptional upregulation of innate immunity.
Assuntos
Proteínas F-Box/metabolismo , Fosfoproteínas/metabolismo , Febre do Vale de Rift/metabolismo , Vírus da Febre do Vale do Rift/metabolismo , Fatores de Transcrição TFII/metabolismo , Proteínas não Estruturais Virais/metabolismo , Fatores de Virulência/metabolismo , Linhagem Celular , Proteínas F-Box/genética , Humanos , Fosfoproteínas/genética , Proteólise , Febre do Vale de Rift/enzimologia , Febre do Vale de Rift/genética , Febre do Vale de Rift/virologia , Vírus da Febre do Vale do Rift/genética , Fator de Transcrição TFIIH , Fatores de Transcrição TFII/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas não Estruturais Virais/genética , Fatores de Virulência/genéticaRESUMO
Rift Valley fever virus (RVFV) is an emerging RNA virus with devastating economic and social consequences. Clinically, RVFV induces a gamut of symptoms ranging from febrile illness to retinitis, hepatic necrosis, hemorrhagic fever, and death. It is known that type I interferon (IFN) responses can be protective against severe pathology; however, it is unknown which innate immune receptor pathways are crucial for mounting this response. Using both in vitro assays and in vivo mucosal mouse challenge, we demonstrate here that RNA helicases are critical for IFN production by immune cells and that signaling through the helicase adaptor molecule MAVS (mitochondrial antiviral signaling) is protective against mortality and more subtle pathology during RVFV infection. In addition, we demonstrate that Toll-like-receptor-mediated signaling is not involved in IFN production, further emphasizing the importance of the RNA cellular helicases in type I IFN responses to RVFV.
Assuntos
RNA Helicases DEAD-box/imunologia , Interferon beta/imunologia , Mucosa/virologia , Febre do Vale de Rift/enzimologia , Febre do Vale de Rift/imunologia , Vírus da Febre do Vale do Rift/fisiologia , Animais , Linhagem Celular , Proteína DEAD-box 58 , RNA Helicases DEAD-box/genética , Células Dendríticas/imunologia , Células Dendríticas/virologia , Feminino , Humanos , Interferon beta/genética , Macrófagos/imunologia , Macrófagos/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout/genética , Mucosa/imunologia , Febre do Vale de Rift/prevenção & controle , Febre do Vale de Rift/virologia , Transdução de Sinais , Receptores Toll-Like/genética , Receptores Toll-Like/imunologiaRESUMO
The cell intrinsic innate immune responses provide a first line of defense against viral infection, and often function by targeting cellular pathways usurped by the virus during infection. In particular, many viruses manipulate cellular lipids to form complex structures required for viral replication, many of which are dependent on de novo fatty acid synthesis. We found that the energy regulator AMPK, which potently inhibits fatty acid synthesis, restricts infection of the Bunyavirus, Rift Valley Fever Virus (RVFV), an important re-emerging arthropod-borne human pathogen for which there are no effective vaccines or therapeutics. We show restriction of RVFV both by AMPK and its upstream activator LKB1, indicating an antiviral role for this signaling pathway. Furthermore, we found that AMPK is activated during RVFV infection, leading to the phosphorylation and inhibition of acetyl-CoA carboxylase, the first rate-limiting enzyme in fatty acid synthesis. Activating AMPK pharmacologically both restricted infection and reduced lipid levels. This restriction could be bypassed by treatment with the fatty acid palmitate, demonstrating that AMPK restricts RVFV infection through its inhibition of fatty acid biosynthesis. Lastly, we found that this pathway plays a broad role in antiviral defense since additional viruses from disparate families were also restricted by AMPK and LKB1. Therefore, AMPK is an important component of the cell intrinsic immune response that restricts infection through a novel mechanism involving the inhibition of fatty acid metabolism.