Viral immune modulators perturb the human molecular network by common and unique strategies

A hallmark of Ebola virus (EBOV) infection is the formation of viral inclusions in the cytoplasm of infected cells. These viral in-clusions contain the EBOV nucleocapsids and are sites of viral replication and nucleocapsid maturation. Although there is growing evidence that viral inclusions create a protected environment that fosters EBOV replication, little is known about their role in the host response to infection. The cellular stress response is an effective antiviral strategy that leads to stress granule (SG) formation and translational arrest mediated by the phosphorylation of a translation initiation factor, the ␣ subunit of eukaryotic initiation factor 2 (eIF2␣). Here, we show that selected SG proteins are sequestered within EBOV inclusions, where they form distinct granules that colocalize with viral RNA. These inclusion-bound (IB) granules are functionally and structurally different from canonical SGs. Formation of IB granules does not indicate translational arrest in the infected cells. We further show that EBOV does not induce formation of canonical SGs or eIF2␣ phosphorylation at any time postinfection but is unable to fully inhibit SG formation induced by different exogenous stressors, including sodium arsenite, heat, and hippuristanol. Despite the sequestration of SG marker proteins into IB granules, canonical SGs are unable to form within inclusions, which we propose might be mediated by a novel function of VP35, which disrupts SG formation. This function is independent of VP35's RNA binding activity. Further studies aim to reveal the mechanism for SG protein sequestration and precise function within inclusions. IMPORTANCEAlthough progress has been made developing antiviral therapeutics and vaccines against the highly pathogenic Ebola virus (EBOV), the cellular mechanisms involved in EBOV infection are still largely unknown. To better understand these intracellular events, we investigated the cellular stress response, an antiviral pathway manipulated by many viruses. We show that EBOV does not induce formation of stress granules (SGs) in infected cells and is therefore unrestricted by their concomitant translational arrest. We identified SG proteins sequestered within viral inclusions, which did not impair protein translation. We further show that EBOV is unable to block SG formation triggered by exogenous stress early in infection. These findings provide insight into potential targets of therapeutic intervention. Additionally, we identified a novel function of the interferon antagonist VP35, which is able to disrupt SG formation.

Section: Discussionsupporting

confidence: 81%

Ebola Virus Does Not Induce Stress Granule Formation during Infection and Sequesters Stress Granule Proteins within Viral Inclusions

Nelson

Schmidt

Deflubé

et al. 2016

“…3B, middle and right panels). These results thus confirm the findings of the proteomics study (22) and establish the F-box protein FBXO3 as a host cell interactor of RVFV NSs.…”

Section: Rapid Degradation Of Nss Targetssupporting

confidence: 79%

“…We have recently performed a proteomic screen for host cell interactors of 70 viral IFN antagonists, including the NSs of the virulent RVFV strain ZH548 (22). Since RVFV NSs inhibits RNA polymerase II-driven mRNA synthesis, it is difficult to express from eukaryotic plasmids.…”

Section: Rapid Degradation Of Nss Targetsmentioning

confidence: 99%

Virulence Factor NSs of Rift Valley Fever Virus Recruits the F-Box Protein FBXO3 To Degrade Subunit p62 of General Transcription Factor TFIIH

Kainulainen

Habjan

Hubel

et al. 2014

Self Cite

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. IMPORTANCERift 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.

“…VP35 contains an SQTQT motif that is required for LC8 interaction (15). However, LC8 binding was not required for, nor did it inhibit, VP35 interferon (IFN) antagonist function (15,21). Therefore, the significance of the VP35-LC8 interaction has remained undefined.…”

mentioning

confidence: 99%

Ebola Virus VP35 Interaction with Dynein LC8 Regulates Viral RNA Synthesis

Luthra

Jordan

Leung

et al. 2015

bEbola virus VP35 inhibits alpha/beta interferon production and functions as a viral polymerase cofactor. Previously, the 8-kDa cytoplasmic dynein light chain (LC8) was demonstrated to interact with VP35, but the functional consequences were unclear. Here we demonstrate that the interaction is direct and of high affinity and that binding stabilizes the VP35 N-terminal oligomerization domain and enhances viral RNA synthesis. Mutational analysis demonstrates that VP35 interaction is required for the functional effects of LC8. E bola virus (EBOV) VP35 is a multifunctional protein critical for both viral innate immune evasion and viral RNA synthesis (1, 2). It contains an N-terminal oligomerization domain and a C-terminal double-stranded RNA (dsRNA) binding domain referred to as the interferon inhibitory domain (IID) (3, 4). VP35 acts as an interferon antagonist, using both dsRNA-binding-dependent and -independent mechanisms, to block the RIG-I-like receptor signaling pathways (5-9). VP35 also participates with the viral large protein (L), the enzymatic component of the RNA-dependent RNA polymerase (RDRP) complex, VP30, and nucleoprotein (NP) in viral RNA synthesis (10, 11). VP35 is critical in this role because it mediates the interaction between L and the RNA template-associated NP (2,(12)(13)(14).VP35 can interact with the highly conserved 8-kDa cytoplasmic dynein light-chain (LC8) protein (15). LC8 is a subunit of the cytoplasmic dynein motor complex, which plays an important role in the microtubule-associated intracellular retrograde transport system (16,17). It also can exist in a soluble form without association with the dynein motor or microtubule (18). LC8 exists as a dimer containing two identical grooves located at opposite faces of the protein dimer interface (19). These grooves bind to proteins containing consensus motifs of either (K/S)XTQT or G (I/V)QVD (20). VP35 contains an SQTQT motif that is required for LC8 interaction (15). However, LC8 binding was not required for, nor did it inhibit, VP35 interferon (IFN) antagonist function (15,21). Therefore, the significance of the VP35-LC8 interaction has remained undefined.Here we characterized the interaction between LC8 and EBOV VP35 and provide evidence that the interaction stabilizes the VP35 N-terminal oligomerization domain to enhance viral RNA synthesis. The VP35-LC8 interaction was first validated by coimmunoprecipitation (co-IP). Briefly, HEK293T cells were transfected with plasmids expressing hemagglutinin (HA)-tagged LC8 (500 ng) and either full-length FLAG-tagged VP35 (1 g) (GenBank accession no. AF086833.2) or a VP35 truncation mutant (1 g) corresponding to the VP35 N-terminal domain (FLAG-VP35-N, residues 1 to 218) or the IID (FLAG-VP35-C, residues 219 to 340). Twenty-four hours posttransfection, anti-FLAG immunoprecipitations were performed. Both wild-type (WT) VP35 and VP35-N coprecipitated with LC8, confirming that VP35 binds LC8 via its N terminus (Fig. 1A). Mutation to alanine of T73 or Q74 within the VP35 71-SQTQT-75 motif disrupted or...