Evasion of Antiviral Immunity through Sequestering of TBK1/IKKε/IRF3 into Viral Inclusion Bodies

Abstract

Viruses have adopted various mechanisms to evade or subvert host antiviral responses initiated by viral RNA or DNA through Toll-like receptor (TLR) (1, 2) and RIG-I-like receptor (RLR) (3-5) signaling pathways. Viral double-stranded RNA (dsRNA) can be sequestered by VP35 of Ebola virus (6) to avoid the activation of TLRs or RLRs. Viral proteins that contain the TIR domain can be interferon (IFN) antagonists such as A46R of vaccinia virus, which blocks upstream IFN signaling by directly targeting MyD88, MAL, TRIF, and TRAM, the TIR domain-containing adaptors (2). RLR sensors such as RIG-I and MDA5 and adaptor IPS1 are targeted by NS1 of influenza virus (7), V protein of paramyxoviruses (3), and the protease precursor protein 3ABC of hepatitis A virus (5). The adaptor protein TRIF of the TLR signaling pathway can be targeted for degradation by viral proteins containing protease activities, such as NS3/4A of flavivirus hepatitis C virus (HCV) (8) and 3C of picornavirus EV71 (9), which can also cleave transcription factor (TF) IRF7, compromising IFN induction. In addition, the N-terminal fragment of cleaved IRF7 interacts with and inhibits IRF3, another TF essential for IFN induction, thereby enhancing viral infection (10). Downstream TFs are also targeted by viral proteins, such as NS1 of West Nile virus, which inhibits TLR3 signaling by preventing IRF3 translocation into the nucleus (11). Between TLR/RLR sensors and downstream TFs, IKK complexes are also targeted by proteins encoded by HCV, vaccinia virus, and rabies virus (12-14), among others.Severe fever with thrombocytopenia syndrome (SFTS) is an emerging febrile illness caused by a novel phlebovirus, SFTS bunyavirus (SFTSV), in the family Bunyaviridae (15). SFTSV is a vector-borne zoonotic arbovirus transmitted to humans by tick bites, causing high fever, loss of white blood cells and platelets, and, in severe cases, multiorgan failure. It is a single-stranded negativesense RNA virus with three genomic segments, L, M, and S (15). The L segment encodes viral RNA polymerase, while the M segment encodes the two viral envelope glycoproteins, Gc and Gn. The S segment is an ambisense RNA of 1,744 bases encoding a nucleoprotein (NP) and a nonstructural protein (NSs). Our recent study demonstrated that NSs inhibits host IFN-␤ and NF-B responses, although the mechanism remains unclear (16). Here we report that NSs forms unique cytoplasmic inclusion bodies (IBs) in infected or transfected cells that appear to play a role in immune evasion through the interaction of NSs and the IKK complex. Our data indicate that SFTSV has adopted a novel host immune evasion strategy involving the sequestration of IKK complex components TBK1, IKKε, and IRF, which are critical for the activation of the IFN signaling pathway, into IBs. MATERIALS AND METHODSCells, viruses, and reagents. African green monkey kidney Vero, HeLa, human embryonic kidney 293T, and MDCK cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovin...

Reverse Genetics System for Severe Fever with Thrombocytopenia Syndrome Virus

“…7B). Again, the staining patterns of NSs were similar for wild-type HB29pp and for the recombinant viruses and in accord with published observations (45)(46)(47)(48).…”

“…For both of these viruses, some if not most of the NSs protein produced locates to the nucleus, and considerable information on the molecular mechanisms involved in inhibiting the IFN response has accrued (53). Work by several groups involving the transient expression of SFTSV NSs has elucidated a novel mechanism used by this virus to antagonize the host cell innate immune response (45)(46)(47)(48)54). SFTSV NSs forms distinct cytoplasmic bodies, as confirmed here (Fig.…”

“…It has been reported previously that the NSs proteins of both Bunyamwera orthobunyavirus (BUNV) and RVFV (37,40) inhibit the viral polymerase as measured by minigenome assays. SFTSV NSs forms distinct punctate structures in the cytoplasm of infected or plasmid-transfected cells (45)(46)(47)(48). The NSs structures have been shown to colocalize with N protein and to be associated with viral RNAs, suggesting that NSs plays a role in the replication of the virus (47).…”

“…7B); NSs was not detected in the nuclei of infected cells. The Rab5-positive cytoplasmic "viroplasm" structures cause the sequestration and spatial isolation of several key components of the cellular innate immune response, such as retinoic acid-inducible gene I (RIG-I), tripartite motif 25 (TRIM25), TANK-binding kinase 1 (TBK1), and interferon regulatory factor 3 (IRF-3), thereby preventing interferon signaling (45)(46)(47)(48)54). SFTSV NSs has also been shown to colocalize with N protein and to be associated with viral RNAs, suggesting that NSs plays a role in the replication of the virus (47).…”

Suppression of type I and type III IFN signalling by NSs protein of severe fever with thrombocytopenia syndrome virus through inhibition of STAT1 phosphorylation and activation

“…Consistent with this, SFTSV NSs protein has been shown to suppress type I IFN production through the interaction with RIG-I and TRIM25, as well as IRF3 kinases TBK1 and IKKe, leading to their sequestration in virus-induced cytoplasmic subdomains separated from mitochondria (Qu et al, 2012;Ning et al, 2014;Santiago et al, 2014;Wu et al, 2014). In addition, SFTSV NSs has recently been found to perturb type I IFN signalling by interacting with STAT2, and thus retaining STAT1 and STAT2 in the cytoplasm (Ning et al, 2015).…”