Rotavirus nonstructural protein 1 antagonizes innate immune response by interacting with retinoic acid inducible gene I

Abstract

Background: The nonstructural protein 1 (NSP1) of rotavirus has been reported to block interferon (IFN) signaling by mediating proteasome-dependent degradation of IFN-regulatory factors (IRFs) and (or) the β-transducin repeat containing protein (β-TrCP). However, in addition to these targets, NSP1 may subvert innate immune responses via other mechanisms.

Results: The NSP1 of rotavirus OSU strain as well as the IRF3 binding domain truncated NSP1 of rotavirus SA11 strain are unable to degrade IRFs, but can still inhibit host IFN response, indicating that NSP1 may target alternative host factor(s) other than IRFs. Overexpression of NSP1 can block IFN-β promoter activation induced by the retinoic acid inducible gene I (RIG-I), but does not inhibit IFN-β activation induced by the mitochondrial antiviral-signaling protein (MAVS), indicating that NSP1 may target RIG-I. Immunoprecipitation experiments show that NSP1 interacts with RIG-I independent of IRF3 binding domain. In addition, NSP1 induces down-regulation of RIG-I in a proteasome-independent way.

Conclusions: Our findings demonstrate that inhibition of RIG-I mediated type I IFN responses by NSP1 may contribute to the immune evasion of rotavirus.

Figure 1

Rotavirus NSP1 inhibits IFN-β promoter activation independent of IRF3 degradation. (A) Scheme of full-length (wt) NSP1 structures of rotavirus OSU and SA11 strains and C-truncated (ΔIRF3 binding domain) NSP1 mutant of rotavirus SA11. (B) Western blot analysis for degradation of IRF3 by OSU NSP1, SA11 NSP1 and NSP1ΔIRF3BD (SA11). 293FT cells were transfected with pCMV-IRF3, pEGFP-OSU NSP1 or pEGFP-SA11 NSP1 or pEGFP-NSP1ΔIRF3BD (SA11) plasmids and cell extracts were assayed 48 h post-transfection for the expression of IRF3. Immunoblots were probed with anti-IRF3 monoclonal antibody (top panel). β-actin was used as a loading control (bottom panel). (C, D, E) Inhibition of virus-induced IFN-β promoter activation by SA11 NSP1 (C), OSU NSP1 (D) or NSP1ΔIRF3BD (SA11) (E). 293FT cells were transfected with pGL3-IFN-β-Luc, pRL-SV40, and increasing amounts of SA11 NSP1, OSU NSP1 or NSP1ΔIRF3BD (SA11) expression plasmids. Cells were infected with Sendai virus for 24 h and assayed for luciferase activities. Data are expressed as folds of activation with standard deviations among triplicate samples.

Figure 2

Rotavirus NSP1 inhibits RIG-I mediated IFN-β promoter activation. 293FT cells were transfected with pGL3-IFN-β-Luc along with plasmids encoding RIG-I (A and D), MDA5 (B and F), MAVS (C and E) and OSU NSP1 or SA11 NSP1ΔIRF3BD. pRL-SV40 was included as a control. At 36 h after transfection, the luciferase activities were measured as described in the Methods section. Results are expressed as folds of activation with standard deviations among triplicate samples.

Figure 3

Analysis of interaction between NSP1 and RIG-I. (A) Immunoprecipitation analysis for the association between OSU NSP1 and RIG-I. 293FT cells were transfected with plasmids encoding full-length Myc-RIG-I, GFP, and GFP-OSU NSP1. At 48 h after transfection, cell lysates were immunoprecipitated (IP) with antibodies against Myc, and detected by SDS-PAGE. Immunoprecipitates and aliquots of cell lysates were then assayed by Western blot (WB) analysis. (B) SA11 NSP1 interaction with RIG-I. 293FT cells were transfected with plasmids encoding Myc-RIG-I, GFP-SA11 NSP1, and GFP separately. Immunoprecipitation and Western blot analysis were performed as described for panel A. (C) Interaction between NSP1ΔIRF3BD of rotavirus SA11 and RIG-I. Transfection, immunoprecipitation and Western blot analysis were performed as described above.

Figure 4

RIG-I is down-regulated by NSP1 at the protein level but is proteasome-independent. (A, B) Western blot analysis of RIG-I down-regulation by NSP1. 293FT cells were transfected with increased amount of pEGFP-OSU NSP1 (A) or pEGFP-SA11 NSP1 (B) and plasmid encoding Myc-RIG-I. Cell extracts were prepared 48 h post-transfection. Immunoblots were probed with anti-Myc monoclonal antibody to detect RIG-I (top panel). β-actin was used as a loading control (bottom panel). (C) Transcription level of RIG-I at different time points after transfection. 293FT cells were co-transfected with SA11-NSP1 and RIG-I plasmids. At different time points after transfection, total RNA extracted from cells was subjected to RT-PCR amplification and electrophoresis for RIG-I, NSP1 and GAPDH (inner control) mRNAs. (D, E) RIG-I is degraded in rotavirus infected cells. MA104 cells were infected with rotavirus SA11 at a m.o.i. of 0.1. Cell extracts were prepared at 0, 4, 8, 12, 24 and 36 h post-infection (p.i). RIG-I protein levels at each time point p.i. were determined by Western blot analyses using an anti-RIG-I antibody. The viral protein VP6 was used as an indicator for rotavirus infection. β-actin was used as a loading control. RIG-I, NSP1, VP6 and GAPDH mRNAs were also checked in parallel for evaluating the transcription level (E). (F, G) Effects of a proteasome inhibitor on NSP1 mediated RIG-I down-regulation. 293FT cells were transfected with Myc-RIG-I, pEGFP-OSU NSP1 (F) or pEGFP-SA11 NSP1 (G). The cells were treated with the proteasome inhibitor MG132 or an equivalent volume of DMSO as described in Methods. Lysates were prepared 36-48 h post-transfection. Immunoblots were probed with anti-Myc to detect myc-tagged RIG-I.

Figure 5

Schematic diagram of the proposed mechanisms by which rotavirus NSP1 subverts host innate immunity. Shown are the targets of NSP1 involved in host innate immune signaling. NSP1, nonstructural protein 1 of rotavirus; RIG-I, retinoic acid inducible gene I;MDA5, melanoma differentiation-associated gene 5;MAVS, mitochondrial antiviral-signaling protein; IPS-1, IFNβ-promoter stimulator 1; IFN, interferon; IRF, interferon transcription factor; Ub, ubiquitin.