Toscana virus NSs protein inhibits the induction of type I interferon by interacting with RIG-I

Abstract

Toscana virus (TOSV) is a phlebovirus, of the Bunyaviridae family, that is responsible for central nervous system (CNS) injury in humans. Previous data have shown that the TOSV NSs protein is a gamma interferon (IFN-β) antagonist when transiently overexpressed in mammalian cells, inhibiting IRF-3 induction (G. Gori Savellini, F. Weber, C. Terrosi, M. Habjan, B. Martorelli, and M. G. Cusi, J. Gen. Virol. 92:71-79, 2011). In this study, we investigated whether an upstream sensor, which has a role in the signaling cascade leading to the production of type I IFN, was involved. We found a significant decrease in RIG-I protein levels in cells overexpressing TOSV NSs, suggesting that the nonstructural protein interacts with RIG-I and targets it for proteasomal degradation. In fact, the MG-132 proteasome inhibitor was able to restore IFN-β promoter activation in cells expressing NSs, demonstrating the existence of an evasion mechanism based on inhibition of the RIG-I sensor. Furthermore, a C-terminal truncated NSs protein (ΔNSs), although able to interact with RIG-I, did not affect the RIG-I-mediated IFN-β promoter activation, suggesting that the NSs domains responsible for RIG-I-mediated signaling and interaction with RIG-I are mapped on different regions. These results contribute to identify a novel mechanism for bunyaviruses by which TOSV NSs counteracts the early IFN response.

Fig 1

Luciferase reporter assay. (A) HEK293FT cells were mock infected or infected with TOSV, UV-inactivated TOSV, or SeV (MOI. 0.05). Forty-eight hours later, the cleared lysates were used to measure the luciferase activities according to the manufacturer's instructions. Relative Firefly luciferase values were normalized with respect to the Renilla luciferase activity (fold induction). Relative quantitation of specific IFN β or RIG-I mRNA is reported on the right side. (B) Luciferase reporter assay on HEK293FT cells, transfected with 0.5 μg of empty plasmid or RIG-I-expressing plasmid, and mock infected or infected with TOSV, UV-inactivated TOSV, or SeV. (C) Luciferase reporter assay on HEK293FT cells transfected with reporter plasmids alone (mock) or in combination with 0.05 μg of FLAG-RIG-I and 0.5 μg of TOSV NSs, N, or empty plasmids. Thirty-six hours later, cells were stimulated with 0.1 μg of poly(I·C) and luciferase activities were measured. Results are given as mean values of several experiments ± standard deviations.

Fig 2

Coimmunoprecipitation assay. HEK293FT cells were cotransfected with FLAG-tagged RIG-I N terminus (RIG-IN) or C terminus (RIG-IC) along with 6×His-tagged TOSV NSs. Clarified lysates (300 μg) were subjected to co-IP by using either anti-FLAG M2 magnetic beads (upper panel) or polyclonal anti-6×His antibody (lower panel). The presence of RIG-I and NSs proteins in the immune complex was revealed by immunoblotting (IB) with anti-FLAG M2 or anti-NSs antibodies.

Fig 3

IFN-β promoter activation was evaluated by luciferase reporter assay in HEK293FT cells transfected with reporter plasmids along with FLAG-tagged RIG-IN, in combination with NSs expressing plasmid or the empty vector. Results are expressed as the average IFN-β promoter fold induction. The error bars represent the standard deviations from the mean values obtained from independent experiments.

Fig 4

Immunoblot detection of RIG-I protein in cells coexpressing TOSV NSs protein (A) or infected by TOSV (B) using anti-FLAG M2 and anti-NSs antibodies, respectively. Anti-N serum was used to evaluate the expression of viral antigen in selected samples.

Fig 5

Effects of the MG-132 proteasome inhibitor were investigated in HEK293FT cells transfected with RIG-I and NSs plasmids. (A) Cells were collected and assessed for indirect immunofluorescence with anti-FLAG M2, anti-6×His, or anti-TOSV N antibodies. (B) The ability of MG-132 to revert the antagonistic effects of the NSs protein was further investigated by luciferase reporter assay evaluating the activation of the IFN-β promoter under different conditions. Luciferase activities were measured in each sample, and results are reported as mean values ± standard deviations from at least three independent experiments.

Fig 6

HEK293FT cells were transfected with RIG-I alone or in combination with full-length NSs or ΔNSs plasmids. Equal amounts of whole-cell lysates were subjected to immunoblotting (A) or to luciferase reporter assay (B). Graph bars represent mean results from different experiments ± standard deviations. (C) HEK293FT transfected with FLAG-tagged RIG-I or RIG-IN, alone or in combination with ΔNSs plasmids, were lysed and precipitated with anti-FLAG M2 magnetic beads. RIG-I and NSs proteins were detected by immunoblotting with anti-FLAG M2 and anti-NSs antibodies.