Activation of the beta interferon promoter by unnatural Sendai virus infection requires RIG-I and is inhibited by viral C proteins

Abstract

As infection with wild-type (wt) Sendai virus (SeV) normally activates beta interferon (IFN-beta) very poorly, two unnatural SeV infections were used to study virus-induced IFN-beta activation in mouse embryonic fibroblasts: (i) SeV-DI-H4, which is composed mostly of small, copyback defective interfering (DI) genomes and whose infection overproduces short 5'-triphosphorylated trailer RNAs (pppRNAs) and underproduces viral V and C proteins, and (ii) SeV-GFP(+/-), a coinfection that produces wt amounts of viral gene products but that also produces both green fluorescent protein (GFP) mRNA and its complement, which can form double-stranded RNA (dsRNA) with capped 5' ends. We found that (i) virus-induced signaling to IFN-beta depended predominantly on RIG-I (as opposed to mda-5) for both SeV infections, i.e., that RIG-I senses both pppRNAs and dsRNA without 5'-triphosphorylated ends, and (ii) it is the viral C protein (as opposed to V) that is primarily responsible for countering RIG-I-dependent signaling to IFN-beta. Nondefective SeV that cannot specifically express C proteins not only cannot prevent the effects of transfected poly(I-C) or (ppp)RNAs on IFN-beta activation but also synergistically enhances these effects. SeV-V(minus) infection, in contrast, behaves mostly like wt SeV and counteracts the effects of transfected poly(I-C) or (ppp)RNAs.

FIG. 1.

IFN-β activation induced by SeV-GFP(+/−) infections. (A) Parallel cultures of MEFs were first transfected with pIFNβ-luc^ff and pTK-luc^r and then infected with increasing amounts of either SeV-GFP(+) alone (which expresses a GFP mRNA from a transgene between the M and F genes) or 20 PFU/cell of SeV-GFP(+) plus increasing amounts of either SeV-GFP(−) (which expresses an anti-GFP mRNA from a transgene in the same location) or SeV-RFP (which expresses an RFP mRNA from a transgene in the same location), as indicated. GFP expression was monitored by fluorescence-activated cell sorter analysis at 20 hpi. Cell extracts were prepared at 20 hpi, and equal amounts were used to determine luciferase activities (below). These transfections were carried out three times with independent virus stocks, with similar results. (B) Cytoplasmic extracts were centrifuged on CsCl density gradients to isolate nonencapsidated (pellet) RNAs. The levels of GFP and anti-GFP mRNAs in 15 μg of CsCl pellet RNA were determined using sense- and antisense-specific primers for RT, followed by quantitative PCR (TaqMan) (see Materials and Methods). (C) Parallel cultures of MEFs were first transfected with the luciferase reporter plasmids plus either an empty vector, one expressing wt E3L(100-190), or one expressing mutant E3L(100-190) (E3L-mut.) and then infected with increasing amounts of SeV-GFP(+) and SeV-GFP(−) as indicated. Cell extracts were prepared at 20 hpi, and equal amounts were used to determine luciferase activities. Equal amounts of cell extracts were also Western blotted using anti-N and anti-HA (below).

FIG. 2.

Relative contributions of mda-5 and RIG-I in sensing SeV infections in MEFs. (A) Parallel cultures of MEFs were first transfected with pIFNβ-luc^ff, pTK-luc^r, and plasmids expressing dominant-negative RIG-I(ΔCARDs) or an empty vector as a negative control. After 24 h, the cells were either transfected with poly(I-C) or infected with either SeV-DI-H4 or SeV-GFP(+/−) (as indicated). Cytoplasmic extracts were prepared after a further 20 h of incubation and used to determine firefly and Renilla luciferase levels and the relative levels of RIG-I(ΔCARDs) (anti-Flag) and viral proteins (anti-P/V/C) by Western blotting (bottom). All transfections were carried out in duplicate, and the range of values obtained is indicated by the error bars. Ctrl., control. (B) Parallel cultures of MEFs were transfected with pIFNβ-luc^ff, pTK-luc^r, and plasmids expressing Flag-tagged RIG-I or mda-5 or these helicases plus RIG-I(ΔCARDs), mda-5(ΔCARDs), SeV V (whose overlapping C ORF was closed by a stop codon), SeV C, or an empty plasmid as a negative control, as indicated. Cytoplasmic extracts were prepared after 40 h of incubation and used to determine firefly and Renilla luciferase levels. All transfections were carried out in duplicate, and the range of values obtained is indicated by error bars. The relative levels of the Flag-RIG-I constructs were determined by Western blotting with anti-Flag, those of the Flag-mda-5 constructs were determined with anti-mda-5, and those of the viral V and C proteins were determined with anti-P/V/C serum (bottom). Vect., vector.

FIG. 3.

SeV V and C inhibition of IFN-β activation induced by SeV infections. (A) Parallel cultures of MEFs were transfected with pIFNβ-luc^ff, pTK-luc^r, and plasmids expressing the SeV V protein, the SeV C protein (actually C^1-23-Tom-C^24-204), or unmodified Tom as a negative control. After 24 h, the cells were infected with either SeV-DI-H4 or SeV-GFP(+/−) (as indicated). Cytoplasmic extracts were prepared after a further 24 h of incubation and used to determine firefly and Renilla luciferase levels. The relative levels of viral P, V, and C proteins were determined by Western blotting with anti-P/C/V serum (bottom). All transfections were carried out in duplicate, and the range of values obtained is indicated by error bars. (B) Parallel cultures of MEFs were transfected with pIFNβ-luc^ff, pTK-luc^r, and plasmids expressing either Tom, RIG-I(ΔCARDs), IAV NS1(1-73), C^1-23-Tom-C^24-204 (C*), or V, as indicated. After 24 h, the cells were infected with increasing amounts of SeV-DI-H4 (1×, 2×, and 4×). Cytoplasmic extracts were prepared after a further 24 h of incubation and used to determine luciferase levels.

FIG. 4.

SeV C^24-204 (or Y1) protein inhibits IFN-β activation induced by DI-H4 infection. Parallel cultures of MEFs were transfected with pIFNβ-luc^ff, pTK-luc^r, and plasmids expressing tomato constructs carrying either the wt (C^1-23) or mutant (P⁸P⁹) C^1-23 fused to their N termini, with and without C^24-204 (or Y1) fused to their carboxy termini, C^24-204 fused to the carboxy terminus alone (Tom-C^24-204), or unmodified Tom as a negative control (ctrl.), as indicated. After 24 h, the cells were infected with SeV-DI-H4 (as indicated). Cytoplasmic extracts were prepared after a further 24 h of incubation and used to determine luciferase levels. The relative levels of the various tomato constructs were determined by Western blotting with anti-dsRED (bottom). All transfections were carried out in duplicate, and the range of values obtained is indicated by error bars.

FIG. 5.

_pppRNA-induced activation of IFN-β. (A) Parallel cultures of MEFs were transfected with pIFNβ-luc^ff and pTK-luc^r, and pRIG-I was also transfected in some cultures, as indicated. After 24 h, the cells were transfected for 3 h with increasing amounts (1 or 3 μg) of either _pppGGG/RNA1, phosphatase-treated GGG/RNA1, _pppGCA/RNA1, or _OHGCA/RNA1, as indicated. Cytoplasmic extracts were prepared 18 h post-RNA transfection and used to determine luciferase levels. (B) Parallel cultures of MEFs were transfected with pIFNβ-luc^ff, pTK-luc^r, and plasmids expressing Tom, RIG-I(ΔCARDs), IAV NS1(1-73), C^1-23-Tom-C^24-204 (C*), or V, as indicated. After 24 h, the cells were transfected with increasing amounts (1 μg and 3 μg) of _pppGGG/RNA1, as indicated. Cytoplasmic extracts were prepared after 3 h of RNA transfection and used to determine luciferase levels. Rel., relative. (C) Same as above (B), except that the cells were transfected with 3 μg of _ppptrailer RNA.

FIG. 6.

RNA-induced activation of IFN-β in cells infected with SeV that cannot express either V or C. Parallel cultures of MEFs were either mock infected or infected with 20 PFU/ml of wt SeV, SeV-V^minus, or SeV-C^minus, as indicated. After 24 h, the cells were transfected with luciferase reporter plasmids and either _pppGGG/RNA1, poly(I-C), or no RNA (untreated). Cytoplasmic extracts were prepared after 18 h of RNA transfection, used to determine luciferase levels (above), and Western blotted to determine the levels of P, V, and C proteins as well as endogenous RIG-I and actin as a loading control. rSeV, recombinant SeV.