RIG-I is cleaved during picornavirus infection

Abstract

The innate immune system senses RNA virus infections through membrane-bound Toll-like receptors or the cytoplasmic proteins RIG-I and MDA-5. RIG-I is believed to recognize the 5'-triphosphate present on many viral RNAs, and hence is important for sensing infections by paramyxoviruses, influenza viruses, rhabdoviruses, and flaviviruses. MDA-5 recognizes dsRNA, and senses infection with picornaviruses, whose RNA 5'-ends are linked to a viral protein, VPg, not a 5'-triphosphate. We previously showed that MDA-5 is degraded in cells infected with different picornaviruses, and suggested that such cleavage might be a mechanism to antagonize production of type I IFN in response to viral infection. Here we examined the state of RIG-I during picornavirus infection. RIG-I is degraded in cells infected with poliovirus, rhinoviruses, echovirus, and encephalomyocarditis virus. In contrast to MDA-5, cleavage of RIG-I is not accomplished by cellular caspases or the proteasome. Rather, the viral proteinase 3C(pro) cleaves RIG-I, both in vitro and in cells. Cleavage of RIG-I during picornavirus infection may constitute another mechanism for attenuating the innate response to viral infection.

Figure 1. Degradation of RIG-I during picornavirus infection

Monolayers of HeLa cells (A, C–F) or SH-SY-5Y cells (B) were infected (MOI 10) with poliovirus (A, B), echovirus (C), rhinovirus type 16 (D), rhinovirus type 1A (E) or EMCV (F). At the indicated times after infection, cell extracts were prepared, fractionated by SDS-PAGE, and RIG-I was detected by western blot analysis. Positions of RIG-I and a putative cleavage product are indicated. Separate bottom panels show detection of EF1α to ensure that equal amounts of protein were applied to each lane.

Figure 2. Effect of protease inhibitors on poliovirus-induced cleavage of RIG-I

Monolayers of HeLa cells were infected with poliovirus (MOI 10) in the absence (A) or presence of Z-VAD-FMK (100 μM) (B) or MG132 (20 μM) (C). At the indicated times after infection, cell extracts were prepared, fractionated by SDS-PAGE, and RIG-I was detected by western blot analysis. Positions of RIG-I and a putative cleavage product are indicated. Separate bottom panels show detection of EF1α to ensure that equal amounts of protein were applied to each lane.

Figure 3. Effect of amino acid changes in polioviral proteinases 2A^pro and 3C^pro on cleavage of RIG-I

Monolayers of HeLa cells were infected with wild-type poliovirus (A), mutant 2A^proY88L (B) or mutant Se1–3C-02 (C) (MOI 10). At the indicated times after infection, cell extracts were prepared, fractionated by SDS-PAGE, and RIG-I was detected by western blot analysis. Positions of RIG-I and a putative cleavage product are indicated. Separate bottom panels show detection of EF1α to ensure that equal amounts of protein were applied to each lane.

Figure 4. Effect of 2A^pro and 3CD^pro on RIG-I in vitro

A cytoplasmic extract from HeLa cells was incubated with purified 2A^pro or 3CD^pro for 6 or 8 hr, fractionated by SDS-PAGE, and RIG-I was detected by western blot analysis. Cytoplasmic extracts from mock-infected HeLa cells (m) and from cells 6 hr after poliovirus infection (pv 6) were included to show the location of uncleaved and cleaved RIG-I, respectively. eIF4GI and PABP (to confirm enzyme activity of 2A^pro and 3CD^pro) and EF1α (loading control) were detected by western blot analysis.

Figure 5. Effect of 2A^pro and 3C^pro on RIG-I in vivo

Plasmids encoding poliovirus 2A^pro or 3C^pro (each with an N-terminal FLAG epitope) or vector alone (v) were introduced into HeLa cells by transformation. At 0 and 24 after DNA-mediated transformation, cell extracts were prepared, fractionated by SDS-PAGE, and RIG-I was detected by western blot analysis. Production of each viral proteinase was confirmed by western analysis using anti-FLAG antibody. eIF4GI (to confirm enzyme activity of 2A^pro) and EF1α (loading control) were detected by western blot analysis.