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. 2007 Apr;81(8):3677-84.
doi: 10.1128/JVI.01360-06. Epub 2007 Jan 31.

MDA-5 is cleaved in poliovirus-infected cells

Paola M Barral  1 , Juliet M Morrison, Jennifer Drahos, Pankaj Gupta, Devanand Sarkar, Paul B Fisher, Vincent R Racaniello
Affiliations

MDA-5 is cleaved in poliovirus-infected cells

Paola M Barral et al. J Virol. 2007 Apr.

Abstract

Infections with RNA viruses are sensed by the innate immune system through membrane-bound Toll-like receptors or the cytoplasmic RNA helicases RIG-I and MDA-5. It is believed that MDA-5 is crucial for sensing infections by picornaviruses, but there have been no studies on the role of this protein during infection with poliovirus, the prototypic picornavirus. Beginning at 4 h postinfection, MDA-5 protein is degraded in poliovirus-infected cells. Levels of MDA-5 declined beginning at 6 h after infection with rhinovirus type 1a or encephalomyocarditis virus, but the protein was stable in cells infected with rhinovirus type 16 or echovirus type 1. Cleavage of MDA-5 is not carried out by either poliovirus proteinase 2Apro or 3Cpro. Instead, degradation of MDA-5 in poliovirus-infected cells occurs in a proteasome- and caspase-dependent manner. Degradation of MDA-5 during poliovirus infection correlates with cleavage of poly(ADP) ribose polymerase (PARP), a hallmark of apoptosis. Induction of apoptosis by puromycin leads to cleavage of both PARP and MDA-5. The MDA-5 cleavage product observed in cells treated with puromycin is approximately 90 kDa, similar in size to the putative cleavage product observed in poliovirus-infected cells. Poliovirus-induced cleavage of MDA-5 may be a mechanism to antagonize production of type I interferon in response to viral infection.

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Figures

FIG. 1.
FIG. 1.
Degradation of MDA-5 during poliovirus infection. Monolayers of HeLa cells (A, B) or CHP100L cells (C) were treated with poly(IC) (20 μg/ml) for 16 h and then infected with poliovirus (MOI, 10) (A, C) or mock infected (B). At the indicated times after infection, cell extracts were prepared and fractionated by SDS-PAGE, and MDA-5 was detected by Western blot analysis. Unlabeled arrows indicate putative cleavage products of MDA-5. The identities of the ∼75-kDa protein and smaller proteins detected by the antibody are not known; the intensities of these proteins vary depending on the batch of anti-MDA-5 antibody used. Separate bottom panels show detection of EF1α to ensure that equal amounts of protein were applied to each lane.
FIG. 2.
FIG. 2.
Effect of picornavirus infection on MDA-5. Monolayers of HeLa cells were treated with poly(IC) (20 μg/ml) for 16 h and then infected with rhinovirus type 16 (A) or type 1a (B), echovirus type 1 (C), or EMCV (D) (MOI, 10). At the indicated times after infection, cell extracts were prepared and fractionated by SDS-PAGE, and MDA-5 was detected by Western blot analysis. The panels at the right show longer exposures of the Western blot to enable detection of putative MDA-5 cleavage products (unlabeled arrows). Separate bottom panels show detection of EF1α to ensure that equal amounts of protein were applied to each lane.
FIG. 3.
FIG. 3.
Poliovirus (PV)-induced cleavage of HA-MDA-5 produced from an adenovirus (Ad) vector. Monolayers of HeLa cells were infected with Ad.mda-5 (MOI, 50) and 16 h later were either mock infected (A) or infected with poliovirus (MOI, 10) (B, C, D). After adsorption of Ad.mda-5, cells were treated with MG132 (20 μM) (C) or poly(IC) (20 μg/ml) (D). At the indicated times after poliovirus infection, cell extracts were prepared and fractionated by SDS-PAGE, and MDA-5 was detected by Western blot analysis using anti-HA antibody. Separate bottom panels show detection of EF1α to ensure that equal amounts of protein were applied to each lane.
FIG. 4.
FIG. 4.
Effect of amino acid changes in viral proteinases 2Apro and 3Cpro on poliovirus-induced degradation of MDA-5. Monolayers of HeLa cells were treated with poly(IC) (20 μg/ml) for 16 h and then infected with wild-type (wt) poliovirus (A), the single mutant 2AproY88L (B) or Se1-3C-02 (C), or the double mutant 2AproY88L+Se1-3C-02 (D) (MOI, 10). At the indicated times after infection, cell extracts were prepared and fractionated by SDS-PAGE, and MDA-5 was detected by Western blot analysis. Unlabeled arrows indicate putative cleavage products of MDA-5. The identity of the ∼75-kDa species in panels A to C is not known; the intensity of this protein varies depending on the batch of anti-MDA-5 antibody used. This protein is not present in panel D. Separate bottom panels show detection of EF1α to ensure that equal amounts of protein were applied to each lane.
FIG. 5.
FIG. 5.
Effect of 2Apro and 3CDpro on MDA-5 in vitro. MDA-5 was produced by in vitro translation in a reticulocyte lysate in the presence of [35S]methionine. The lysate was subsequently incubated with purified 2Apro or 3CDpro and fractionated by SDS-PAGE, and [35S]methionine labeled proteins were detected by phosphorimaging. PABP (to confirm enzyme activity) and β-actin (loading control) were detected by Western blot analysis of a separate gel. The plasmid encoding MDA-5 was omitted from the reaction in lane 1.
FIG. 6.
FIG. 6.
Effect of proteinase inhibitors on poliovirus-induced MDA-5 cleavage. Monolayers of HeLa cells were treated with poly(IC) (20 μg/ml) for 16 h and then infected with poliovirus (MOI, 10) in the absence of inhibitor (A) or in the presence of MG132 (20 μM) (B), Z-VAD-FMK (100 μM) (C), or epoxomicin (10 μM) (D). At the indicated times after infection, cell extracts were prepared and fractionated by SDS-PAGE, and MDA-5 was detected by Western blot analysis. Unlabeled arrows indicate a putative cleavage product of MDA-5. The identity of the protein migrating faster than the 75-kDa marker in panel B is not known, but this protein was not consistently observed. Separate bottom panels show detection of EF1α to ensure that equal amounts of protein were applied to each lane.
FIG. 7.
FIG. 7.
Effect of proteinase inhibitors on poliovirus yields in HeLa cells. Monolayers of HeLa cells were infected with poliovirus (MOI, 10) in the absence or presence of MG132 (20 μM), Z-VAD-FMK (100 μM), or epoxomicin (10 μM). At the indicated times after infection, virus titers were determined by plaque assay on HeLa cell monolayers.
FIG. 8.
FIG. 8.
Cleavage of PARP during picornavirus infection. Monolayers of HeLa cells were treated with poly(IC) (20 μg/ml) (A, B, D, F, G) for 16 h and then mock infected (G) or infected with poliovirus (A, B, C), rhinovirus type 16 (D, E), or EMCV (F) (MOI, 10). In panel B, Z-VAD-FMK (100 μM) was included during infection. At the indicated times after infection, cell extracts were prepared and fractionated by SDS-PAGE, and PARP was detected by Western blot analysis.
FIG. 9.
FIG. 9.
Cleavage of MDA-5 and PARP in cells induced to undergo apoptosis by treatment with puromycin. Monolayers of HeLa cells were treated with poly(IC) (20 μg/ml) for 16 h, and then puromycin (puro) (10 μM) was added to induce apoptosis. At the indicated times after addition of puromycin to the culture medium, cell extracts were prepared and fractionated by SDS-PAGE, and MDA-5 or PARP were detected by Western blot analysis. The unlabeled arrow indicates a putative ∼90-kDa cleavage product of MDA-5. The identity of the protein migrating faster than the 75-kDa marker is not known.
FIG. 10.
FIG. 10.
Induction of IFN-β RNA in poliovirus-infected cells. Monolayers of HeLa cells were either mock treated, treated with poly(IC) (20 μg/ml) or Z-VAD-FMK (100 μM) for 16 h, or infected with wild-type (WT) or mutant polioviruses (MOI, 10). At 1, 3, and 5 h after infection, RNA was extracted from cells and analyzed for IFN-β expression by quantitative real-time PCR, using SYBR green to detect dsDNA. RNA levels were normalized to β-actin.
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