SFPQ is differentially cleaved during HRV16 or poliovirus infection of HeLa cells; cleavage is independent of caspase activity and is carried out by recombinant 3CD <i>in vitro</i>.

(A) HeLa cells were mock- or HRV16-infected (MOI 10), cell lysates were generated at the indicated times, and lysates were subjected to Western blot analysis. SFPQ cleavage products are indicated (cp*). HRV16 2C and its precursor 2BC were used as indicators of infection and GAPDH was used as a loading control. (B) HeLa cells were mock- or poliovirus-infected (MOI 10) followed by the generation of cell lysates at the indicated times, and which were then subjected to Western blot analysis. The single SFPQ cleavage product is indicated as in (A). Poliovirus 3A and its precursor 3AB served as markers of infection and GAPDH was used as above. Poliovirus and HRV16 infections were both carried out at 34°C. (C) HeLa cells were infected with HRV16 in the presence or absence of 50 μM zVAD-FMK after which lysates were generated at the indicated times and subjected to Western blot analysis. SFPQ cleavage was observed with or without zVAD-FMK. HRV16 3D/3CD and GAPDH were used as above. Cleavage product of poly(ADP-ribose) polymerase 1 (PARP) is indicated (cp*). Data are representative of at least two independent experiments. (D) HeLa cell nuclear extract was incubated with bovine serum albumin (BSA) or different forms of recombinant HRV16 3CD and subjected to Western blot analysis. Cleavage of SFPQ (cp*) was observed in the presence of wild type (WT) 3CD and a form of 3CD containing a mutation in the 3C/3D autoproteolysis site (uncleavable, μ10). Catalytically inactive 3CD (C146A) did not cause cleavage of SFPQ. Hpi: hours post infection; ns: non-specific.