Cleavage of poly(A)-binding protein by coxsackievirus 2A protease in vitroand in vivo: Another mechanism for host protein synthesis shutoff?

Infection of cells by picornaviruses of the rhinovirus, aphthovirus, and en terovirus groups results in the shutoff of host protein synthesis but allow s viral protein synthesis to proceed. Although considerable evidence sugges ts that this shutoff is mediated by the cleavage of eukaryotic translation initiation factor eIF4G by sequence-specific viral proteases (2A protease i n the case of coxsackievirus), several experimental observations are at var iance with this view. Thus, the cleavage of other cellular proteins could c ontribute to the shutoff of host protein synthesis and stimulation of viral protein synthesis. Recent evidence indicates that the highly conserved 70- kDa cytoplasmic poly(A)-binding protein (PABP) participates directly in tra nslation initiation. We have now found that PABP is also proteolytically cl eaved during coxsackievirus infection of HeLa cells. The cleavage of PABP c orrelated better over time with the host translational shutoff and onset of viral protein synthesis than did the cleavage of eIF4G. In vitro experimen ts with purified rabbit PABP and recombinant human PABP as well as in vivo experiments with Xenopus oocytes and recombinant Xenopus PABP demonstrate t hat the cleavage is catalyzed by 2A protease directly. N- and C-terminal se quencing indicates that cleavage occurs uniquely in human PABP at (482)VANT STQTM down arrow GPRPAAAAAA(500), separating the four N-terminal RNA recogn ition motifs (80%) from the C-terminal homodimerization domain (20%). The N -terminal cleavage product of PABP is less efficient than full-length PABP in restoring translation to a PABP-dependent rabbit reticulocyte lysate tra nslation system. These results suggest that the cleavage of PABP may be ano ther mechanism by which picornaviruses alter the rate and spectrum of prote in synthesis.