MUTAGENESIS OF THE COXSACKIE B3 VIRUS 2B 2C CLEAVAGE SITE - DETERMINANTS OF PROCESSING EFFICIENCY AND EFFECTS ON VIRAL REPLICATION/

The enterovirus 2B/2C cleavage site differs from the common cleavage s ite motif AxxQ down arrow G by the occurrence of either polar residues at the P1' position or large aliphatic residues at the P4 position. T o study (i) the putative contribution of these aberrant residues to th e stability of precursor protein 2BC (ii) the determinants of cleavage site specificity and efficiency of 3C(pro), and (iii) the importance of efficient cleavage at this site for viral replication, a mutational analysis of the coxsackie B3 virus (CBV3) 2B/2C cleavage site (AxxQ d own arrow N) was performed. Neither replacement of the P1' asparagine with 3 serine or a glycine nor replacement of the P4 alanine with a va line significantly affected 2B/2C cleavage efficiency, RNA replication , or virus growth, The introduction of a P4 asparagine, as can be foun d at the CBV3 3C/3D cleavage site, caused a severe reduction in 2B/2C cleavage and abolished virus growth. These data support the idea that a P4 asparagine is an unfavorable residue that contributes to a slow t urnover of precursor protein 3CD but argue that it is unlikely that th e aberrant 2B/2C cleavage site motifs serve to regulate 2B/2C processi ng efficiency and protein 2BC stability. The viability of a double mut ant containing a P4 asparagine and a P1' glycine demonstrated that a P 1' residue can compensate for the adverse effects of an unfavorable P4 residue. Poliovirus (or poliovirus-like) 2B/2C cleavage site motifs w ere correctly processed by CBV 3C(pro), albeit with a reduced efficien cy, and yielded viable viruses. Analysis of in vivo protein synthesis showed that mutant viruses containing poorly processed 2B/2C cleavage sites were unable to completely shut off cellular protein synthesis. T he failure to inhibit host translation coincided with a reduced abilit y to modify membrane permeability, as measured by the sensitivity to t he unpermeant translation inhibitor hygromycin B. These data suggest t hat a critical level of protein 2B or 2C, or both, may be required to alter membrane permeability; and, possibly as a consequence, to shut o ff host cell translation.