The C-terminal residues of poliovirus proteinase 2Apro are critical for viral RNA replication but not for cis- or trans-proteolytic cleavage

Abstract

Poliovirus proteinase 2Apro is an essential enzyme involved in cleavages of viral and cellular proteins during the infectious cycle. Evidence has been obtained that 2Apro is also involved in genome replication. All enteroviruses have a negatively charged cluster of amino acids at their C terminus (EE/D E/DAMEQ–NH2), a common motif suggesting function. When aligned with enterovirus sequences, the 2Apro proteinase of human rhinovirus type 2 (HRV2) has a shorter C terminus (EE…Q–NH2) and, indeed, the HRV2 2Apro cannot substitute for poliovirus 2Apro to yield a viable chimeric virus. Here evidence is provided that the C-terminal cluster of amino acids plays an unknown role in poliovirus genome replication. Deletion of the EEAME sequence from poliovirus 2Apro is lethal without significantly influencing proteinase function. On the other hand, addition of EAME to HRV2 2Apro, yielding a C terminus of this enzyme of EEEAMEQ, stimulated RNA replication of a poliovirus/HRV2 chimera 100-fold. The novel role of the C-terminal sequence motif is manifested at the level of protein function, since silent mutations in its coding region had no effect on virus proliferation. Poliovirus type 1 Mahoney 2Apro could be provided in trans to rescue the lethal deletion EEAME in the poliovirus variant. Encapsidation studies left open the question of whether the C terminus of poliovirus 2Apro is involved in particle formation. It is concluded that the C terminus of poliovirus 2Apro is an essential domain for viral RNA replication but is not essential for proteolytic processing.