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

Poliovirus proteinase 2A(pro) is an essential enzyme involved in cleavages of viral and cellular proteins during the infectious cycle. Evidence has be en obtained that 2A(pro) is also involved in genome replication. All entero viruses have a negatively charged cluster of amino acids at their C terminu s (E-E/(E)(D)/(D)AMEQ-NH2), a common motif suggesting function. When aligne d with enterovirus sequences, the 2A(pro) proteinase of human rhinovirus ty pe 2 (HRV2) has a shorter C terminus (EE...Q-NH2) and, indeed, the HRV2 2A( pro) cannot substitute for poliovirus 2A(pro) to yield a viable chimeric vi rus. Here evidence is provided that the C-terminal cluster of amino acids p lays an unknown role in poliovirus genome replication. Deletion of the EEAM E sequence from poliovirus 2A(pro) is lethal without significantly influenc ing proteinase function. On the other hand, addition of EAME to HRV2 2A(pro ), yielding a C terminus of th is enzyme of EEEAMEQ, stimulated RNA replica tion of a poliovirus/HRV2 chimera 100-fold. The novel role of the C-termina l sequence motif is manifested at the level of protein function, since sile nt mutations in its coding region had no effect on virus proliferation. Pol iovirus type 1 Mahoney 2A(pro) could be provided in trans to rescue the let hal deletion EEAME in the poliovirus variant. Encapsidation studies left op en the question of whether the C terminus of poliovirus 2A(pro) is involved in particle formation. It is concluded that the C terminus of poliovirus 2 A(pro) is an essential domain for viral RNA replication but is not essentia l for proteolytic processing.