GENETIC-ANALYSIS OF MENGOVIRUS PROTEIN 2A - ITS FUNCTION IN POLYPROTEIN PROCESSING AND VIRUS REPRODUCTION

To examine the functional requirements of mengovirus 2A for virus repr oduction, a series of mutants with overlapping deletions within the 2A region of mengovirus, and two chimeric constructs in which 2A is repl aced either by Theiler's murine encephalomyelitis virus (TMEV) 2A or b y coxsackie B3 virus (CBV3) 2A(pro) were generated, In vitro polyprote in synthesis showed that in both deletion mutants and the TMEV 2A chim eric construct, viral 3C protease (3C(pro))-mediated cleavage at the V P1-2A junction was disturbed, which resulted in decreased formation of mature capsid proteins and accumulation of the P1-2A precursor, 2A(pr o)-mediated processing of the chimeric VP1-2A(pro) junction was highly efficient, Although the resulting L-P1 precursor was cleaved at the L -VP4 junction, further processing of the P1 precursor was abrogated, T wo deletion mutant viruses and a TMEV 2A chimeric virus were obtained after transfection. The CBV 2A(pro) construct did not result in viable virus, Deletion mutant virus production was less than 3% compared to wild-type virus production, whereas chimeric virus production was redu ced to 25%. Although inhibition of host-cell translation was identical in wild-type and mutant virus-infected cells, viral protein and RNA s ynthesis were reduced in cells infected with mutant virus, independent ly of the impaired P1-2A processing, It is concluded that mengovirus 2 A may play a functional role in either virus translation or replicatio n, and that the functional aspects of mengovirus and TMEV 2A cannot be exchanged, The results also confirm that the processing cascade of L- P1-2A occurs sequentially and is probably regulated by subsequent conf ormational transitions of the cleavage products after each proteolytic event, The sequential release of L and 2A may be essential in the con text of their function in virus replication.