To examine the functional requirements of mengovirus 2A for virus reproduction, a series of mutants with overlapping deletions within the 2A region of mengovirus, and two chimeric constructs in which 2A is replaced either by Theiler's murine encephalomyelitis virus (TMEV) 2A or by coxsackie B3 virus (CBV3) 2Apro were generated. In vitro polyprotein synthesis showed that in both deletion mutants and the TMEV 2A chimeric construct, viral 3C protease (3Cpro)-mediated cleavage at the VP1-2A junction was disturbed, which resulted in decreased formation of mature capsid proteins and accumulation of the P1-2A precursor. 2Apro-mediated processing of the chimeric VP1-2Apro 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. Two deletion mutant viruses and a TMEV 2A chimeric virus were obtained after transfection. The CBV 2Apro 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 reduced to 25%. Although inhibition of host-cell translation was identical in wild-type and mutant virus-infected cells, viral protein and RNA synthesis were reduced in cells infected with mutant virus, independently of the impaired P1-2A processing. It is concluded that mengovirus 2A may play a functional role in either virus translation or replication, 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 conformational transitions of the cleavage products after each proteolytic event. The sequential release of L and 2A may be essential in the context of their function in virus replication.


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