The yeast Saccharomyces cerevisiae has been used as a system to obtain PV 2A pro variants [75] . The fact that this protease is very toxic for yeast has been exploited to generate 2A pro variants devoid of this cytotoxicity. Using this approach, several PV 2A pro unable to cleave eIF4G have been obtained. The characterization of these mutants revealed a region in 2A pro involved in the interaction with substrates but none of the mutations were found in the catalytic triad. A parallelism has been observed between the ability of these PV 2A pro variants to block protein synthesis and to cleave eIF4G [76] . PV mutants in 2A gene that lack trans but not cis proteolytic activity have been also identified. Normal Journal of Biomedicine and Biotechnology processing of the viral polyprotein is observed upon infection with these PV variants, whereas eIF4G remains intact in these cells [77] . Interestingly, RNA replication of those mutant viruses is hampered, suggesting that there is a correlation between PV RNA replication and the trans activity of 2A pro . It is controversial whether PV 2A pro contributes directly to viral replication or not. Although for many years it was thought that PV 2A pro plays a direct role in PV replication, more recent studies have shown that a full-length dicistronic PV construct lacking 2A pro is capable to give rise to progeny viruses. Moreover, virus yields of PV variants lacking the P1 coding region is partially restored when P1 is expressed in trans, suggesting that cleavage of the viral polyprotein by PV 2A pro is not essential for viral replication. [78] . However, it is known that 2A pro is important for inducing the cytophatic effect and for avoiding the inhibition of PV replication in interferon (IFN) α treated cells [79] . In agreement with the idea that 2A pro participates in viral RNA replication, a fraction of this protease localizes in PV replicative foci although the majority of 2A pro is associated with the matrix structure in the cytoplasm of infected cells [80] . However, the presence of 2A pro in the proximity of replication complexes does not demonstrate that it participates directly in the replication process. 