The multiple roles of the viral proteinase 2A in poliovirus replication have been difficult to assess because, to date, it has not been possible to isolate and characterize a viral genome with an inactive 2A(pro). We have previously reported that a poliovirus replicon containing an inactive 2A(pro) by virtue of a change at amino acid 109 from a cysteine to a serine (C109S) was replication competent when transfected into cells previously infected with vaccinia virus (R. Pal-Ghosh and C. D. Morrow, J. Virol. 67:4621-4629, 1993). To further develop this system, we have used a poliovirus replicon which contains the human immunodeficiency virus type 1 (HIV-1) gag gene positioned between nucleotides 1174 and 2470 of the poliovirus genome and have engineered a second mutation within this replicon to change the codon for amino acid 109 of the 2A(pro) from cysteine to serine (2AC109S). Transfection of this replicon into cells previously infected with vaccinia virus results in the replication and expression of a protein with a molecular mass consistent with that of a P1-HIV-1 Gag-2A fusion protein. Using a recently described complementation system which relies on the capacity of a recombinant vaccinia virus (VV-P1) to provide the capsid precursor (P1) in trans (D.C. Ansardi, D.C. Porter, and C. D. Morrow, J. Virol. 67:3684-3690, 1993; and D.C. Porter, D.C. Ansardi, W. S. Choi, and C. D. Morrow, J. Virol. 67:3712-3719, 1993), we have encapsidated this replicon containing the 2AC109S mutation. By using reverse transcription PCR, we demonstrated that after 15 serial passages the encapsidated replicon still contained the 2AC109S mutation. Infection of cells with a stock of encapsidated replicon, either in the presence or in the absence of vaccinia virus, resulted in the expression of the P1-HIV-1 Gag-2A fusion protein. Expression of the P1-HIV-1 Gag fusion protein in cells infected with the encapsidated replicon containing the 2AC109S mutation was reduced compared with the expression of P1-HIV-1 Gag in those cells infected with a replicon containing a wild type 2A gene. The protein expression and replication of the replicon RNA in cells containing the 2AC109S mutation was maintained for a longer period of time than for the replicons containing the wild-type 2A gene, possibly because of a reduced cytopathic effect. Coinfection of cells with the encapsidated replicon containing the 2AC109S mutation and wild-type poliovirus resulted in the further processing of the P1-HIV-1 Gag-2A fusion protein to produce the P1-HIV-1 Gag fusion protein, demonstrating that the protease activity of 2A can be supplied in part in trans. This study is the first to describe a poliovirus genome (or replicon) that contains an enzymatically inactive P2 or P3 gene product. On the basis of this study, we conclude that the protease activity of 2A is not essential for viral RNA replication, although 2A is required for high-level replication and expression.