ENCAPSIDATION AND SERIAL PASSAGE OF A POLIOVIRUS REPLICON WHICH EXPRESSES AN INACTIVE 2A PROTEINASE

The multiple roles of the viral proteinase 2A in poliovirus replicatio n have been difficult to assess because, to date, it has not been poss ible to isolate and characterize a viral genome with an inactive 2A(pr o). We have previously reported that a poliovirus replicon containing an inactive 2A(pro) by virtue of a change at amino acid 109 from a cys teine to a serine (C109S) was replication competent when transfected i nto cells previously infected with vaccinia virus (R. Pal-Ghosh and C. D. Morrow, J. Virol. 67:4621-4629, 1993). To further develop this sys tem, we have used a poliovirus replicon which contains the human immun odeficiency virus type 1 (HIV-1) gag gene positioned between nucleotid es 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 i n the replication and expression of a protein with a molecular mass co nsistent with that of a P1-HIV-1 Gag-2A fusion protein. Using a recent ly 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. 6 7:3684-3690, 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 re plicon containing the 2AC109S mutation. By using reverse transcription PCR, we demonstrated that after 15 serial passages the encapsidated r eplicon still contained the 2AC109S mutation. Infection of cells with a stock of encapsidated replicon, either in the presence or in the abs ence 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 c ells infected with the encapsidated replicon containing the 2AC109S mu tation was reduced compared with the expression of P1-HIV-1 Gag in tho se cells infected with a replicon containing a wild type 2A gene. The protein expression and replication of the replicon RNA in cells contai ning the 2AC109S mutation was maintained for a longer period of time t han for the replicons containing the wild-type 2A gene, possibly becau se of a reduced cytopathic effect. Coinfection of cells with the encap sidated replicon containing the 2AC109S mutation and wild-type poliovi rus resulted in the further processing of the P1-HIV-1 Gag-2A fusion p rotein to produce the P1-HIV-1 Gag fusion protein, demonstrating that the protease activity of 2A can be supplied in part in trans. This stu dy is the first to describe a poliovirus genome (or replicon) that con tains an enzymatically inactive P2 or P3 gene product. On the basis of this study, we conclude that the protease activity of 2A is not essen tial for viral RNA replication, although 2A is required for high-level replication and expression.