A POLIOVIRUS MINIREPLICON CONTAINING AN INACTIVE 2A PROTEINASE IS EXPRESSED IN VACCINIA VIRUS-INFECTED CELLS

It has been difficult to evaluate the role of individual viral protein s in poliovirus replication because a suitable complementation system has not yet been developed. To approach this problem, we constructed a chimeric human immunodeficiency virus type 2 (HIV-2)-gag-poliovirus m inireplicon in which regions of the gag gene of HIV-2 were inserted in the poliovirus genome between nucleotides 1174 and 2470. Transfection of this chimeric RNA into HeLa cells results in the replication of th e minireplicon and expression of an HIV-2-gag-P1 fusion protein which can be immunoprecipitated with antibodies to HIV-2-gag. Expression of the HIV-2-gag-Pl fusion protein was dependent on replication of the ch imeric RNA genome. Although the chimeric HIV-2-gag-poliovirus RNA geno me replicated in poliovirus-infected cells, transfection of the chimer ic HIV-2-gag-poliovirus genome into vaccinia virus-infected cells resu lted in increased replication as measured by analysis of chimeric RNA. The increase in replication correlated with an increase in the expres sion of the HIV-2-gag-P1 fusion protein in vaccinia virus-infected cel ls. To characterize this system, we constructed a mutation in the 2A g ene to change a cysteine at amino acid 109 to a serine. Expression of the HIV-2-gag-P1 fusion protein was not detected when the HIV-2-gag-po liovirus genome containing the 2A mutation was transfected into HeLa c ells, demonstrating the mutation was lethal for replication. When the chimeric genome was transfected into poliovirus-infected cells, no RNA replication or expression of the HIV-2-gag-Pl fusion protein was obse rved. In contrast, transfection of this genome into vaccinia virus-inf ected cells resulted in replication of the chimeric RNA and expression of two proteins with larger molecular masses than the HIV-2-gag-P1 pr oteins, possibly representing HIV-2-gag-P1-2A and HIV-2-gag-P1-2ABC fu sion proteins. The transfection of the chimeric HIV-2-gag-poliovirus g enome containing the 2A mutation into poliovirus-vaccinia virus coinfe cted cells resulted in the expression and partial processing of the tw o larger HIV-2-gag-P1 fusion proteins to give the correct molecular ma ss for the HIV-2-gag-P1 fusion protein. The 2A mutation was reconstruc ted back into the full-length infectious cDNA of poliovirus. Transfect ion of this cDNA into vaccinia virus-infected cells followed by immuno precipitation with anticapsid antibodies demonstrated the presence of two proteins with molecular masses larger than P1, possibly PI-2A and Pl-2ABC fusion proteins. The results of this study suggest the protein ase activity of 2A(pro) is not required for RNA replication in vaccini a virus-infected cells and demonstrate the use of poliovirus minirepli cons as a means to study poliovirus RNA replication.