ENCAPSIDATION OF GENETICALLY-ENGINEERED POLIOVIRUS MINIREPLICONS WHICH EXPRESS HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 GAG AND POL PROTEINS UPON INFECTION

The use of recombinant viruses for the expression of a wide array of f oreign proteins has become commonplace during the last few years. Rece ntly, we have described the construction and characterization of chime ric human immunodeficiency virus type 1 (HIV-1)-poliovirus genomes in which the gag and pol genes of HIV-1 have been substituted for the VP2 and VP3 capsid genes of the P1 capsid precursor region of poliovirus. Transfection of these RNAs into tissue culture cells results in repli cation of the RNA genome and expression of HIV-1-P1 fusion proteins (W . S. Choi, R. Pal-Ghosh, and C. D. Morrow, J. Virol. 65:2875-2883, 199 1). Here we report on the encapsidation and amplification of the minir eplicons to obtain sufficient quantities for biological characterizati on. To do this, HIV-1-poliovirus minireplicon genomes containing the g ag or pol gene were transfected into cells previously infected with a recombinant vaccinia virus (VV-P1) which expresses the poliovirus caps id precursor protein, P1 (D. C. Ansardi, D. C. Porter, and C. D. Morro w, J. Virol. 65:2088-2092, 1991). The chimeric minireplicons replicate d and expressed the appropriate HIV-1-P1 fusion proteins as determined by immunoprecipitation with HIV-1-specific antibodies. The encapsidat ed genomes were isolated by ultracentrifugation. Reinfection of cells with the encapsidated chimeric RNA genomes resulted in expression of t he HIV-1-Gag-P1 or HIV-1-Pol-P1 fusion protein. Serial passaging of th e encapsidated chimeric HIV-1-poliovirus genomes was accomplished by c oinfecting cells with the encapsidated minireplicons and VV-P1, result ing in stocks of the encapsidated minireplicons. Northern (RNA) blot a nalysis of passaged material revealed that no detectable deletions of the chimeric genomes occurred during 14 serial passages. Infection of cells by the encapsidated minireplicons was blocked by antipoliovirus antibodies. Coinfection of cells with encapsidated minireplicons and t ype 1 Sabin poliovirus resulted in encapsidation of the chimeric genom es by wild-type poliovirus as measured by immunoprecipitation of the H IV-1-P1 fusion proteins with HIV-1-specific antibodies. The results of this study demonstrate the encapsidation of poliovirus minireplicons which express foreign proteins and point to the future use of this sys tem as a potential vaccine vector.