ENCAPSIDATION OF POLIOVIRUS REPLICONS ENCODING THE COMPLETE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 GAG GENE BY USING A COMPLEMENTATION SYSTEM WHICH PROVIDES THE P1 CAPSID PROTEIN IN TRANS

Poliovirus genomes which contain small regions of the human immunodefi ciency virus type 1 (HIV-1) gag, pol, and env genes substituted in fra me for the P1 capsid region replicate and express HIV-1 proteins as fu sion proteins with the P1 capsid precursor protein upon transfection i nto cells (W.S. Choi, R. Pal-Ghosh, and C. D. Morrow, J. Virol. 65:287 5-2883, 1991). Since these genomes, referred to as replicons, do not e xpress capsid proteins, a complementation system was developed to enca psidate the genomes by providing P1 capsid proteins in trans from a re combinant vaccinia virus, VV-P1. Virus stocks of encapsidated replicon s were generated after serial passage of the replicon genomes into cel ls previously infected with VV-P1 (D. C. Porter, D. C. Ansardi, W. S. Choi, and C. D. Morrow, J. Virol. 67:3712-3719, 1993). Using this syst em, we have further defined the role of the PI region in viral protein expression and RNA encapsidation, In the present study, we constructe d poliovirus replicons which contain the complete 1,492-bp gag gene of HIV-1 substituted for the entire P1 region of poliovirus. To investig ate,whether the VP4 coding region was required for the replication and encapsidation of poliovirus RNA, a second replicon in which the compl ete gag gene was substituted for the VP2, VP3, and VP1 capsid sequence s was constructed, Transfection of replicon RNA with and,without the V P4 coding region into cells resulted in similar levels of expression o f the HIV-1 Gag protein and poliovirus 3CD protein, as indicated by im munoprecipitation using specific antibodies. Northern (RNA) blot analy sis of RNA from transfected cells demonstrated comparable levels of RN A replication for each replicon, Transfection of the replicon genomes into cells infected with VV-P1 resulted in the encapsidation of the ge nomes; serial passage in the presence of VV-P1 resulted in the generat ion of virus stocks of encapsidated replicons. Analysis of the levels of protein expression and encapsidated replicon RNA from virus stocks after 21 serial passages of the replicon genomes with VV-P1 indicated that the replicon which contained the VP4 coding region was present at a higher level than the replicon which contained a complete substitut ion of the P1 capsid sequences. These differences in encapsidation, th ough, were not detected after only two serial passages of the replicon s with VV-P1 or upon coinfection and serial passage with type 1 Sabin poliovirus. The results of this study demonstrate that the entire P1 r egion of the poliovirus genome can be substituted with a foreign gene without significantly affecting the capacity of the genome to replicat e or be encapsidated.