ANTIVIRAL ACTIVITY OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PROTEASE INHIBITORS IN A SINGLE-CYCLE OF INFECTION - EVIDENCE FOR A ROLE OF PROTEASE IN THE EARLY PHASE

The antiviral activities of two substrate-based inhibitors of human im munodeficiency virus type 1 (HIV-1) protease, UK-88,947 and Ro 31-8959 , were studied in acute infections. H9 and HeLaCDC-4LTR/beta-gal cells were infected either with HTV-1(IIIB) or a replication-defective viru s, HIV-gpt(HXB-2). Both inhibitors were capable of blocking early step s of HIV-1 replication if added to cells prior to infection. Partial i nhibition was also obtained by addition of inhibitor at the time of or as late as 15 min after infection. The inhibitors were ineffective if added 30 min postinfection. The inhibitory effects were studied by cD NA analysis,vith PCR followed by Southern blot hybridization and by in fectivity assays allowing quantitation of HIV-1 in a single cycle of r eplication. When UK-88,947-treated H9 cells were coinfected with HIV-1 and human T-cell leukemia virus type I only the replication of HIV-I was inhibited, demonstrating viral specificity. Pretreating the infect ious virus stocks with the inhibitors also prevented replication, indi cating that the inhibitors block the action of the viral protease and not a cellular protease. A panel of primer sets aas used to analyze cD NA from cell lysates by PCR amplification at 4 and 18 h postinfection. Four hours after infection, viral specific cDNA was detected with all of the four primer pairs used: R/U5, nef/U3, 5' gag, and long termina l repeat (LTR)/gag. However, after 18 h, only the R/U5 and nef/U3 prim er pairs and not the 5' gag or LTR/gag primer pair were able to allow amplification of cDNA. The results suggest a crucial role of HN-I prot ease in the early phase of viral replication. Although it is not clear what early steps are affected by the protease, it is likely that the target is the NC protein, as referred from our previous reports of the in situ cleavage of the nucleocapsid (NC) protein by the viral protea se inside lentiviral capsids. The results suggest that it is not the i nhibition of initiation and progression of reverse transcription but t he stability of full-size unintegrated cDNA which is affected in the p resence of protease inhibitors. Alternatively, the cleavage of the NC protein may be required for the proper formation of preintegration com plex and/or for its transport to the nucleus.