CHARACTERIZATION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 (HIV-1) PARTICLES THAT EXPRESS PROTEASE-REVERSE TRANSCRIPTASE FUSION PROTEINS

We have selectively mutagenized specific residues at the junction betw een the protease (PR) and reverse transcriptase (RT) genes of human im munodeficiency virus type 1 (HIV-1) to study the effects of PR-RT fusi on proteins in the context of a full-length, infectious proviral con s truct. Mutant viruses derived from COS-7 cells transfected with this c on struct were analyzed in regard to each of viral replication, matura tion; and infectivity. Immunoblot analysis revealed that the mutation prevented cleavage between the PR and RT proteins and that both existe d as a PR-RT fusion protein in each of cellular and viral lysates. Int erestingly, intracellular PR that existed within the PR-RT fusion prot ein remained functionally active, whereby HIV-1 precursor proteins wer e processed efficiently. Furthermore, the RT component of the fusion p rotein also retained its enzymatic activity as shown in RT assays. Ele ctron microscopy revealed that the mutant viruses containing the PR-RT fusion protein possessed wild-type morphology. These viruses also dis played wild-type sensitivities to inhibitors of each of the HIV-1 PR a nd RT activities. However, viruses containing the PR-RT fusion protein were 20 times less infectious than wild-type viruses. This defect was further pronounced when mutated Gag-Pol proteins were overexpressed a s a consequence of an additional mutation that interfered with framesh ifting. Thus, unlike cleavage site mutations at the N terminus of PR, a cleavage site mutation between PR and RT did not affect the enzymati c activities of either PR or RT and viruses containing PR-RT fusion pr oteins were viable. (C) 1998 Academic Press.