Background: In the maturation step of the HIV-1 lifecycle, the virus-encoded protease (PR) must process the structural polyprotein Gag to generate infectious virus particles. Previous reports found that cleavage of the p15NC maturation intermediate accelerates when RNA is present. Our studies have shown RNA-enhancement is substrate-independent. We hypothesize that enhancement results from an interaction between PR and RNA.
Methods: In vitro proteolysis reactions were performed +/- RNA with globular substrates derived from Gag or a 12-amino acid peptide. HIV PRs of multiple lineages were used to investigate whether RNA-enhancement is general to HIV. Alternative nucleic acids were also employed. Gel-shift assays were used to examine interactions between proteins and nucleic acids.
Results: Addition of RNA to two-substrate in vitro proteolysis reactions containing Gag derivatives p15NC and MA/CA accelerated cleavage of both substrates. In reactions with only MA/CA, the rate increased 80x (see figure). Cleavage of a peptide substrate, too small to interact with RNA and be simultaneously cleaved, was accelerated 20x. These data argue that RNA enhancement is substrate-independent. RNA enhanced the activity of Subtype B, C, and AE PRs; however, the HIV-2 PR was unaffected. Select short DNA oligonucleotides (~20 bases) capable of forming homomultimers could increase PR activity, but to a lesser degree than long, heteropolymeric RNA. The PR preferentially interacted with the multimeric forms of oligos in gel-shift assays, implicating the DNA complexes as necessary. Dimer stability is unaffected by RNA, since the tethered-dimer was equivalent to the wild-type PR in our assays. RNA lowered the Km by 5-10x, and raised the Vmax 2-5x.
Conclusions: RNA can directly interact with HIV-1 PRs in vitro, dramatically increasing their catalytic activity. This interaction likely relies on electrostatic forces, rather than sequence, since multimer formation was the principle determinant of effectiveness for DNA oligonucleotides. Mechanistically, RNA likely affects PR function post-dimerization, since RNA equivalently enhanced the wild type and tethered-dimer PRs. The differences in Km and Vmax show that RNA increases the PR’s affinity for its substrate and its turnover capacity. These data raise the possibility that PR cleavage during assembly is regulated in part by the juxtaposition of the viral PR and virion-packaged RNA.