Gag-Pol Transframe Domain p6* Is Essential for HIV-1 Protease-Mediated Virus Maturation

HIV-1 protease (PR) is encoded by pol, which is initially translated as a Pr160^gag-pol polyprotein by a ribosomal frameshift event. Within Gag-Pol, truncated p6gag is replaced by a transframe domain (referred to as p6* or p6pol) located directly upstream of PR. p6* has been proposed as playing a role in modulating PR activation. Overlapping reading frames between p6* and p6gag present a challenge to researchers using genetic approaches to studying p6* biological functions. To determine the role of p6* in PR activation without affecting the gag reading frame, we constructed a series of Gag/Gag-Pol expression vectors by duplicating PR with or without p6* between PR pairs, and observed that PR duplication eliminated virus production due to significant Gag cleavage enhancement. This effect was mitigated when p6* was placed between the two PRs. Further, Gag cleavage enhancement was markedly reduced when either one of the two PRs was mutationally inactivated. Additional reduction in Gag cleavage efficiency was noted following the removal of p6* from between the two PRs. The insertion of a NC domain (wild-type or mutant) directly upstream of PR or p6*PR did not significantly improve Gag processing efficiency. With the exception of those containing p6* directly upstream of an active PR, all constructs were either noninfectious or weakly infectious. Our results suggest that (a) p6* is essential for triggering PR activation, (b) p6* has a role in preventing premature virus processing, and (c) the NC domain within Gag-Pol is not a major determinant of PR activation.