A virus-like particle assembly system for probing the HIV-1 Gag-Pol dimerization domain: supporting evidence for reverse transcriptase involvement in protease activation by influencing Gag-Pol/Gag-Pol interaction.

Abstract

HIV-1 protease (PR) activation is triggered by Gag-Pol dimerization. We previously reported that reverse transcriptase (RT) amino acid substitution mutations resulted in p66/51RT heterodimer instability associated with impaired PR activation, and that treatment with efavirenz (EFV, an RT dimerization enhancer) increased PR activation, suggesting RT involvement. However, the contribution of RT to PR activation via the promotion of Gag-Pol dimerization has not been corroborated. To determine whether RT/RT interaction affects Gag-Pol dimerization, RT amino acid substitution mutations known to impair PR activation were cloned into a p6gag-containing construct, Gagp6-Pol, which assembles and releases virus-like particles (VLPs) when PR is inactivated. To map domains involved in Gag-Pol/Gag-Pol interaction, the major Gag assembly domain, with or without additional p6*, PR, or integrase (IN) deletions, was removed from Gagp6-Pol. Resulting constructs were transiently expressed in HEK293T cells. Sucrose density gradient fractionation and electron microscopy results suggest that p6gag-containing RT could form VLPs with lower densities and smaller sizes compared to wild-type particles. RT-PCR results suggest that p6-RT is capable of viral RNA packaging. RT-destabilizing amino acid mutations associated with PR-mediated virus processing deficiencies were found to be capable of reducing Gagp6-Pol VLP yields and attenuating EFV enhancement of Gagp6-Pol VLP assembly. Our results support the proposal that impaired RT stability or RT/RT interaction can disrupt Gag-Pol/Gag-Pol interaction, leading to impaired PR activation. This Gagp6-Pol VLP assembly system offers a potential assay method for probing domains involved in Gag-Pol/Gag-Pol interaction.

Importance: HIV-1 protease (PR) activation for mediating virus particle processing is essential for virus infectivity. As part of our attempt to determine whether Gag-Pol dimerization triggers PR activation, we found that RT point mutations that impair RT heterodimer stability and virus particle processing markedly reduced VLP assembly efficiencies in a p6gag-containing Gag-Pol expression vector (designated Gagp6-Pol). Further, these unstable RT point mutations markedly inhibited the facilitating effect of an RT dimerization enhancer on Gagp6-Pol VLP assembly. Our data support the proposal that RT/RT interaction contributes to PR activation by promoting Gag-Pol/Gag-Pol interaction, thus suggesting that targeting Gag-Pol dimerization may serve as an alternative HIV/AIDS treatment strategy. A Gag-Pol VLP assembly assay might be usable for probing the potential impacts of Gag-Pol dimerization on PR activation.