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
Year: 2025
Type: article
Abstract: , 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.
Source: Journal of Virology
DOI: 10.1128/jvi.02236-24
Authors: Shih-Han Hsieh, Kuo-Jung Huang, Chin-Tien Wang
Language: English
Topic: HIV Research and Treatment
Subfield: Virology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: diamond
OpenAlex ID: W4408343984