Investigating the Role HIV Viral gRNA and Gag-Pol Incorporation in HIV-1 Maturation

Project Abstract: HIV is a continual threat to public health. Despite advances to prevent infection and slow disease progression, no definitive cure exists for this deadly virus. Existing treatments for HIV target essential viral proteins and enzymes, such as protease, reverse transcriptase, integrase, and viral capsid interactions, all of which contribute to the successful delivery of an infectious virus to the next host cell. While the major roles of these enzymes are well understood, the mechanism of their incorporation during viral assembly as part of the Gag-Pol polyprotein has yet to be defined. Preliminary data from the Saffarian Lab shows a mix of mature and immature Virus-Like Particles (VLPs) from expression of an NL43 backbone with a Psi (Ѱ) packaging signal deletion, NL43(Ѱ: Δ(105-278)&Δ(301-332)), in HEK293 cells. The Psi packaging signal deletion reduces viral gRNA incorporation during assembly, but the effect of the deletion on maturation efficiency has not been defined. This finding highlights the need for further investigation of the mechanism of Gag-Pol incorporation and the possible role of viral gRNA in maturation efficiency. This proposal aims to test two hypothesis to address these unknowns: Hypothesis 1- An unknown Gag-Pol packaging mechanism is disrupted by removal of the psi packaging signal, (Δ(105-278)&Δ(301-332)), resulting in VLPs lacking sufficient Gag-Pol for maturation. Hypothesis 2- There is a role for gRNA in protease activation, and virions that did not mature did not have sufficient gRNA or cellular RNA’s incorporated. Aim 1 will investigate the mechanism of Gag-Pol incorporation by examining the fluorescence of labeled Gag-Pol proteins in purified HIV VLPs. Aim 2 will uncover the role of viral gRNA in virion maturation efficiency by leveraging lentiviral vector systems to selectively incorporate gRNA domains into minimally viable VLPs. These VLPs will be examined for maturation efficiency using advanced Cryo-Electron Tomography and subtomogram averaging. These experiments will provide training in advanced fluorescent light microscopy, Cryo-Electron Tomography, and complimentary biochemical assays. Additionally, exposition of the proposal findings will offer experience in oral and written scientific communication by attending and presenting at conferences and publishing in high quality journals. These skills will serve the fellow as an independent researcher following the conclusion of the fellowship. Finally, the findings of this proposal stand to elucidate essential HIV maturation mechanisms, opening avenues for drug targets and advanced treatment for HIV.