Elucidating oligomerization state and composition of Gag:GagPol:RNA complexes during membrane trafficking and nucleation

• Applicant: Professor Dr. Don C. Lamb
• Subject Area: Virology ; Biophysics
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 550627869

Although HIV-1 has been extensively studied, there is no cure for AIDS. In addition, the development of viral resistance during treatment underscores the need for new therapeutic targets.. A better molecular understanding of the assembly and maturation processes could help to develop specific intervention strategies. During viron assembly, Gag:GagPol multimerization is most likely linked to vRNA incorporation, assembly kinetics and, at later stages, to viral protease activation. Thus, it is important to reveal the composition of Gag:GagPol multimers during assembly in the cytosol, membrane trafficking and assembly nucleation. These viral processes could eventually be exploited as new targets for anti-retroviral drug development. Building on our expertise with respect to quantitative fluorescence spectroscopy and microscopy, we propose to study the early stages of HIV-1 viral particle assembly focusing on the role of Gag:GagPol:vRNA interactions. In particular, we will investigate the cytosolic multimerization of Gag:GagPol complexes, analyze the temporal order of membrane interactions and discern the stoichiometry of Gag:GagPol:RNA complexes when they arrive as nucleators at the plasma membrane. Additionally, we aim to explore the influence of cellular factors on these processes. To accomplish this, we will develop a dual-labeled GagPol molecule to directly monitor the frameshift and ratio of Gag to GagPol in living cells and nascent HIV-1 particles. Various image correlation methods will be employed to investigate cytosolic interactions of Gag:GagPol, including the presence of GagPol in small aggregates of Gag, as we discovered previously. To follow further steps in HIV-1 assembly, we will measure assembly at the plasma membrane using total internal reflection fluorescence microscopy. With a third color, we can follow the interaction of Gag:GagPol with the viral RNA or various host proteins. The culmination of these goals will lead to an extensive model of Gag and GagPol multimerization throughout the early steps of the HIV-1 viral cycle, encompassing cytosolic dynamics to virion release.

• DFG Programme: Research Grants