BPS2026 – Real-time fluorescence imaging of retrovirus particle biogenesis in T cells

Retroviral particle assembly is driven by the Gag structural polyprotein. Real-time fluorescence measurements of virus particle biogenesis in living cells are challenging due to the structured background fluorescence from monomeric Gag protein in the cytoplasm. As a result, most studies have been confined to the bottom plasma membrane (PM) of adherent cells along the coverslip using TIRF microscopy. We have recently combined double helix point spread function (DHPSF) imaging with deep learning to image particle assembly throughout the cell. This imaging approach has revealed intriguing differences in the kinetics and mobility of Gag assemblies in U2OS cells at the adherent bottom PM compared to that of the nonadherent top PM. While U2OS cells are convenient cells for use in fluorescence imaging due to their large, flat morphology, they are not the natural target cells for HIV-1 or HTLV-1 (i.e., CD4 + T cells). Here, we implement DHPSF imaging and deep learning to study retroviral particle assembly in T cells. The large spherical size and non-adherent nature of T cells cells pose new technical challenges that include the immobilization of T cells on glass slides or in a 3D matrix and a thorough interrogation of their compatibility for 3D fluorescence imaging of biogenesis. Updated imaging and deep learning protocols are being adapted for use with T cells. Finally, we are analyzing how cellular polarization influences Gag localization and particle assembly kinetics by using an engineered platform to induce polarization in transfected cells. This work has been supported by grants from the National Institutes of Health (R21 DE032878; R01 AI177264; R01 GM151775).