Retrovirus particle assembly and release are crucial steps in the viral lifecycle, but many details of these complex processes remain incompletely understood. There are numerous virus and host cell factors that coordinate the trafficking of the Gag structural polyprotein to virus assembly sites. Virus particle biogenesis is difficult to observe in real time due to the large fluorescence background signal and long observation times required, which make the observation throughput slow and tedious. To address these challenges, we have applied an automated, multiplexed approach that combines the double helix point spread function, deep learning-based image analysis, fluorescence fluctuation spectroscopy, and total internal reflection microscopy to track the biogenesis of individual Gag puncta along with the concentration of cytoplasmic Gag monomers simultaneously. Differences were observed in Gag puncta biogenesis when HIV-1 Gag was expressed alone vs. that in the context of a full-length molecular clone. Furthermore, a subset of released HIV-1 particles remains nonspecifically associated with the cell. This approach has been extended to study other human retroviruses, i.e., HIV-2 and HTLV-1. This work has been supported by grants from the National Institutes of Health (R21 DE032878; R01 AI177264; R01 GM151775).
Kohler et al. (2026) studied this question.