Cryoelectron tomography of HIV-1 cell-cell transmission conjugates reveals a secluded environment for viral assembly and transfer

Cell-cell transmission of HIV-1 (CCT) is a highly efficient mode of intrahost transmission that provides viral particles broad protection from antiretroviral agents including antiretroviral drugs, host restriction factors, and broadly neutralizing antibodies. Interestingly, the exact factors that grant viral particles increased protection and efficiency during intrahost spread remain unclear. Using a T cell co-culturing system and correlative cryoelectron tomography (cryoET) workflow, we investigated the architecture of HIV-1 CCT sites in a near-native, in situ context to address why CCT is efficient and grants this resistance. Contrary to previous models which suggest large, virus-packed sites, our 3D reconstructions reveal that CCT occurs within small intercellular spaces containing on average 1-5 viral particles. Surrounding these spaces are long, tight membrane interfaces that seclude the viral particles from the remaining extracellular environment. These observations suggest a model where spatial isolation may limit accessibility of antiretroviral agents to the virus-containing spaces thereby conferring protection to viral particles during transmission. To functionally validate these structural insights, we developed a quantitative flow cytometry strategy that decouples CCT conjugate formation from successful CCT infection through the analysis of multicell events. This methodology allows us to systematically compare the impact different molecules such as Env and CD4 have on HIV-1 CCT in a quantitative and population-level manner. Using this approach, we determined that initial HIV-1 CCT conjugate formation is partially dependent on CD4, but the Env involved in conjugate formation may differ from Env used for viral entry. Overall, these findings may prompt a rethinking of intervention strategies for HIV-1 CCT and have further implications with cytoplasmic trafficking, nuclear import, and host restriction factors.