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 this increased protection and efficiency during intrahost spread remains 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 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, narrow membrane interfaces that seclude the virus 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 molecular determinants such as Env and CD4 have on HIV-1 CCT in a high-throughput and population-level manner. Using this approach, we determined that initial HIV-1 CCT conjugate formation is dependent on HIV-1 Envelope (Env) and cellular CD4; however, CCT was less impacted by anti-CD4 blockage as compared to free virus particle transmission only. This partial inhibition of HIV-1 CCT conjugate formation points towards other protein-protein interactions being involved with facilitating the formation of cell-cell conjugates. Overall, these results taken together suggest that successful CCT may require fewer viral particles and that ultrastructure makes a significant contribution to the broad protection and infection efficiency of HIV-1 CCT. These findings may prompt a rethinking of intervention strategies for HIV-1 CCT and have further implications for cytoplasmic trafficking, nuclear import, and host restriction factors.
Jensen, J., Cunha, M., Adhikary, B., Rodriguez, Z., Moe, I., Beldin, J., Lyumkis, D., Mendonca, L.
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