Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Tuesday Speaker Abstracts

HIV-1 PREFERENTIALLY FUSES WITH PH-NEUTRAL VESICLES IN CELL LINES AND PRIMARY CD4 + T-CELLS

Manish B Sharma 1 ; Mariana Marin 1 ; Gregory B. Melikian ; 1 Emory University, Pediatrics, Atlanta, GA, USA

The HIV-1 envelope glycoprotein (Env) binds the CD4 receptor and coreceptors, CXCR4 or CCR5, on the cell surface to trigger viral fusion. The mechanism of Env-mediated fusion is well characterized but the preferred cellular sites of viral fusion leading to productive infection remain controversial. To elucidate the HIV-1 entry pathways into cells, a novel virus triple labeling approach was developed. HIV-1 pseudoviruses were co-labeled with (1) Gag-iCherry, a fluid phase marker that is released into the cytoplasm upon virus fusion; (2) ecliptic pHluorin (EcpH) that is quenched at mildly acidic pH; and (3) the lipophilic dye DiD that enables discrimination between fusion with the plasma membrane vs fusion with the endosome based upon the extent of dye dilution upon redistribution to respective membranes. Pseudoviruses containing all three markers were imaged in live HeLa-derived cells, CEM T-cell line and activated primary human CD4 + T-cells. Strikingly, ~80-90% of pseudoviruses fused with pH neutral vesicles but not the plasma membrane or acidified endosomes of cell lines and primary cells. To further delineate the mechanism of productive virus uptake by different cells, distinct endocytic pathways were blocked using EIPA (inhibitor of macropinocytosis), Pitstop2 (clathrin inhibitor) and Dynasore (dynamin inhibitor). Pitstop2 and Dynasore potently inhibited HIV-1 infection in all cell types tested, while EIPA inhibited infection only in primary CD4 + T-cells. These findings reveal the existence of alternative HIV-1 entry pathways into different cell types, with a major fraction of productive infections in primary CD4 + T-cells occurring through macropinocytosis. Together, our results support the role of HIV-1 endocytosis in productive infection and reveal a strong preference for fusion with pH-neutral endocytic compartments in cell lines and primary CD4 + T-cells. This work was supported by the NIH R37 AI150453 to GBM.

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