HIV-1 Entry in SupT1-R5, CEM-ss, and Primary CD4+ T Cells Occurs at the Plasma Membrane and Does Not Require Endocytosis

Cytoplasmic entry of HIV-1 requires binding of the viral glycoproteins to the cellular receptor and coreceptor, leading to fusion of viral and cellular membranes. Early studies suggested that productive HIV-1 infection occurs by direct fusion at the plasma membrane. Endocytotic uptake of HIV-1 was frequently observed but was considered to constitute an unspecific dead-end pathway. More recent evidence suggested that endocytosis contributes to productive HIV-1 entry and may even represent the predominant or exclusive route of infection. We have analyzed HIV-1 binding, endocytosis, cytoplasmic entry, and infection in T-cell lines and in primary CD4(+) T cells. Efficient cell binding and endocytosis required viral glycoproteins and CD4, but not the coreceptor. The contribution of endocytosis to cytoplasmic entry and infection was assessed by two strategies: (i) expression of dominant negative dynamin-2 was measured and was found to efficiently block HIV-1 endocytosis but to not affect fusion or productive infection. (ii) Making use of the fact that HIV-1 fusion is blocked at temperatures below 23 degrees C, cells were incubated with HIV-1 at 22 degrees C for various times, and endocytosis was quantified by parallel analysis of transferrin and fluorescent HIV-1 uptake. Subsequently, entry at the plasma membrane was blocked by high concentrations of the peptidic fusion inhibitor T-20, which does not reach previously endocytosed particles. HIV-1 infection was scored after cells were shifted to 37 degrees C in the presence of T-20. These experiments revealed that productive HIV-1 entry occurs predominantly at the plasma membrane in SupT1R5, CEM-ss, and primary CD4(+) T cells, with little, if any, contribution coming from endocytosed virions. IMPORTANCE HIV-1, like all enveloped viruses, reaches the cytoplasm by fusion of the viral and cellular membranes. Many viruses enter the cytoplasm by endosomal uptake and fusion from the endosome, while cell entry can also occur by direct fusion at the plasma membrane in some cases. Conflicting evidence regarding the site of HIV-1 fusion has been reported, with some studies claiming that fusion occurs predominantly at the plasma membrane, while others have suggested predominant or even exclusive fusion from the endosome. We have revisited HIV-1 entry using a T-cell line that exhibits HIV-1 endocytosis dependent on the viral glycoproteins and the cellular CD4 receptor; results with this cell line were confirmed for another T-cell line and primary CD4(+) T cells. Our studies show that fusion and productive entry occur predominantly at the plasma membrane, and we conclude that endocytosis is dispensable for HIV-1 infectivity in these T-cell lines and in primary CD4(+) T cells.