Lentivirus and Pseudovirus production LV in HEK-293T: 75 ng shRNA plasmid + 37,5 ng packaging or 75 ng of 3:1 (psPAX2:pMD2.G) mix in 24-well plate Pseudovirus in HEK-293T/17: 3 µg Env + 4 µg pR8DEnv + 2 µg BlaM-Vpr + 0.5 µg pcRev in 10 cm dish LV in HEK-293T/17: 4 µg pooled shRNA plasmids or ORF-Rab5 + 3 µg psPAX2 + 1.5 µg pMDG-VSVG plasmid in 10 cm dish

The HIV-1 entry pathway into permissive cells has been a subject of debate. Accumulating evidence, including our previous single virus tracking results, suggests that HIV-1 can enter different cell types via endocytosis and CD4/coreceptor-dependent fusion with endosomes. However, recent studies that employed indirect techniques to infer the sites of HIV-1 entry into CD4+ T cells have concluded that endocytosis does not contribute to infection. To assess whether HIV-1 enters these cells via endocytosis, we probed the role of intracellular trafficking in HIV-1 entry/fusion by a targeted shRNA screen in a CD4+ T cell line. We performed a screen utilizing a direct virus-cell fusion assay as readout and identified several host proteins involved in endosomal trafficking/maturation, including Rab5A and sorting nexins, as factors regulating HIV-1 fusion and infection. Knockdown of these proteins inhibited HIV-1 fusion irrespective of coreceptor tropism, without altering the CD4 or coreceptor expression, or compromising the virus' ability to mediate fusion of two adjacent cells initiated by virus-plasma membrane fusion. Ectopic expression of Rab5A in non-permissive cells harboring Rab5A shRNAs partially restored the HIV-cell fusion. Together, these results implicate endocytic machinery in productive HIV-1 entry into CD4+ T cells.