Abstract
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.
Highlights
As an enveloped virus, HIV-1 delivers its genome to the cytoplasm by fusing the viral membrane to the host cell membrane
Since a large fraction of hits obtained by the previous genome-wide RNAi screens were factors involved in transcriptional regulation, nuclear transport, biosynthesis and signaling pathways that are unlikely to affect HIV-1 fusion, we focused on proteins involved in endocytosis, membrane trafficking, and lipid metabolism (Figure 1A)
The shRNA screen for host factors involved in HIV-1 fusion with CEM.CCR5 cells performed in this study revealed several proteins involved in endocytosis and membrane trafficking as essential factors for productive virus entry
Summary
HIV-1 delivers its genome to the cytoplasm by fusing the viral membrane to the host cell membrane. Free HIV-1 entry and virus-mediated cell-cell fusion, occurring as a result of virus fusion at the plasma membrane, exhibited drastically different efficiencies and requirements for actin dynamics in adherent cells and in CEM cells [13]. We probed whether endocytic trafficking contributes to HIV-1 fusion with CD4+ T cells by performing a targeted short hairpin (shRNA) screen for host factors involved in intracellular membrane transport. Our results demonstrate that inhibition of HIV-1 fusion with T cells depleted of endocytic trafficking factors is not caused by the altered expression of CD4 or coreceptors, or by the compromised ability to fuse with the plasma membrane. The finding that unperturbed intracellular transport processes play a significant role in productive HIV-1 entry into a CD4+ T cell line supports the notion that a major fraction of viruses infects these cells by fusing with endosomes
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