Involvement of Clathrin-Mediated Endocytosis in Human Immunodeficiency Virus Type 1 Entry

Abstract

Productive entry of human immunodeficiency virus (HIV) is believed to occur by direct fusion at the plasma membrane. Endocytic uptake of HIV particles has been observed in several studies but is considered to be nonproductive, leading to virus degradation in the lysosome. We show here that endocytosis contributes significantly to productive HIV entry in HeLa cells by using trans dominant-negative mutants of dynamin and Eps15. Inducible expression of a dominant-negative mutant of dynamin in a CD4-positive HeLa cell line reduced HIV infection by 40 to 80%. This effect was independent of the infectious dose and was observed for three different isolates. Analysis of reverse transcription products by real-time PCR and of virus entry by delivery of a virion-associated Vpr-␤-lactamase fusion protein revealed a similar reduction, indicating that the block occurred at the entry stage. A strong reduction of HIV entry was also observed upon transient transfection of a different trans dominant-negative variant of dynamin, and this reduction correlated with the relative inhibition of transferrin endocytosis. Expression of a dominant-negative variant of Eps15, which is specific for clathrin-dependent endocytosis, reduced HIV entry in HeLa cells by ca 95%, confirming the role of endocytosis for productive infection. In contrast, no effect was observed for a dominant-negative variant of caveolin. We conclude that dynamin-dependent, clathrin-mediated endocytosis can lead to productive entry of HIV in HeLa cells, suggesting this pathway as an alternative route of virus entry.

Virus trafficking – learning from single-virus tracking

“…Experiments tracking Reoviruses, transferrin, LDL and labelled CCPs revealed a random initiation behaviour of coated pits, which are only stabilized after cargo binding 30 . For HIV, although direct fusion with the plasma membrane is the main pathway to deliver the capsid into the cytoplasm, a smaller fraction of viruses also infect cells by clathrin-mediated endocytosis 32 . Besides clathrin-or caveolin-mediated endocytosis, various viruses, including Polyoma virus and Herpes Simplex virus (HSV) [33][34][35] , exploit other clathrin-and caveolinindependent entry pathways.…”

HIV-1–cellular interactions analyzed by single virus tracing

“…Co-receptor recognition then leads to conformational changes within the viral transmembrane glycoprotein gp41, which mediates membrane fusion. While productive HIV-1 entry through direct fusion of the virion with the plasma membrane has been well established, we have recently shown that endocytotic uptake of HIV-1 can also contribute to productive infection (Daecke et al 2005;Fackler and Peterlin 2000;Maurin et al 2007). It is difficult to differentiate between different parallel viral-entry pathways or to dissect subsequent steps of the entry process using bulk biochemical and virological analyses, which have been widely used to investigate the HIV-1 entry process.…”

High-Throughput HIV–Cell Fusion Assay for Discovery of Virus Entry Inhibitors

“…42,51,52 The principle of this assay is illustrated in Figure 1. HIV-1 pseudoviruses containing b-lactamase fused to the N-terminus of the HIV-1 Vpr (BlaM-Vpr) are produced by coexpressing the BlaM-Vpr and HIV-1 backbone in producer cells, as described in Miyauchi et al 42 HIV-1 Vpr is required to direct BlaM-Vpr incorporation into the viral core.…”