HIV-1 Entry into T-cells Is Not Dependent on CD4 and CCR5 Localization to Sphingolipid-enriched, Detergent-resistant, Raft Membrane Domains

Abstract

The contribution of raft domains to human immunodeficiency virus (HIV) 1 entry was assessed. In particular, we asked whether the CD4 and CCR5 HIV-1 receptors need to associate with sphingolipid-enriched, detergent-resistant membrane domains (rafts) to allow viral entry into primary and T-cell lines. Based on Triton X-100 solubilization and confocal microscopy, CD4 was shown to distribute partially to rafts. In contrast, CCR5 did not associate with rafts and localized in nonraft plasma membrane domains. HIV-1-receptor partitioning remained unchanged upon viral adsorption, suggesting that viral entry probably takes place outside rafts. To directly investigate this possibility, we targeted CD4 to nonraft domains of the membrane by preventing CD4 palmitoylation and interaction with p56 lck . Directed mutagenesis of both targeting signals significantly prevented association of CD4 with rafts, but did not suppress the HIV-1 receptor function of CD4. Collectively, these results strongly suggest that the presence of HIV-1 receptors in rafts is not required for viral infection. We show, however, that depleting plasma membrane cholesterol inhibits HIV-1 entry. We therefore propose that cholesterol modulates the HIV-1 entry process independently of its ability to promote raft formation.

The Effects of HIV-1 Nef on CD4 Surface Expression and Viral Infectivity in Lymphoid Cells Are Independent of Rafts

“…Rafts were initially proposed to act as platforms for virus entry, facilitating interactions between CD4 co-receptors, and incoming virions (44, 46 -48). This role has been, however, recently questioned, because CD4 molecules unable to associate with rafts still allow virus entry (49,50). There is also mounting evidence that rafts are important for HIV-1 assembly and budding.…”

“…Efficient CD4 association with rafts requires palmitoylation, but also interaction with the tyrosine kinase p56Lck (49,50,70). CD4 palmitoylation occurs on two membrane proximal residues (49,71) and also requires a short cluster of positively charged residues within the cytoplasmic tail (50). HIV-1 Env glycoproteins are palmitoylated on two intracellular cysteines (56,72).…”

“…At least two proteins involved in HIV replication are known to be palmitoylated, the cellular receptor CD4 and the viral Env glycoprotein. Efficient CD4 association with rafts requires palmitoylation, but also interaction with the tyrosine kinase p56Lck (49,50,70). CD4 palmitoylation occurs on two membrane proximal residues (49,71) and also requires a short cluster of positively charged residues within the cytoplasmic tail (50).…”

Analysis of proteins copurifying with the cd4/lck complex using one-dimensional polyacrylamide gel electrophoresis and mass spectrometry: comparison with affinity-tag based protein detection and evaluation of different solubilization methods

“…Also, a part of CD4 has been reported to participate in detergent-insoluble microdomains [18] and this compartment together with any possible associating molecules, is likely to escape detection in a method employing cell lysis in cold Triton X-100. To reduce contamination with abundant proteins from the transcription/translation machinery and to generally increase solubility of membrane proteins a detergent combination was used for cell lysis that had been reported to efficiently solubilize proteins from the NMDA receptor complex [19].…”

Vav exchange factor counteracts the HIV‐1 Nef‐mediated decrease of plasma membrane GM1 and NF‐AT activity in T cells

“…Although recent data demonstrate that HIV entry into T cells is not dependent on raft membrane domains [12], some authors suggest that HIV virus may utilize raft domains on the host cell plasma membrane for allowing budding, signaling and infection [13,14]. The HIV Nef protein may play a prominent role in these events.…”