The Enhancement of Virus Release Activity of HIV-1 Vpu is Dependent on the AP-3 Pathway

Beth Noble, Children's Hospital, Los Angeles
Biomedical and Clinical Sciences

The assembly and release of retroviruses from human cells is inhibited by a dominant restriction factor. Different retroviruses have evolved mechanisms to counteract this factor, and this enhancement of virus release (EVR) activity is a property of the HIV-1 Vpu protein, the HIV-2 Env protein and the MLV Env protein. Although the EVR activity of Vpu was first described in 1989, the mechanism by which it enhances virus release is still unknown and represents a significant gap in our knowledge of HIV biology.

Previously, we have described a HeLa cell line (HeLa-T17) that has lost the ability to respond to Vpu, while remaining sensitive to stimulation by the HIV-2 Env. Confocal analysis revealed that the distribution of Vpu in these cells is aberrant, with a concentrated perinuclear distribution that colocalized with markers for the TGN and Golgi. To identify the defect in T17 cells we performed microarray analysis on wild-type and T17 HeLa cells, and observed that two different alkaline phosphatase (ALP) genes were highly up-regulated in the T17 cells. In yeast, ALP traffics from the Golgi to the large central vacuole through an interaction with AP-3, suggesting that the phenotype of T17 cells could arise from a defect in the equivalent mammalian pathway. To test this hypothesis, we performed RNAi knockdown of AP-3 in HeLa cells and found that this recapitulated the loss of Vpu activity. We also observed the same aberrant distribution of Vpu. Interestingly, in both T17 and AP-3 knock-down cells, the distribution of the CD63 marker was also altered. CD63 is present in late endosomes and lysosomes and uses AP-3 to traffic to these compartments from the TGN.

Previous studies have mapped the EVR function of Vpu to its membrane-spanning domain (MSD), a region known to have ion channel activity. Although no model has emerged to explain how ion channel activity could enhance HIV release this early observation has tended to focus studies on the MSD alone. We have now also found that the cytoplasmic tail of Vpu is essential for EVR activity, and mapped this to a [DE]XXXLq motif. Such sequences have previously been shown to interact with AP-1, AP-2, and AP-3 subunits. Mutation of residues within this motif both modified the protein's intracellular location and abrogated EVR activity, further suggesting that interaction with the correct cellular trafficking pathway is essential for Vpu's activity.