Association of Vpu-Binding Protein with Microtubules and Vpu-Dependent Redistribution of HIV-1 Gag Protein

Abstract

The efficient exit of HIV-1 particles from cells requires the action of the viral encoded protein Vpu. Vpu-binding protein (Ubp) is a cellular protein that interacts with both Vpu and the major structural component of the viral capsid (Gag) and appears to affect the efficiency of particle exit. Elucidation of the function of Ubp and characterization of the spatial distribution of Ubp may provide information pertinent to understanding the role of Ubp in virus replication. To investigate the subcellular location of Ubp, and to see whether Vpu affects the intracellular distribution of Gag, we carried out immunofluorescence localization in conjunction with confocal microscopy. Based on this analysis Ubp is present in both the nucleus and the cytoplasm. In the cytoplasm, Ubp appeared to be associated with microtubules as evidenced by cofluorescence with tubulin in the absence and in the presence of colchicine. However, cytoskeletal isolation and detergent extraction of cells resulted in association of Ubp with the soluble fractions, indicating that Ubp is not in tight association with microtubules. Moreover, flotation gradient analysis demonstrated that Ubp is cytoplasmic and not stably associated with the plasma membrane. Interestingly, expression of Vpu in cells resulted in redistribution of both Ubp and Gag to a location near the periphery of the cell. The effect of Vpu on both Ubp and Gag protein has implications for Vpu-mediated particle exit from cells.

Dynamic Fluorescent Imaging of Human Immunodeficiency Virus Type 1 Gag in Live Cells by Biarsenical Labeling

“…Our FlAsH-labeled imaging of Gag expressed from a fulllength HIV-1 pol Ϫ molecular clone found that Gag assembling into virions was distributed similar to Gag-TC and Gag. Thus, the expression of other viral proteins that are involved in assembly and have been previously implicated in Gag localization, primarily Env (36) and Vpu (22), the presence of cisacting RNA sequences (68), or levels of Gag expression did not appreciably alter the Gag localization pattern in HeLa cells.…”

“…While Gag is sufficient for particle formation, other elements in HIV-1 might influence Gag localization and assembly. HIV-1 Env can direct HIV-1 budding to the basolateral surface in certain polarized cell systems (36) and Vpu, which increases virus release (3), can influence Gag localization (22). Also, RNA signals in the genomic RNA might target assembly by RNA trafficking (68).…”

Control of Androgen Receptor Signaling in Prostate Cancer by the Cochaperone Small Glutamine–Rich Tetratricopeptide Repeat Containing Protein α

“…Interaction of aSGT with the HIV-1 encoded Vpu protein facilitates redistribution of HIV Gag to points of accumulation at the plasma membrane, resulting in more efficient budding of viral particles from infected cells (31). aSGT may also aid in the transport of the growth hormone receptor from the endoplasmic reticulum to the plasma membrane (34), and the secretion and activity of myostatin (32).…”

“…Cytoplasmic aSGT is associated with microtubules and actin filaments, but does not form an integral part of the cytoskeleton (31). Microtubule-targeting by aSGT could contribute to cytoplasmic retention of apo-AR, and may facilitate efficient exchange of aSGT for dynein-linked TPR proteins following ligand binding and the subsequent receptor redistribution to the nucleus.…”

“…This hypothesis is consistent with the emerging dependence of steroid receptors on an intact cytoskeleton for nuclear translocation (10,12). Importantly, the redistribution of aSGT observed with the collapse of the microtubule network during cell division and by chemical means (31), suggests that microtubule-targeting agents will disrupt aSGT regulation of its clients. This may explain the efficacy of the chemotherapeutic agent, docetaxel, in patients with PCa following the failure of conventional androgen ablation (41).…”