Lentiviruses encode a number of unique accessory proteins that are important for viral replication and pathogenesis, but are not encoded by other retroviruses. The versatility of HIV accessory proteins arise from their ability to function as adaptor molecules that connect various viral and cellular proteins to pre-existing cellular pathways, modulate these pathways and control processes important for viral replication. Thus, protein-protein interactions are an important aspect of the functioning of these proteins. The Vpu protein of HIV-1 is known to bind CD4 and βTRCP, the latter being a component of the E3 ubiquitin ligase complex. This association is instrumental in dislocation of CD4 from the ER, its ubiquitination and subsequent degradation by the proteosome . The other function of Vpu is to promote the release of progeny viruses from infected cells. This appears to be dependent upon the ability of Vpu to form oligomeric complexes with an ion channel activity in cellular membranes .
The nature of the Vpu oligomer is important due to its functional significance in virion release. An earlier study used chemical cross-linking to demonstrate the oligomerization of Vpu . Here we have used various genetic, biochemical and biophysical approaches to characterize the oligomerization of Vpu in vitro and in vivo. Molecular dynamic simulations and conductance studies have shown that the Vpu TM domain is sufficient for its ion channel activity  and the pattern of channel activity is characteristic of the self-assembly of conductive oligomers in the membrane . This suggested that the TM domain of Vpu would also be required for its oligomerization. We provide here direct evidence that the hydrophobic N-terminal TM domain, and not the charged cytoplasmic domain, is critical for Vpu oligomerization. The two-hybrid and MBP pull-down analyses further showed that full-length Vpu proteins as well as their TM domains derived from two different HIV-1 subtypes interacted efficiently with each other. The isolated TM domains also showed stable interaction with the full-length Vpu protein. Molecular-dynamic simulations of ion channels formed by the Vpu TM domain predict the most favourable channel assembly to be a pentamer, but higher and lower oligomeric species were also predicted [23, 28]. Recently, Becker et al  have used synthetic proteins containing a carrier template to which four or five peptides corresponding to the Vpu TM domain were attached, to demonstrate ion channel formation by oligomerization of the TM domain. In our analysis, multimeric species, including pentamers were evident on gel electrophoresis of the in vitro synthesized Vpu proteins and their TM domains. Further, molecular sizing of in vitro synthesized Vpu by gel permeation chromatography clearly showed it to assemble into a pentameric species. Thus, while complementing earlier studies [23, 28, 29] we provide direct evidence for pentamerization of the full-length Vpu protein as well as its TM domain. In vitro synthesized Vpu proteins, in the absence of other cellular components, demonstrated a stable equilibrium between monomers and pentamers. This demonstrates the inherent ability of Vpu to oligomerize and pentamers appear to be the thermodynamically most stable form of these oligomers.
Earlier studies have shown Vpu to be localized to the perinuclear region of the cell that includes the ER and Golgi [16, 20]. Using CD4-Vpu fusion proteins and endoglycosidase H resistance, an earlier study has provided evidence for Vpu movement beyond the ER . A similar fusion protein was also used to tease out the apoptotic pathway . Recently, the imaging of a Vpu-EGFP fusion protein has also localized it to the ER, Golgi and plasma membrane . So, there is evidence that Vpu has the ability to be transported to post-ER membranes. Several models have been proposed for a role of the Vpu channel in the budding of new virions . It has been suggested that oligomerization of Vpu at the ER could form conducting channels leading to a collapse in the membrane potential across the ER cisternae and acceleration of membrane fusion and protein traffic in the exocytic pathway. Alternatively, at the ER/mitochondrial junctions, Vpu is proposed to collapse of the mitochondrial membrane potential and promote apoptosis. It is also possible that Vpu channels in the plasma membrane may attenuate the cell resting potential, promoting the fusion and release of new virions. We have used confocal microscopy and FRET analysis to test these models. Using transfected fluorescent protein-tagged Vpu fusion proteins and subcellular markers we show that Vpu localizes to the ER and Golgi regions but not to the mitochondria. In the absence of its mitochondrial localization, it would be difficult to support a direct effect of Vpu on the mitochondrial pore transition complex or transmembrane potential . One possibility is the effect of Vpu channels on Ca2+ release from its intracellular stores in the ER . The FRET analysis in this study showed no oligomerization of Vpu associated with the ER, arguing against the role of ER directly or indirectly in this process.
Oligomerization of Vpu was observed by FRET in structures that were distal to the ER. While these structures could not be identified positively in the same cell due to the technical limitations, separate stainings in two different cell types (COS-1 and U2-OS) suggested that these structures might either be Golgi or vesicles associated with exocytic protein transport. The Vpu ion channel activity would be detrimental to ER function, inducing ER toxicity, stress and apoptosis of the infected cell. This would go against the plan of survival of productively infected cells followed by retroviruses and so nicely exemplified by HIV . It would therefore make sense for Vpu oligomerization to occur downstream of the ER, enroute to the plasma membrane. Our FRET analysis in live cells supports this model. Since the cytoplasmic and not the transmembrane domain of Vpu is required for CD4 relocation from the ER and its subsequent degradation , this scheme would not affect the CD4 downmodulation function of Vpu. However, contrasting results have recently been presented wherein Vpu with a scrambled transmembrane domain was unable to downmodulate CD4 from the surface of transfected cells . Whether this is due to the inability of mutant Vpu to oligomerize, or due to an altered protein structure or its arrangement in the membrane, remains to be seen.