Oligomeric polymorphism of HIV-1 Vpu protein in lipid environment and in solution

Abstract

The HIV-1 encoded protein Vpu forms an oligomeric ion channel/pore in membranes and interacts with multiple host proteins to support virus lifecycle. However, Vpu molecular mechanisms are currently not well understood. The structures of full-length Vpu in its monomeric and oligomeric forms are unknown, although both the monomer and oligomer are deemed important. Here, we report on the diversity of Vpu oligomeric structures and how the environment affects the Vpu oligomer formation. We produced a uniquely designed MBP-Vpu chimera protein in E. coli in soluble form. We subjected this protein to analytical size exclusion chromatography (SEC) and negative staining electron microscopy (nsEM). Strikingly, we found that MBP-Vpu forms stable oligomers in solution, presumably driven by Vpu transmembrane domain self-association. Our coarse modeling suggests that these oligomers are pentamers, in agreement with the pentameric membrane-bound Vpu. To the best of our knowledge, this is the first observation of Vpu self-association out of lipid membrane environment. We further found that MBP-Vpu oligomer stability decreases when the protein was reconstituted in lipid membrane mimetics, such as β-DDM, and mixtures of lyso PC/PG or DHPC/DHPG. In these cases, significant oligomer heterogeneity was observed with oligomeric order lesser than that of MBP-Vpu oligomer in solution, but larger oligomers were observed as well. Importantly, we found that in lyso PC/PG, above certain protein concentration, MBP-Vpu forms linear array-like structures, which is also novel. Thus, our studies provide unique information about Vpu protein quaternary organization by capturing multiple Vpu oligomeric structures, which we believe are physiologically relevant.

Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu

“…Within the literature on Vpu, there is still ongoing debate on its oligomeric state. ,− There is currently no structural information on the full length Vpu forming an oligomer, but there has been one structure solved of full-length Vpu in its monomeric form. ,, Some previous studies have suggested that Vpu assembles into fixed pentameric complexes, whereas other studies found that Vpu assembles into a broader range of oligomers. ,− Interestingly, some previous studies suggested that higher solution ionic strengths drive Vpu to assemble into large oligomeric complexes …”

Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu

“…37,53,54 Some previous studies have suggested that Vpu assembles into fixed pentameric complexes, 55 whereas other studies found that Vpu assembles into a broader range of oligomers. 32,[56][57][58] Interestingly, some previous studies suggested that higher solution ionic strengths drive Vpu to assemble into large oligomeric complexes. 56 Additionally, the electrophysiology assays performed revealed that single channels of Vpu had different magnitudes of ion current across the bilayer (Figure S-7).…”