Bacteria-Based Analysis of HIV-1 Vpu Channel Activity

Abstract

HIV-1 Vpu is a small, single-span membrane protein with two attributed functions that increase the virus' pathogenicity: degradation of CD4 and inactivation of BST-2. Vpu has also been shown to posses ion channel activity, yet no correlation has been found between this attribute and Vpu's role in viral release. In order to gain further insight into the channel activity of Vpu we devised two bacteria-based assays that can examine this function in detail. In the first assay Vpu was over-expressed, such that it was deleterious to bacterial growth due to membrane permeabilization. In the second and more sensitive assay, the channel was expressed at low levels in K+ transport deficient bacteria. Consequently, Vpu expression enabled the bacteria to grow at otherwise non permissive low K+ concentrations. Hence, Vpu had the opposite impact on bacterial growth in the two assays: detrimental in the former and beneficial in the latter. Furthermore, we show that channel blockers also behave reciprocally in the two assays, promoting growth in the first assay and hindering it in the second assay. Taken together, we investigated Vpu's channel activity in a rapid and quantitative approach that is amenable to high-throughput screening, in search of novel blockers.

Potential Viroporin Candidates From Pathogenic Viruses Using Bacteria-Based Bioassays

“…The secretion was subsequently directed by the signal peptide of MBP, placing the amino terminus of the viral protein in the bacterial periplasm. Similar constructs were shown to be successful with other viroporins such as Influenza M2 [58,59,60] and HIV vpu [61]. Finally, membrane incorporation could be confirmed using a genetic screen upon expression of the chimera in mal E - bacteria.…”

Vpu Protein: The Viroporin Encoded by HIV-1

“…Other recent studies have proposed alternative cellular models that are suitable for screening of inhibitors of full-length Vpu. Notably, the K+ dependent growth stimulation caused by Vpu protein in S. cerevisiae and also in Escherichia coli provides efficient cellular platforms to develop high–throughput screening assays [ 41 , 113 ]. Finally, Xenopus oocytes have also been used for drug testing for anti-Vpu activity by voltage clamp assays [ 114 ].…”

Pathophysiological Consequences of Calcium-Conducting Viroporins

“…Increased viroporin activity allows hygromycin B to enter the cell, thereby causing decreased 35 S-methionine incorporation (4, 22). Other small molecules that have been used include β-galactosidase substrates, fluorescent molecules (FITC dextran, propidium iodide), and fluorescent redox sensors (4, 23–27). Mammalian cell permeability assays are conducted in a similar fashion, such that virus infection or viroporin expression increases the cell’s permeability to small molecules.…”

“…Therefore, to demonstrate specificity to viroporin activity rather than cell stress or cytotoxicity from overexpression of transmembrane proteins, experiments must also be done to test for activity of the candidate viroporin bearing mutations of the amphipathic α-helix and clustered basic residues (28). These relatively simple assays serve as an important foundation for understanding viroporin structure/function relationships, as a rapid means for screening for mutations that disrupt viroporin activity, and as a basis for drug screens to identify compounds that may act as viroporin channel inhibitors (23).…”

“…This is typically accomplished using one of the permeability assays presented above. Additionally, a small molecule inhibitor of viroporin activity (typically identified using one of the bacterial or liposome permeability assays described above) can be used to demonstrate specificity for viroporin function (23). Second, it is critical to use electrophysiology and/or fluorescent imaging to demonstrate that the viroporin domain is responsible for ion conductance or alteration of ion gradients in cells, and that this activity is prevented by the same mutations or drugs that abolish viroporin activity in the permeability assays.…”