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 possess 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.