Exploring the Mechanisms by which Small Molecule Nef Inhibitors Suppress HIV‐1 Infectivity

Abstract

Nef is an HIV‐1 accessory factor essential for viral pathogenesis and immune escape of HIV‐infected cells. Recently, our group reported the discovery of small molecule Nef antagonists that reverse Nef‐mediated downregulation of MHC‐I and restore the anti‐HIV cytotoxic T cell response. These compounds also inhibit Nef's ability to enhance the inherent infectivity of HIV‐1 virions, although their mechanism of action in this regard is not known. Recent studies have identified the host cell factor SERINC5 (S5) as a suppressor of HIV‐1 infectivity that is countermanded by Nef. Here we investigated whether our Nef inhibitors suppress viral infectivity through a S5‐dependent mechanism. We first confirmed that expression of S5 within viral producer cells results in Nef‐reversible inhibition of HIV‐1 infectivity. For these experiments, we co‐transfected 293T cells with wild‐type and ΔNef HIV‐1 proviral DNA along with various amounts of a S5 expression plasmid. The infectivity of each resulting virus was then quantified using the luciferase‐based HIV‐1 reporter cell line, TZM‐bl. As expected, co‐expression of S5 resulted in suppression of HIV‐1 infectivity. This S5 effect was reduced by Nef in cells producing wild‐type HIV‐1, but not in cells producing the ΔNef virus. Co‐expression of Nef from a separate expression plasmid during the production of the ΔNef virus completely reversed S5 inhibition of viral infectivity, demonstrating Nef dependence in this system. To determine whether our inhibitors block Nef‐enhanced infectivity in a S5‐specific manner, we treated cells producing wild‐type or ΔNef virus in the presence or absence of S5 with either DMSO or the diphenylpyrazolo Nef inhibitor, B9 (3 μM). This compound inhibited the infectivity of wild‐type but not ΔNef HIV‐1 at the producer cell level, consistent with direct action on Nef. However, the effect of the compound was independent of S5 expression, suggesting that mechanisms in addition to S5 may contribute to inhibition of HIV‐1 infectivity by B9 through Nef.Support or Funding InformationT32 AI65380 (R.P.S.)R01 AI057083, R42 GM112516 (T.E.S.)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.