Tetherin antagonism by Nef depends on clathrin and dynamin 2.
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Abstract
<p>(A) Dominant-negative mutants of AP180 and Dyn2 were tested for disruption of Nef-mediated tetherin downregulation. 293T cells expressing HA-tagged rhesus tetherin were transfected with constructs expressing Nef, or empty vector, and constructs expressing AP180C, Dyn1K44A, Dyn2K44A and Dyn2. Surface expression of tetherin was measured by HA staining, and the percentage of tetherin expressed on the cell surface was determined by dividing the MFI of tetherin on cells expressing the dominant-negative mutants by the MFI of tetherin on cells transfected with empty vectors. (B) The dominant-negative mutants of AP180 and Dyn2 were also tested for their effects on Nef-mediated virus release. 293T cells were transfected with SIV<sub>mac</sub>239 or SIV<sub>mac</sub>239 Δ<i>nef</i> proviral DNA, and constructs expressing rhesus tetherin, and either AP180C, Dyn1K44A, Dyn2K44A, Dyn2 or empty vector. Virus release was measured by SIV p27 antigen-capture ELISA and expressed as the percentage of maximal release in the absence of tetherin. (C) Protein expression for Nef, tetherin, AP180C, Dyn1K44A, Dyn2K44A and Dyn2 was verified by western blot analyses using endogenous β-actin as a loading control. (D) Replication of wild-type SIV (WT SIV) versus SIV Δ<i>nef</i> in the presence and absence of Dynasore. 221 T cells were infected with 20 ng p27 of wild-type SIV or SIV Δ<i>nef</i>. Twenty-four hours post-infection, cells were treated with IFNα (100 U). Eight hours later, Dynasore (20 µM) was added to one of the cultures. Supernatants were collected at the indicated time points and virus replication was determined by SIV p27 antigen-capture ELISA.</p