2.1. Plasmids, Antibodies, and Chemicals

The full-length, infectious HIV-1 molecular clone pNL4-3 and the Vpu-defective counterparts pNL4-3delVpu and pNL4-3Udel have been described previously [60,61,62]. pNL4-3delVpu and pNL4-3Udel were kindly provided by K. Strebel (National Institutes of Health (NIH), Bethesda, MD, USA). Vectors expressing tetherin derivatives bearing an N-terminal hemagglutinin (HA) epitope tag or an HA tag inserted at residue 154 in the extracellular CC domain of tetherin have been described previously [1,15] and were generously provided by P. Bieniasz (The Rockefeller University, New York, NY, USA). PCR-based mutagenesis was used to introduce Ala substitutions at either one or both of two Asn residues (N65A, N92A, and N65,92A) that are targets for N-linked glycosylation. Anti-HA antiserum, kifunensine, and tunicamycin were purchased from Sigma (St. Louis, MO, USA). Alexa Fluor 488 or 594-conjugated secondary antibodies were from Invitrogen (Grand Island, NY, USA). Anti-Vpu, anti-tetherin, and anti-HIV-1 immunoglobulins (Ig) were obtained from the NIH AIDS Research and Reference Reagent Program.