The RT coding region of the pol gene of plasmids encoding the HIV-1NL4–3 with a deletion in env and the luciferase gene in place of nef (NLdE-luc) (48 (link)) or full-length HIV-1NL4–3 or HIV-1NL4-BAL proviruses were amplified by PCR and were subcloned into pCR2.1-TOPO (Life Technologies). RT mutations E138K, V179I, Y181C, Y181V, E138K/V179I, V179I/Y181C, and V179I/Y181V were introduced into these plasmids using the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies). After verification by Sanger sequencing, the mutated fragment was cloned into the plasmids.
WT and mutant viruses were produced by transfection of 293T cells using Lipofectamine 2000 (Thermo Fisher Scientific). NLdE-luc viruses were pseudotyped with VSV-G, using the pLVSV-G plasmid. Virus-containing supernatants were harvested 48h after transfection.
WT and mutant viruses were produced by transfection of 293T cells using Lipofectamine 2000 (Thermo Fisher Scientific). NLdE-luc viruses were pseudotyped with VSV-G, using the pLVSV-G plasmid. Virus-containing supernatants were harvested 48h after transfection.
