Quikchange 2 xl site directed mutagenesis protocol

To test the replication capacity of NL4.3 viruses expressing wild-type HIV-1_JR-FL Env or the adapted virus Envs, we introduced mutations into the HIV-1_NL4.3(JR-FL) proviral DNA and transfected them into target cells. Site-directed mutagenesis was performed using the QuikChange II XL Site-Directed Mutagenesis protocol (Agilent) and the PfuUltra Hotstart DNA Polymerase (Agilent). Approximately 4×10⁴ Cf2Th-CD4/CCR5 or R5-Low cells preincubated with 50 or 100 ng/mL doxycycline were transfected with 100 μg proviral DNA and passaged for 3 weeks. Supernatants of each cell sample were collected regularly throughout the culture period and evaluated for RT activity.

The vesicular stomatitis virus G (VSV-G)-encoding plasmid was previously described ^{86 (link)}. The plasmid pSVIIIenv expressing the full-length HIV-1_YU2 Env (WT and its S375W counterpart) and the Tat-expressing plasmid (pLTR-Tat) were previously reported ^{20 (link),50 (link)}. The infectious molecular clones (IMCs) HIV-1_{NL4.3.ADA.GFP WT} and HIV-1_{NL4.3.ADA.GFP S375H}, as well as the SHIV construct SHIV₄₀₁₀₀ were also previously described ^{15 (link),51 (link),87 (link)}. The following mutations (H375S, H61Y, Q105H, V108I, N474D, I475M and K476R) were introduced individually or in combination into the SHIV₄₀₁₀₀ construct using the QuikChange II XL site-directed mutagenesis protocol (Agilent Technologies, Santa Clara, CA, USA) to generate the SHIV_{40100 LM-HS} construct. The presence of the desired mutations was determined by automated DNA sequencing