HIV-1 Env Plasmid Construction and Characterization

The vesicular stomatitis virus G (VSV-G)-encoding plasmid was previously described [20 (link)]. The sequence of full-length clade B HIV-1_JRFL Env and clade A HIV-1_BG505 (N332) were codon-optimized (GenScript) and cloned into the pcDNA3.1(-) expression plasmid (Invitrogen, Rockford, IL, USA) [21 (link),22 (link)]. The plasmids expressing full-length S375W or cleavage-deficient (Cl-) (R508S/R511S) HIV-1_JRFL Env were previously described [9 (link)]. The R508S/R511S mutations were introduced into the full-length HIV-1_JRFL Env S375W by overlapping the PCR to generate the S375W Cl- mutant. The presence of the desired mutations was confirmed by automated DNA sequencing. pSVIIIenv-expressing full-length HIV-1_YU2 Env and Tat-expressing plasmid pLTR-Tat were previously described [23 (link)]. Transmitted/Founder (T/F) and chronic infectious molecular clones (IMCs) of patients CH058, CH077, RHGA, STCO1, and ZM246F-10 were inferred, constructed, and biologically characterized, as previously described [24 (link),25 (link),26 (link),27 (link)]. The IMCs encoding for the HIV-1 reference strains JRFL, YU2, and BG505 (T332N) were described elsewhere [28 (link),29 (link),30 (link),31 (link)]. Expression plasmids for CH040, RHGA, STCO1, CH198, and ZM246F-10 Env were generated by PCR amplification, inserting the respective Env genes into pCAGGS via EcoRI/XmaI.