FIG. 1.

Construction of recombinant RV-expressing SHIV 89.6P envelope proteins. (A) Schematic representation of membrane-associated trimeric gp160. The gp120 and gp41 ecto- and TM domains (ED and TM) are fused in frame to the CD of RV G protein (RVG). The envelope protein described herein is derived from the pathogenic SHIV-89.6P strain. The schematic was adapted from Binley and Moore (5). (B) BNSP-333 is an RV vaccine strain-based vector containing a minimal RV transcription unit between the N and P genes and an arginine-to-glutamic acid substitution at position 333 of RV G protein. A BNSP vector without the 333 mutation was the target to introduce the cDNA sequence encoding SHIV-89.6P-RVG envelope by using the BsiWI and NheI sites, resulting in BNSP-89.6P-RVG. BNSP-333 was the target for introduction of 89.6P-RVG SOS envelope to create BNSP-333-89.6P-RVG-SOS. In the SOS construct, site-directed mutagenesis was used to introduce cysteine residues at positions 499 (Ala→Cys) and 603 (Thr→Cys) of 89.6P gp160, which allows an intermolecular disulfide bond to form between gp120 and the gp41 ectodomain. All viruses were recovered by standard methods. The presence of the 333 mutation has been shown to have no effect on virus growth or immunogenicity (51).