Full length env sequencing

SIVmac239 gp160 sequences were obtained from infected plasma samples as previously described (3, 53). Viral RNA was isolated from plasma samples using a QIAamp viral RNA kit (Qiagen). Isolated RNA was reverse transcribed to cDNA using SuperScript III reverse transciptase (Invitrogen). cDNA synthesis was performed using 1× RT buffer, 0.5 mM dNTP (New England BioLabs), 5 mM dithiothreitol (DTT), 40 units RNaseOut (Invitrogen), 200 units of SuperScript III reverse transcriptase, and 0.25 mM of SIVEnvR1 antisense primer (5’ TGT AAT AAA TCC CTT CCA GTC CCC CC 3’). RNA, SIVEnvR1, and dNTPs were heated at 65°C for 5 minutes followed by 1 minute at 4C. RT Buffer, dNTP, DTT, and RNaseOut were then added and the reaction mixture was incubated for one hour at 50°C followed by 55°C for one hour. The reaction mixture was heat inactivated for 15 minutes at 70°C. 1 uL of RNase H (New England Biolabs) was added and the reaction mixture was incubated for 20 minutes at 37°C. SIVmac239 gp160 sequences were PCR amplified using the Platinum Taq (Invitrogen) with the following mixture conditions: 1× buffer, 2 mM MgCl2, 0.2 mM dNTP, 0.2 uM of sense and antisense primer, and 0.025 units of Platinum Taq polymerase. For the first round of PCR, the primers used were SIVEnvF1 (5’ CCT CCC CCT CCA GGA CTA GC 3’) and SIVEnvR1. The primers used for the second round were SIVEnvF2 (5’ GTT TCT TTA TAA TAG ACA TGG AGA CAC CCT TGA GGG AGC 3’) and SIVEnvR2 (5’ GTT TCT TAT GAG ACA TGT CTA TTG CCA ATT TGT A 3’). Cycle conditions were 94°C for 2 minutes, then 35 cycles of 94°C for 15 seconds, 55°C for 30 seconds, and 68°C for 4 minutes, and a final extension of 68°C for 10 minutes. 2 uL of the first PCR was used as the template for the second PCR which used the same conditions, but run for a total of 45 cycles. After the second PCR, 2.5 units of Platinum Taq polymerase were added and the reaction mixture was incubated at 72°C for 15 minutes. The PCR product was gel extracted and eluted with 20 uL of elution buffer (Qiagen). The gel purified PCR product was then cloned into the pcDNA3.1/V5-His vector by Topo TA cloning per manufacturer’s instructions. Cloned vectors were then transformed using Top10 competent cells. Sequencing of the SIVmac239 env was performed using the following primers: SIVEnvF3 (5’ GGT AAT CAT ATC TAT AAT AGA CAT GGA GAC AC 3’), mac239EnvSeqF1 (5’ GGG GAA CAA CTC AGT GCC TAC C 3’), mac239EnvSeqF3 (5’ GCA CCT CCA GGT TAT GCT TTG C 3’), mac239EnvSeqF5 (5’ GCA GAG GAG AGT TCC TCT AC 3’), mac239EnvSeqF7 (5’ GCA ACA GCT GTT GGA CGT GG 3’), mac239EnvSeqF9 (5’ CCA GCA GAC CCA TAT CCA ACA GG 3’). Sequences were aligned to wild-type SIVmac239 gp160 to identify mutations using the Los Alamos Highlighter tool (https://www.hiv.lanl.gov/content/sequence/HIGHLIGHT/highlighter_top.html).