These cassettes contained the env genes of transmitted/founder HIV-1 cloned into pcDNA3.1 and contained the entire gp160 open reading frame, including 59 sequence extending to the Rev start codon. A portion of the amino acid alignments of the leader peptides of these sequences are shown in Table 1. Complete nucleotide sequences for these envelopes are accessible through GenBank , as are all transmitted and chronic envelopes used in the initial analysis that defined the position 12 signature. Characteristics of the study subjects at the time at which viruses were isolated, including patient viral load and Fiebig stage are detailed in Table S1 and in reference. Six of these envelopes were selected at random from the entire cohort because their leader peptide sequences contained a position 12 signature histidine or a similarly basic arginine at position 12. The remaining eight envelopes were specifically chosen because they are atypical in the transmitted envelope cohort in that they contain a variety of non-canonical residues at this position, including glutamine, glycine, asparagine, or a gap in the alignment. There was no phylogenetic clustering of envelopes bearing the signature distinct from those lacking the signature , suggesting that the ABT-263 phenotypic similarities among signature containing envelopes could not be explained by a shared evolutionary history. We began our exploration of the effects of the position 12 polymorphism on HIV biology by examining envelope translation. We transiently transfected Jurkat T cells with the 14 transmitted envelope Foretinib side effects constructs described previously. Forty-eight hours after transfection, we compared expression by Western blot,. Comparable transfection efficiencies were confirmed by cotransfection with a GFP expression plasmid. We observed higher levels of steady-state envelope expression by envelopes with a basic residue at position 12 in comparison to those lacking the signature. Using band densitometry to compare relative expression levels, we found that, on average, signature envelope expression was 2.5-fold higher than non-signature envelope expression; the difference in signal intensity between the two groups was highly significant. A trivial explanation for the apparent differences in expression between these groups of envelope constructs might be differences in affinity of the anti-gp120 antibody for the different envelope proteins. To control for this, we generated epitope-tagged versions of three signature and three non-signature envelopes utilizing a carboxy-terminus V5 epitope tag encoded within the pcDNA3.1 expression vector.