We probed Western blots of transfections of these constructs with an anti-V5 antibody, and again observed a trend to reduced BEZ235 expression in non-signature envelopes. The acutely transmitted envelopes described above were all isolated from different patients, and hence have significant sequence heterogeneity throughout the envelope protein. To demonstrate that differences in expression were due specifically to polymorphisms at position 12, we arbitrarily selected three basic residue-bearing envelopes, AA01, AA02 and AA03, and used sitedirected PCR mutagenesis to convert the histidine or arginine residue into a glutamine, a relatively common amino acid variant found at this position. We cloned these position 12 envelope mutants into pcDNA3.1, and confirmed the integrity of the fulllength envelopes by sequencing. We expressed these position 12 mutant envelopes, and the parent envelopes, in Jurkat cells and observed an over 60% decrement in expression associated with the point mutations. We performed the converse experiment using three nonsignature envelopes, AC01, AC02 and AC03, and replaced the glutamine or alignment gap at position 12 of these leader peptides with histidine. We observed an approximately three-fold increase in steady-state protein expression of these envelopes after mutation of this residue , confirming that a basic position 12 residue is important for optimal expression of envelope in lymphocytes. Non-signature leader peptide polymorphisms diminish ER targeting A plausible explanation for the differences in steady-state protein expression among position 12 variants is that position 12 histidine or arginine is critical for the trafficking efficiency of the leader peptide in Vismodegib certain cell types, and loss of this basic residue results in misdirected nascent peptide and loss of fully synthesized envelope. In general, the success of appropriate targeting of secreted or membrane-bound proteins to the endoplasmic reticulum appears to vary significantly between leader peptides, with misdirected proteins locating to the cytoplasm where they are degraded. Furthermore, polymorphisms within endoplasmic reticulum targeting sequences are relevant for disease states: an inherited mutation in the leader peptide sequence of factor VII has been shown to result in mistargeting of the nascent polypeptide to the cytoplasm and a reduction in overall expression of the mature Factor VII protein; this endoplasmic reticulum-trafficking abnormality underlies the heritable coagulation disorder associated with this mutation.