Intact mitochondria were incubated with proteinase to find the mitochondrial outside membrane necessary protein Mfn1

In the case of the HLA-B*44 restricted PR 34-42 epitope EEMNLPGRW, the escape mutation PR D35E is predicted by the MHC binding predictor, NetMHCPan, to have a dramatic negative impact on peptide affinity to HLA-B*4402, possibly diminishing the presentation potential of the epitope on HLA- B*4402. Using statistical methods to infer mutations significantly associated with CTL immunity escape is a challenging task as most public available sequences are rarely annotated with the patient’s HLA genotype. This is especially true of sequences obtained from patients that are not treatment naı¨ve. Fortunately, other researchers have found relationships between amino acid substitutions in HIV-1 proteins and HLA allotypes and can potentially be used to provide HLA annotation from HIV-1 protein sequences. Here, it was discovered that there are possible interactions between a common and important protease inhibitor resistance mutation, L90M, and the HLA subtypes B*15, B*48 and potentially A*32. Using the aforementioned data correlating relating amino acid substitutions with HLA subtype, patients were assigned HLA subtypes and the frequencies of L90M were compared between sequence sets of patients that are HLA B*15/ B*48/A*32 positive and those that are not. It is true that binding affinity does not necessarily constitute an HLA binder to be an epitope, but affinity does improve the stability of the peptide-HLA complex and increases the chance of contact with the appropriate CTL T-Cell Receptor. Inferring a mechanism of escape cannot easily be accomplished computationally. Other researchers have shown in detail general mechanisms of escape that can include diminished interaction of a presented CTL epitope with a complementary cytotoxic T-cell receptor. Another possible method of escape, is the induction of a highly active proteasomal cleavage site within the epitope. The mechanism behind the CTL immunity escape of T9F provided by the PR I93L mutation is unknown. Indeed, it is shown that the CTL escape provided by the PR I93L has higher avidity in terms of ELISPOT analysis. However, as shown in Table 6, I93L has a significant impact on proteasomal cleavage prediction score for position 93 in PR. This may cause the premature destruction of the epitope before it is loaded onto HLA class I. A significantly higher predicted proteasomal cleavage score for PR 92 was obtained by the Q92K substitution, but this score is very close to the threshold of 0.500 and could very well be a false positive. In conjunction with I93L, the proteasomal score was further increased to 0.735. However, the co-occurrence of Q92K and I93L is severely diminished in HIV PR sequences. It seemed unlikely that another cleavage site would be necessary in close proximity of a highly probable proteasomal cleavage site. No significant diminished cooccurrence of Q92R and I93L was observed. Both the Q92K/R mutations did not have a profound negative impact on HLA binding affinity and did not change the rank percentage from the NetCTLPan results. It was therefore concluded a likely mechanism of escape would be through attenuated or abrogated interaction with a complementary cytotoxic T-cell receptor. This enrichment of substitutions internal to the predicted epitopes provide further evidence of a putative epitope in the region PR 89-97. Further evidence are deduced from NetChop Quinolinic acid results that suggest a highly probable proteasomal cleavage site near the Cterminus of M9L, which is essential, since trimming of proteasomal fragments usually only occur at the N-terminus of the peptide. The predicted creation of a proteasomal cleavage site by Q92K, which is internal to both LM9L and M10F may be an escape mutation. Alternatively, when considering Q92R, it may attenuate interaction between the epitope and complementary cytotoxic Tcell receptor. Even though the existence of LM9L and M10F may be proven with subsequent studies, there is no indication here of the immunogenicity of the epitope. The L90M mutation does appear to occur at lower frequencies, but the reason for the lack of complete elimination need to be investigated. Various issues, including, but not limited to, immunological hierarchies and false assignment of a sequence set to HLA subtypes B*15+ and B*48 could play a role. The contribution of L90M to viral fitness may also outweigh the immunological pressure induced by L90M. Here no experimental evidence is supplied, but the multitude of statistical analysis and epitope prediction results do support the conclusion that there is potential for interaction between protease inhibitor resistance mutations and immunological selective pressure in hosts positive for HLA*B*15 and HLA-B*48 subtypes. Further research into validation of the results presented here are encouraged. It should be emphasized that if either M9L or M10F is shown in subsequent studies to indeed be immunogenic CTL epitopes, it could be prudent to include PIs in treatment regiments such as Saquinavir or Nelfinavir for which L90M is a major resistance mutation.

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