Distinguishing functional amino acid covariation from background linkage disequilibrium in HIV protease and reverse transcriptase.

Correlated amino acid mutation analysis has been widely used to infer functional interactions between different sites in a protein. However, this analysis can be confounded by important phylogenetic effects broadly classifiable as background linkage disequilibrium (BLD). We have systematically separated the covariation induced by selective interactions between amino acids from background LD, using synonymous (S) vs. amino acid (A) mutations. Covariation between two amino acid mutations, (A,A), can be affected by selective interactions between amino acids, whereas covariation within (A,S) pairs or (S,S) pairs cannot. Our analysis of the pol gene--including the protease and the reverse transcriptase genes--in HIV reveals that (A,A) covariation levels are enormously higher than for either (A,S) or (S,S), and thus cannot be attributed to phylogenetic effects. The magnitude of these effects suggests that a large portion of (A,A) covariation in the HIV pol gene results from selective interactions. Inspection of the most prominent (A,A) interactions in the HIV pol gene showed that they are known sites of independently identified drug resistance mutations, and physically cluster around the drug binding site. Moreover, the specific set of (A,A) interaction pairs was reproducible in different drug treatment studies, and vanished in untreated HIV samples. The (S,S) covariation curves measured a low but detectable level of background LD in HIV.