Despite the use of combination antiretroviral drugs for the treatment of HIV-1 infection, the emergence of drug resistance remains
a problem. Resistance may be conferred either by a single mutation or a concerted set of mutations. The involvement
of multiple mutations can arise due to interactions between sites in the amino acid sequence as a consequence of the need to
maintain protein structure. To better understand the nature of such epistatic interactions, we reconstructed the ancestral sequences
of HIV-1’s Pol protein, and traced the evolutionary trajectories leading to mutations associated with drug resistance.
Using contemporary and ancestral sequences we modelled the effects of mutations (i.e. amino acid replacements) on protein
structure to understand the functional effects of residue changes. Although the majority of resistance-associated sequences
tend to destabilise the protein structure, we find there is a general tendency for protein stability to decrease across HIV-1’s
evolutionary history. That a similar pattern is observed in the non-drug resistance lineages indicates that non-resistant mutations,
for example, associated with escape from the immune response, also impacts on protein stability. Maintenance of optimal
protein structure therefore represents a major constraining factor to the evolution of HIV-1
