Constraints

The high rate of HIV-1 evolution contributes to immune escape, enables the virus to escape drug therapy, and may underlie the difficulty of producing an effective vaccine. Identifying constraints on HIV evolution is therefore of prime importance. To investigate this problem, we examined the relationships between sequence diversity, selection, and protein structure. We found that while there was an increase in sequence diversity over time, this variation had a tendency to be limited to specific structural regions. When individual sites were analyzed, there was, in contrast, substantial and widespread evolutionary constraint over gag and env. This constraint was present even in the highly variable envelope proteins. The evolutionary significance of an individual site is indicated by the change in selection pressure along the time course: increasing entropy indicates that the site is evolving predominantly in a more “clock”-like manner, low entropy values with no increase indicate a high degree of constraint, and high entropy values indicate a lack of constraint. Few sites display high degrees of turnover. Mapping these sites onto the three-dimensional protein structure, we found a significant difference between evolutionary rates for regions buried in the core of the protein and those on the surface. This constraint did not change over the time period analyzed and was not subtype dependent, as similar results were found for subtypes B and C. This link between sequence and structure not only demon-strates the limits of recent HIV-1 evolution but also highlights the origins of evolutionary constraint on vira