Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality

Abstract

Viruses often encode proteins with multiple functions due to their compact genomes. Existing approaches to identify functional residues largely rely on sequence conservation analysis. Inferring functional residues from sequence conservation can produce false positives, in which the conserved residues are functionally silent, or false negatives, where functional residues are not identified since they are species-specific and therefore non-conserved. Furthermore, the tedious process of constructing and analyzing individual mutations limits the number of residues that can be examined in a single study. Here, we developed a systematic approach to identify the functional residues of a viral protein by coupling experimental fitness profiling with protein stability prediction using the influenza virus polymerase PA subunit as the target protein. We identified a significant number of functional residues that were influenza type-specific and were evolutionarily non-conserved among different influenza types. Our results indicate that type-specific functional residues are prevalent and may not otherwise be identified by sequence conservation analysis alone. More importantly, this technique can be adapted to any viral (and potentially non-viral) protein where structural information is available.

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

“…High-throughput genetics have been applied to a number of viral, bacterial, and cellular proteins (16, 33–38, 111, 112). Here, point mutations were randomly introduced into segment 2 of influenza A/WSN/33 virus through error-prone PCR.…”

“…Here, point mutations were randomly introduced into segment 2 of influenza A/WSN/33 virus through error-prone PCR. To provide a more accurate quantification of the fitness effect of single mutations, we employed the “small-library” method that we recently developed (16). Nine small libraries were generated to cover all of segment 2 (see Fig.…”

“…A mutation might be deleterious because of structural or functional constraints (16, 42). We have recently demonstrated that coupling high-throughput genetics with mutant stability predictions can identify residues that are dominated by functional constraints (16).…”

“…It is also challenging for conservation-based methods to assess structural and functional constraints and to assign functionality at the single-residue level (Fig. 1) (16). Therefore, a more direct and systematic method needs to be used for the accurate identification and annotation of functional residues.…”

“…We previously developed a method to systematically identify functional residues by coupling experimental fitness measurement with protein stability prediction (16). Here, we extended this method to annotate functional residues in combination with structural comparison of homologous proteins.…”