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

Wu NC, Olson CA, Du Y, Le S, Tran K, Remenyi R, Gong D, Al-Mawsawi LQ, Qi H, Wu TT, Sun R - PLoS Genet. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, United States of America,; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Structure-function relationship of residue 281.(A) The interaction of influenza A PA with the RNA phosphate backbone located between base 3 and 4 is shown. RNA is colored in green. PA is colored in cyan. Hydrogen bonds are represented by dotted black lines. Numbering of residue position is based on A/WSN/33. Conversion of residue position numbering is described in S3 Table. (B) The interaction of influenza B PA with the RNA phosphate backbone located between base 3 and 4 is shown. RNA is colored in green. PA is colored in cyan. Hydrogen bonds are represented by dotted black lines. Numbering of residue position is based on A/WSN/33. Conversion of residue position numbering is described in S3 Table.
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pgen.1005310.g007: Structure-function relationship of residue 281.(A) The interaction of influenza A PA with the RNA phosphate backbone located between base 3 and 4 is shown. RNA is colored in green. PA is colored in cyan. Hydrogen bonds are represented by dotted black lines. Numbering of residue position is based on A/WSN/33. Conversion of residue position numbering is described in S3 Table. (B) The interaction of influenza B PA with the RNA phosphate backbone located between base 3 and 4 is shown. RNA is colored in green. PA is colored in cyan. Hydrogen bonds are represented by dotted black lines. Numbering of residue position is based on A/WSN/33. Conversion of residue position numbering is described in S3 Table.

Mentions: We aimed to further investigate the structural basis of type-specific functional residues. The RNA binding function is required for viral replication and is conserved among type A and B influenza viruses. In the validation above, substituting lysine [K] to isoleucine [I] at residue 281 completely abolished the polymerase activity. This highlights the importance of the hydrogen bond formed between K281 and the RNA phosphate backbone in the influenza A virus (Fig 7A and boxed in S14 Fig). However, PA K281 is not conserved between type A and B influenza viruses. All influenza B viruses carry an alanine [A] at residue 281, which is unable to form a hydrogen bond with the RNA backbone. The critical hydrogen bond mediated by K281 in influenza A virus is replaced by the main chain of G569 in the influenza B virus (Fig 7B and boxed in S15 Fig). In fact, structural analysis indicates that type A [64] and B [72] influenza viruses display different hydrogen bonding patterns between PA and the viral RNA promoter (S14 Fig and S15 Fig). Thus, conserved functions may not necessarily require conserved functional residues.


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

Wu NC, Olson CA, Du Y, Le S, Tran K, Remenyi R, Gong D, Al-Mawsawi LQ, Qi H, Wu TT, Sun R - PLoS Genet. (2015)

Structure-function relationship of residue 281.(A) The interaction of influenza A PA with the RNA phosphate backbone located between base 3 and 4 is shown. RNA is colored in green. PA is colored in cyan. Hydrogen bonds are represented by dotted black lines. Numbering of residue position is based on A/WSN/33. Conversion of residue position numbering is described in S3 Table. (B) The interaction of influenza B PA with the RNA phosphate backbone located between base 3 and 4 is shown. RNA is colored in green. PA is colored in cyan. Hydrogen bonds are represented by dotted black lines. Numbering of residue position is based on A/WSN/33. Conversion of residue position numbering is described in S3 Table.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4489113&req=5

pgen.1005310.g007: Structure-function relationship of residue 281.(A) The interaction of influenza A PA with the RNA phosphate backbone located between base 3 and 4 is shown. RNA is colored in green. PA is colored in cyan. Hydrogen bonds are represented by dotted black lines. Numbering of residue position is based on A/WSN/33. Conversion of residue position numbering is described in S3 Table. (B) The interaction of influenza B PA with the RNA phosphate backbone located between base 3 and 4 is shown. RNA is colored in green. PA is colored in cyan. Hydrogen bonds are represented by dotted black lines. Numbering of residue position is based on A/WSN/33. Conversion of residue position numbering is described in S3 Table.
Mentions: We aimed to further investigate the structural basis of type-specific functional residues. The RNA binding function is required for viral replication and is conserved among type A and B influenza viruses. In the validation above, substituting lysine [K] to isoleucine [I] at residue 281 completely abolished the polymerase activity. This highlights the importance of the hydrogen bond formed between K281 and the RNA phosphate backbone in the influenza A virus (Fig 7A and boxed in S14 Fig). However, PA K281 is not conserved between type A and B influenza viruses. All influenza B viruses carry an alanine [A] at residue 281, which is unable to form a hydrogen bond with the RNA backbone. The critical hydrogen bond mediated by K281 in influenza A virus is replaced by the main chain of G569 in the influenza B virus (Fig 7B and boxed in S15 Fig). In fact, structural analysis indicates that type A [64] and B [72] influenza viruses display different hydrogen bonding patterns between PA and the viral RNA promoter (S14 Fig and S15 Fig). Thus, conserved functions may not necessarily require conserved functional residues.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, United States of America,; Molecular Biology Institute, University of California, Los Angeles, Los Angeles, California, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus