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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

Structural analysis of putative functional residues.(A) The location of a putative functional subdomain is shown on the structure of the influenza polymerase heterotrimeric complex (PDB: 4WSB) [64]. For PA, residues were colored as according to the scheme presented in Fig 4. A putative host determinant residue, S552, is colored in magenta. Note, residue 559 carries an arginine [R] instead of a lysine [K] on the PA of A/WSN/33. (B) The effects of different PA point mutations on influenza polymerase activity were measured using an influenza A virus-inducible luciferase reporter assay [63]. Error bar represents the standard deviation of three biological replicates. (C) The expression level of each C-terminal Flag-tagged PA mutant or WT was tested by immunoblot analysis. The expression level of actin was served as a loading control.
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pgen.1005310.g005: Structural analysis of putative functional residues.(A) The location of a putative functional subdomain is shown on the structure of the influenza polymerase heterotrimeric complex (PDB: 4WSB) [64]. For PA, residues were colored as according to the scheme presented in Fig 4. A putative host determinant residue, S552, is colored in magenta. Note, residue 559 carries an arginine [R] instead of a lysine [K] on the PA of A/WSN/33. (B) The effects of different PA point mutations on influenza polymerase activity were measured using an influenza A virus-inducible luciferase reporter assay [63]. Error bar represents the standard deviation of three biological replicates. (C) The expression level of each C-terminal Flag-tagged PA mutant or WT was tested by immunoblot analysis. The expression level of actin was served as a loading control.

Mentions: Our functional profile identified a subset of PA residues that interact with PB1 (S8A Fig), PB2 (S8B Fig), and the viral RNA promoter (S9 Fig). Moreover, six out of the 10 validated functional residues participate in these interaction interfaces: − D426, E427, and F681 interacted with PB1; L470 interacted with PB2; K281 and R512 interacted with the viral RNA promoter. Our data also identified functional residues that were not involved in polymerase complex formation or RNA binding activity. For example, E430 did not interact with either PB1, PB2, or the viral RNA promoter (S10 Fig). This is consistent with our data that E430 is involved in a non-polymerase function. In addition, a putative functional subdomain independent of the polymerase-interacting surface was identified in our functional profiling data. This putative functional subdomain is composed of a series of charged or polar residues − D286, N412, K413, R454, D529, K559, and K635. Interestingly, this patch of functional residues was adjacent to residue 552, which has been shown to be a host-specific determinant [65]. This indicates a possible biological significance of the putative functional subdomain we identified. Consistently, substitutions at positions D286, N412, K413, R454, D529, and K635 were shown to abolish the polymerase activity in our validation experiment (Fig 5B-5C), further confirming the functional importance of this subdomain in viral replication.


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)

Structural analysis of putative functional residues.(A) The location of a putative functional subdomain is shown on the structure of the influenza polymerase heterotrimeric complex (PDB: 4WSB) [64]. For PA, residues were colored as according to the scheme presented in Fig 4. A putative host determinant residue, S552, is colored in magenta. Note, residue 559 carries an arginine [R] instead of a lysine [K] on the PA of A/WSN/33. (B) The effects of different PA point mutations on influenza polymerase activity were measured using an influenza A virus-inducible luciferase reporter assay [63]. Error bar represents the standard deviation of three biological replicates. (C) The expression level of each C-terminal Flag-tagged PA mutant or WT was tested by immunoblot analysis. The expression level of actin was served as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4489113&req=5

pgen.1005310.g005: Structural analysis of putative functional residues.(A) The location of a putative functional subdomain is shown on the structure of the influenza polymerase heterotrimeric complex (PDB: 4WSB) [64]. For PA, residues were colored as according to the scheme presented in Fig 4. A putative host determinant residue, S552, is colored in magenta. Note, residue 559 carries an arginine [R] instead of a lysine [K] on the PA of A/WSN/33. (B) The effects of different PA point mutations on influenza polymerase activity were measured using an influenza A virus-inducible luciferase reporter assay [63]. Error bar represents the standard deviation of three biological replicates. (C) The expression level of each C-terminal Flag-tagged PA mutant or WT was tested by immunoblot analysis. The expression level of actin was served as a loading control.
Mentions: Our functional profile identified a subset of PA residues that interact with PB1 (S8A Fig), PB2 (S8B Fig), and the viral RNA promoter (S9 Fig). Moreover, six out of the 10 validated functional residues participate in these interaction interfaces: − D426, E427, and F681 interacted with PB1; L470 interacted with PB2; K281 and R512 interacted with the viral RNA promoter. Our data also identified functional residues that were not involved in polymerase complex formation or RNA binding activity. For example, E430 did not interact with either PB1, PB2, or the viral RNA promoter (S10 Fig). This is consistent with our data that E430 is involved in a non-polymerase function. In addition, a putative functional subdomain independent of the polymerase-interacting surface was identified in our functional profiling data. This putative functional subdomain is composed of a series of charged or polar residues − D286, N412, K413, R454, D529, K559, and K635. Interestingly, this patch of functional residues was adjacent to residue 552, which has been shown to be a host-specific determinant [65]. This indicates a possible biological significance of the putative functional subdomain we identified. Consistently, substitutions at positions D286, N412, K413, R454, D529, and K635 were shown to abolish the polymerase activity in our validation experiment (Fig 5B-5C), further confirming the functional importance of this subdomain in viral replication.

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