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. 2012;8(11):e1003102.
doi: 10.1371/journal.pgen.1003102. Epub 2012 Nov 29.

Selective pressure causes an RNA virus to trade reproductive fitness for increased structural and thermal stability of a viral enzyme

Moshe Dessau  1 , Daniel Goldhill, Robert C McBride, Paul E Turner, Yorgo Modis
Affiliations

Selective pressure causes an RNA virus to trade reproductive fitness for increased structural and thermal stability of a viral enzyme

Moshe Dessau et al. PLoS Genet. 2012.

Erratum in

Abstract

The modulation of fitness by single mutational substitutions during environmental change is the most fundamental consequence of natural selection. The antagonistic tradeoffs of pleiotropic mutations that can be selected under changing environments therefore lie at the foundation of evolutionary biology. However, the molecular basis of fitness tradeoffs is rarely determined in terms of how these pleiotropic mutations affect protein structure. Here we use an interdisciplinary approach to study how antagonistic pleiotropy and protein function dictate a fitness tradeoff. We challenged populations of an RNA virus, bacteriophage Φ6, to evolve in a novel temperature environment where heat shock imposed extreme virus mortality. A single amino acid substitution in the viral lysin protein P5 (V207F) favored improved stability, and hence survival of challenged viruses, despite a concomitant tradeoff that decreased viral reproduction. This mutation increased the thermostability of P5. Crystal structures of wild-type, mutant, and ligand-bound P5 reveal the molecular basis of this thermostabilization--the Phe207 side chain fills a hydrophobic cavity that is unoccupied in the wild-type--and identify P5 as a lytic transglycosylase. The mutation did not reduce the enzymatic activity of P5, suggesting that the reproduction tradeoff stems from other factors such as inefficient capsid assembly or disassembly. Our study demonstrates how combining experimental evolution, biochemistry, and structural biology can identify the mechanisms that drive the antagonistic pleiotropic phenotypes of an individual point mutation in the classic evolutionary tug-of-war between survival and reproduction.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Evolution of Φ6 under thermal pressure.
A) Survival of evolved virus lineages as a function of heat shock temperature after a thermal selection with 20 50°C-heat shocks every five generations, or identical passage without heat shock. B) Viruses evolved under heat shock showed a ‘bull's eye’ phenotype in which plaques appeared partially turbid due to residual bacterial growth within the plaque (closed arrows). Normal plaques from wildtype viruses mixed into the sample are labeled with open arrows. C) Survival of a virus genotype with mutation V207F only is greater than the wildtype virus at elevated temperatures. Each point is the mean percent survival (± std. err.) of 3 to 5 survival assays conducted for the strain, and error bars too small to be visualized are omitted for clarity. *** indicates statistical significance at P<0.0001, and ** is statistical significance at P = 0.004. These results qualitatively agree with those in panel (A) comparing evolved treatment and control populations, with a significant statistical advantage apparent for the V207F mutant at all temperatures tested. D) Bull's eye phenotype of a virus genotype with mutation V207F only. E) The V207F mutation in P5 causes a fitness disadvantage at 25°C relative to wildtype virus; bars indicate means (±95% C.I.). *** indicates statistical significance at P≈0.006.
Figure 2
Figure 2. Thermal and proteolytic stabilities of P5wt and P5V207F.
A) Thermal melting of P5wt and P5V207F measured by circular dichroism (CD) spectrometry at 220 nm. P5wt and P5V207F began to unfold cooperatively at 50–55°C (left panel). Melting temperatures were calculated from the second derivative of the CD melting curves (right panel). The V207F mutation increases the melting temperature of P5 by 7.6°C. B) Limited proteolysis of P5ΔV8wt and P5ΔV8V207F with V8 (Glu-C) protease monitored by SDS-PAGE. Some minor proteolytic products (marked by asterisks) are visible in P5ΔV8wt. C) Thermal melting curves of P5ΔV8wt and P5ΔV8V207F, with melting temperatures calculated as in (A). D) Differential Scanning Calorimetry (DSC) of P5 and P5ΔV8 proteins. Peaks indicate a 6°C difference between the melting points of wildtype and mutant P5.
Figure 3
Figure 3. P5 adopts a lysozyme fold and the V207F mutation fills a cavity.
A) P5 adopts a lysozyme superfamily fold with an N-terminal lobe (NTL, in dark blue) and a C-terminal lobe (CTL, light blue) connected by a central helix (cyan). The predicted catalytic residue Glu95 lies in the substrate binding cleft between the two lobes. The NTL differs from other lysozyme structures in its overall organization and relative orientation to the CTL. The three N-terminal residues of P5 form an unusual crystal packing interaction. Residues 53–59 are disordered (dashed line). Residue 207 is marked with a red circle. B) Close-up of residue 207. The Phe207 side chain in P5ΔV8V207F (orange) fills a cavity (marked by an arrow) that is unoccupied in P5ΔV8wt (light blue), with no significant changes in the protein backbone (see also Figure S3).
Figure 4
Figure 4. The structure of P5 bound to a glycan suggests lytic transglycosylase activity.
A) Overall structure of P5ΔV8wt bound to chitotetraose. The 2Fo - Fc electron density map for the ligand is shown contoured at 1 σ. Residues 199–220 are disordered. Helix α7 and the following linker are in slightly different positions than in the unliganded structure (see also Figure S3). The four NAG residues of chitotetraose bind to subsites A–D. B) Close-up of the active site. A water molecule is observed between Glu95 and the NAG in subsite D, supporting a lytic transglycosylase (LT) activity, with Glu95 as the catalytic acid/base. C) Superposition of ligand-bound P5ΔV8wt (yellow) and apo-P5ΔV8wt (orange) onto the structure of the E. coli slt70 LT (brown) containing a glycan product in subsites E and F (PDB 1QTD). The geometry and electrostatics of the P5 substrate-binding surface (D) are similar to those of slt70 (E). P5 residues displaced by the chitotetraose ligand are shown in grey with a semi-transparent surface.
Figure 5
Figure 5. Cell wall lysis activity of P5wt and P5V207F.
A) To measure the cell lysis activities of P5wt and P5V207F the turbidity of chloroform-treated E. coli was measured as absorbance at 450 nm at 25°C. P5wt and P5V207F have the same maximum cell lysis rates. B) Linear relationship between the maximal rate of decrease in absorbance and the enzyme concentration.
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