Deviations from additivity.

<p>Phage were divided into three bins based on their fitness effects in homozygous coinfections: deleterious (<i>s</i><−0.3, <i>n</i> = 5), slightly deleterious (0.3<<i>s</i><0, <i>n</i> = 6), or beneficial (<i>s</i>>0, <i>n</i> = 5). For mutations in each bin, boxplots show either the marginal effects of mutations in homozygous coinfections (½ s) or the fitness effects of mutations in heterozygous coinfections (<i>hs</i>). <i>p</i>-values resulted from paired Welch’s <i>t</i>-tests that tested the additive expectation that these two quantities were equal (i.e. <i>hs</i> = ½ <i>s</i>). * Indicates the only statistically significant comparison between homozygous and heterozygous effects of mutations (<i>p</i> = 0.0041).</p

Dominance Effects of Deleterious and Beneficial Mutations in a Single Gene of the RNA Virus ϕ6

<div><p>Most of our knowledge of dominance stems from studies of deleterious mutations. From these studies we know that most deleterious mutations are recessive, and that this recessivity arises from a hyperbolic relationship between protein function (i.e., protein concentration or activity) and fitness. Here we investigate whether this knowledge can be used to make predictions about the dominance of beneficial and deleterious mutations in a single gene. We employed a model system – the bacteriophage φ6 – that allowed us to generate a collection of mutations in haploid conditions so that it was not biased toward either dominant beneficial or recessive deleterious mutations. Screening for the ability to infect a bacterial host that does not permit infection by the wildtype φ6, we generated a collection of mutations in P3, a gene involved in attachment to the host and in phage particle assembly. The resulting collection contained mutations with both deleterious and beneficial effects on fitness. The deleterious mutations in our collection had additive effects on fitness and the beneficial mutations were recessive. Neither of these observations were predicted from previous studies of dominance. This pattern is not consistent with the hyperbolic (diminishing returns) relationship between protein function and fitness that is characteristic of enzymatic genes, but could have resulted from a curve of increasing returns.</p></div

Mutation identity.

a<p>Substitutions are labeled relative to their position in P3.</p>b<p>Two additional host range mutants had the same substitution.</p>c<p>One additional host range mutant had the same substitution.</p

Coinfection data.

<p><i>a</i> – Mean and SEM values calculated from the frequency of mutants produced by individual heterozygous coinfections; *indicate intervals that do not include 0.50.</p><p><i>b –</i>Not available.</p

Fitness effects of mutations in heterozygous (<i>hs</i>) and homozygous (<i>s</i>) coinfections.

<p>Data are means ± standard errors of the mean. Regions where mutations have recessive effects (0< <i>h</i><0.5) are shown in gray and additive effects (<i>h</i> = 0.5) are shown as a dotted line. The solid line is the reduced major-axis regression line and falls primarily in the recessive region. Mutations are represented as circles if they were accumulated in the φ6_mindich background and as triangles if they were accumulated in φ6_37-F41. Removal of the two mutants that did not have mutations in P3 (white-filled circles) barely affected the major-axis regression (dashed line). Effects of P3 mutations that were obtained and measured in an alternative, higher fitness genetic background are shown with dark gray circles.</p

The relationship between protein function and fitness alters the complementation of mutations affecting protein function.

<p>Regions where mutations have recessive effects (0< <i>h</i><0.5) are shown in gray and additive effects (<i>h</i> = 0.5) are shown as a dashed line. The Physiological Theory predicts that (A) the hyperbolic relationship between protein function and fitness results in additive mutation effects only when the wildtype fitness is near zero (B). At moderate (C) and high (D) wildtype fitnesses, deleterious and beneficial mutations are predicted to be recessive and dominant, respectively. The sigmoidal fitness function predicted for proteins that display cooperative binding (E) causes a stronger dependence of dominance coefficients on wildtype fitness. For instance, the sigmoidal relationship yields recessive beneficial mutations when wildtype fitness is low (F), but dominant beneficial mutations when wildtype fitness is moderate (G) and high (H).</p

The hyperbolic relationship between enzyme concentration ([<i>E</i>]) and fitness is predicted to determine the dominance of mutations affecting enzyme concentration.

<p>When fitness of the wildtype is near the plateau of the hyperbolic curve, (A) mutations that substantially reduce enzyme concentration are predicted to be recessive and (B) mutations that slightly reduce enzyme concentration have additive effects. (C) When fitness of the wildtype is lower, it is possible to accumulate mutations that substantially increase enzyme concentration. These mutations are predicted to be dominant over the wildtype allele. These predictions are for mutations that alter enzyme concentration, but can be extended to include mutations that alter other components of protein function, namely protein activity.</p

Burst size measures for homozygous mutant (aa), heterozygous (Aa) and homozygous wildtype (AA) coinfections are shown as columns of points and their means are show as lines.

<p>Two of the host range mutants (HR2 and HR23) did not have mutations in P3, but differences in their mean homozygous effects suggest that they are different mutations.</p

Compartmentalization can persist and evolve independently within the CSF over time.

<p>Neighbor-joining phylogenetic trees showing how compartmentalization can: (A) be persistent with multiple clonal expansions allowing recombination; (B) consist of sequential transient clonal expansions; and (C) be established with a transmitted variant. <i>env</i> sequences from the CSF are labeled with circles (C, colors designated in figure) and <i>env</i> sequences from the blood plasma are labeled with triangles (P, colors designated in figure). Days p.i. are noted. Bootstrap values ≥ 50 are indicated (*) at the appropriate nodes to highlight the more significant branch points. Genetic distance is indicated at the top of each phylogenetic tree (0.001, number of nucleotide substitutions per site between <i>env</i> sequences.) Compartmentalized populations are indicated at the appropriate node by an open circle and emphasized using a blue bar. BEAST-generated TMRCAs of the entire viral population are noted adjacent to the subject ID, and the TMRCAs of the different compartmentalized linages (subject 9040 and 9021) and transmitted parental lineages (subject 7146) are also noted.</p

Subject population virologic, clinical and phylogenetic characteristics.

<p>^aTime point(s) beyond 2 years p.i. analyzed for subject 9018 and 9040 were not included in any overall population analyses.</p><p>^bEstimated.</p><p>^c,dVL, viral load; HIV-1 RNA (log₁₀ copies/ml).</p><p>^eCells/μl.</p><p>^fCSF white blood cell (WBC) count, cell/μl.</p><p>^gCSF/plasma albumin ratio.</p><p>^hThree statistical analyses of genetic compartmentalization between viral populations in the blood plasma and CSF: Slatkin-Maddison test (SM) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref018" target="_blank">18</a>], Wright’s measure of population subdivision (F_st) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref019" target="_blank">19</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref020" target="_blank">20</a>] and the Nearest-neighbor statistic (S_nn) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref021" target="_blank">21</a>]. <i>P</i> values <0.05 indicated statistically significant genetic compartmentalization.</p><p>ⁱHIV-1 population characteristics in the CSF compartment (compart). Eq, equilibrated blood plasma and CSF populations; Cp (compartmentalized), significant compartmentalization by three compartmentalization analyses; Ap, clonal amplification of ≥3 variants detected in the CSF.[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref018" target="_blank">18</a>]</p><p>^jTMRCA (Time to Most Recent Common Ancestor) of the entire viral population, analyzed by BEAST [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004720#ppat.1004720.ref015" target="_blank">15</a>]. An asterisk (*) indicates transmission of > 1 variant.</p><p>^kTMRCA for the compartmentalized (Comp) CSF population for compartmentalized subjects.</p><p>^lPatient 9018 diagnosed with neurosyphilis at indicated date.</p><p>^mSignificant compartmentalization scores were due to a compartmentalized lineage in the plasma. After removing this plasma lineage, the remaining plasma and CSF sequences were equilibrated.</p><p>Subject population virologic, clinical and phylogenetic characteristics.</p