Mutation-prone tandem-repeat motifs are conserved in regions of the unexpressed cassettes that correspond to the antigenic loop domains.

<p><b>A</b>) Highly-mutable tandem-repeat motifs were identified in the majority of the regions of the cassettes that correspond to the antigenically important loop domains in each strain despite little sequence conservation among strains. Polymorphic sites, denoted with ambiguities following the IUPAC standard, occur primarily in the second codon position. Although the tandem-repeat motifs are similar within the <i>vls</i> unexpressed cassettes of each strain, the number of repeats varies due to insertion and deletion (indel) mutations. <b>B</b>) Tandem-repeat motifs are associated with high frequencies of indel mutations in antigenic loop regions (shaded). The association between tandem repeats and indel mutations in the regions corresponding to the antigenic loop domains is represented as the frequency of sites with gaps in the nucleotide alignment of unexpressed cassettes in strain N40. Indels are present at significantly lower frequencies in antigenic loop regions of the unexpressed cassettes that do not contain tandem repeats (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003766#ppat.1003766.s001" target="_blank">Fig. S1</a>) and are absent in the conserved alpha helical regions that are devoid of tandem-repeat motifs. <b>C</b>) An alignment of antigenic loop region 2 of the N40 unexpressed cassettes illustrates the 3 bp tandem-repeat motifs common to antigenic loop regions of all strains.</p

Strong diversifying selection is localized to the antigenically important regions of the unexpressed cassettes.

<p><b>A</b>) The six variable regions of the unexpressed cassettes are expressed as loop structures (yellow) exposed on the surface of the bacterium in all <i>B. burgdorferi</i> strains examined. The conserved alpha helical regions (grey) encode structural alpha helices in the expressed protein. Structural models of the expressed VlsE protein are shown for three evolutionarily divergent strains of <i>B. burgdorferi</i>: B31 (crystal structure reported by Eicken et al. 2002), JD1 (predicted structure), and N40 (predicted structure) <b>B</b>) Codon-by-codon analyses of the unexpressed cassettes identified a high frequency of positively selected codons (highlighted in orange) in the regions that correspond to the antigenically-important loop domains (demarcated by black boxes). The majority of codons in the regions that correspond to the alpha helical domains are under stabilizing selection (highlighted in purple). One translated cassette sequence from each of the three evolutionarily divergent strains is shown. There is strong statistical support for positive selection in 28–43% of the codons in regions of the cassettes homologous to the antigenic loop domains of <i>vlsE</i> compared to only 0–5% in regions homologous to the alpha helical domains of <i>vlsE</i>.</p

Strong selection for amino acid diversity among the <i>vls</i> unexpressed cassettes.

<p><b>A</b>) The frequency of non-synonymous differences per non-synonymous site (dN) is significantly greater than the frequency of synonymous differences per synonymous site (dS) in the regions of the cassettes that correspond to the antigenic loop domains (ALR) of each strain suggesting strong positive selection. In contrast, synonymous and non-synonymous differences occur at similar frequencies in the regions of the unexpressed cassettes that correspond to the conserved alpha helical domains (αHR). <b>B</b>) The ratio of non-synonymous to synonymous polymorphisms (dN/dS) in the unexpressed cassettes of each strain is far greater than one in antigenic loop regions (ALR), suggesting diversifying selection, and slightly less than one in alpha helical regions, suggesting purifying selection or neutral evolution. Values are reported as the mean of all pair-wise comparisons of unexpressed cassettes within each strain averaged over all 12 strains (± standard error). Summaries of the statistical tests of diversifying selection are provided in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003766#ppat.1003766.s007" target="_blank">Table S2</a>.</p

Evolvability of <i>vlsE</i> is tightly correlated with sequence diversity among the unexpressed <i>vls</i> cassettes.

<p>The level of sequence diversity at individual sites among the unexpressed <i>vls</i> cassettes was tightly correlated with the rate of sequence change at the corresponding sites in <i>vlsE</i> during experimental infections (R² = 0.67, F(1,200) = 403.6, p<0.0001) (data from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003766#ppat.1003766-Coutte1" target="_blank">[21]</a>). Thus, increasing diversity among the unexpressed cassettes will result in a corresponding increase in the antigenic evolvability at VlsE. Sequence diversity among the unexpressed cassettes was calculated as the entropy (bits) at each site in the 15 unexpressed cassette sequences; the rate of sequence change in the <i>vlsE</i> variants during experimental infections in mice was calculated as the entropy (bits) at each site of the 113 expressed antigen sequences. The diversity at <i>vlsE</i> in these data, from immunodeficient mice, results primarily from mutational inputs from the <i>vls</i> cassettes.</p

raw data Figure 1C

Height (mm) at which each individual larva (n=16) was found at the indicated time points. At each time point survival was monitored (alive/dead =A/D)

raw_data_Fig1D

Height (mm) at which each individual larva (n=32) was found at the indicated time points. At each time point survival was monitored (alive/dead =A/D)

raw data Figure 1B

Height (mm) at which each individual larva (n=16) was found at the indicated time points . At each time point survival was monitored (alive/dead =A/D)

Variation between different replicates in movement assays of mock- and WT-infected larvae at one, two and three dpi.

<p>Minimum, maximum and mean (± SE) distances moved of all replicates are displayed.</p

The <i>ptp</i> gene is present in all Alphabaculovirus group I NPVs.

<p>Bayesian phylogeny of baculoviruses based on the <i>lef-8</i> gene. GenBank accession numbers are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046933#pone.0046933.s003" target="_blank">Table S3</a>. Numbers in bold indicate maximum likelihood bootstrap values based on 100 replicates, while plain numbers depict Bayesian posterior probabilities. Only values ≥50 are indicated for both analyses. The bar at the bottom indicates a branch length of 10% distance. Baculoviruses possessing a <i>ptp</i> gene are marked by a black dot, while baculoviruses possessing a <i>ptp2</i> gene are marked by a black diamond.</p

The AcMNPV <i>ptp</i> gene is expressed in the WT-, repair- and catmut-infected larvae.

<p>RT-PCR analysis on mock- (M), WT- (W), Δ<i>ptp</i>- (Δ), repair- (R) and catmut-infected (C) larvae. Expression of the AcMNPV <i>ptp</i> gene, the AcMNPV <i>ie1</i> gene and the host <i>Se</i>-<i>eIF5A</i> gene was analyzed. For each RT sample, a PCR without RT step (non-RT) was performed in parallel. For each primer pair, a no-template control was processed (-). The GeneRuler 100 bp ladder (Fermentas) was included in the agarose gel to estimate PCR fragment sizes.</p