The effects of historical KoRV polymorphisms on protein structure.

<p>Superimpositions are shown between the present day consensus KoRV (Pci-SN265) protein structure and ancient KoRV variants. Amino acid variations between these sequences mapped on the protein models are shown in red and with arrows. The models are shown in cartoon ribbon representations (left panels) and as semi-transparent surfaces (right panels). The atoms of the variable amino acid residues are in line representations to view the side chains. In all comparisons the Pci-SN265 consensus was used as the reference sequence. (A) The model of the Pci-SN265 Gag protein is superimposed with the models of variants found in archival koalas um3435 and maex1738. (B) The model of the Pci-SN265 Pol protein is superimposed with variants found in QMJ6480, 582119, MCZ8574, Um3435, and maex1738. (C) The model of the Pci-SN265 Env protein is superimposed with the model of variants found in MCZ_12454 and um3435. For all three polypeptides, the structural differences predicted are attributed to changes in the polarity, charge, and atom conformations and are largely localized onto flexible loop regions.</p

Alignment of modern and museum koala retrovirus sequences, showing positions of proviral genes and proteins.

<p>Upper Panel: Character states matching the reference sequence (AF151794) are indicated in light grey, while mismatches (position 312) or polymorphisms (all other positions) are shown as black hatch marks. The infectious clone KV522 (AB721500) is the first sequence below the reference. The aligned sequences all display open reading frames for viral <i>gag</i>, <i>pol</i>, and <i>env</i> regions, except that polymorphisms at three positions in the museum samples code for a stop codon that would disrupt an open reading frame; these are indicated by red hatch marks. Green lines represent polymorphisms that could be placed in phase in overlapping sequence reads. Lower Panel: The coded proteins are indicated, following the divisions proposed by Hanger et al. (2000) relative to the polymorphism alignment. The positions of the SRLPIY domain potentially involved in viral infectivity of the GAG protein and p10 domain (Gag assembly and nuclear export signal, respectively) are indicated. Likewise, the Env motif CETTG, and p15E transmembrane envelope protein are indicated. Regions known to be divergent in Japanese isolates, KoRV-B and KoRV- C, and KoRV-D <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095633#pone.0095633-Xu1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095633#pone.0095633-Shimode1" target="_blank">[40]</a>, are indicated by orange, purple, and green arrows, respectively.</p

Estimates of Tajima’s D^*.

<p>*The analysis involved 9 KoRV sequences. Codon positions included were 1st+2nd+3rd. All positions containing gaps or missing data were eliminated. There were 1565 positions for <i>gag</i>, 3384 for <i>pol</i>, 1980 for <i>env</i>, and 6929 positions for all (concatenated coding sequences) in the final dataset. Abbreviations: m  =  number of sequences; S = Number of segregating sites; ps = S/m; Θ = ps/a1; π = nucleotide diversity; D = Tajima test statistic.</p

Structural superimpositions of Pci-SN265 (green), AF151794 (red), and KV522 (gold) KoRV Gag, Pol, and Env protein structures, demonstrating the overall similarity of the structures.

<p>Amino acid variations across these three sequences are mapped on the protein models (arrows). The structural differences predicted are attributed to changes in the polarity, charge, and atom conformations. The models are shown in cartoon (left panels) and semi-transparent surface (right panels) representations. The atoms of the variable amino acid residues are shown in line representations to view the side chains. In all comparisons the Pci-SN265 consensus sequence was used as the reference.</p

Codon based Z tests of selection.

<p>The test statistic dN-dS is shown above the diagonal. dN and dS are the values of non-synonymous and synonymous substitutions per site, respectively. The Nei-Gojobori method was used to calculate synonymous and nonsynonymous substitutions. The probability of rejecting the null hypothesis of strict-neutrality (dN  =  dS) is shown below the diagonal). Values of P less than 0.05 are highlighted in bold. Values were not significant after Bonferroni correction. The variance of the difference was computed using the bootstrap method (500 replicates). Numbers listed for columns represent the same KoRV sequences numbered in the rows. The first two KoRV sequences are from GenBank; the other KoRVs are consensus sequences from the current study.</p

Koala sample information.

<p>Koala sample numbers were based on studbook numbers (“SN”) for zoos, and specimen numbers for each museum. Wild modern samples were from the National Cancer Institute (NCI), and have NCI codes. North Australian samples are from Queensland; NSW is New South Wales. Collection dates for archival samples were confirmed by K.H. from museum records; date ranges listed are as exact as possible given museum records. Plus sign indicates successful attempt; minus sign indicates attempted unsuccessfully; blank indicates not attempted. Prior <i>env</i> sequences refer to those derived from PCR and reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095633#pone.0095633-AvilaArcos1" target="_blank">[6]</a>. Flanking sequences for modern koala samples are from Ishida et al. (submitted).</p