Discovery of STL polyomavirus, a polyomavirus of ancestral recombinant origin that encodes a unique T antigen by alternative splicing

Abstract

The family Polyomaviridae is comprised of circular double-stranded DNA viruses, several of which are associated with diseases, including cancer, in immunocompromised patients. Here we describe a novel polyomavirus recovered from the fecal microbiota of a child in Malawi, provisionally named STL polyomavirus (STLPyV). We detected STLPyV in clinical stool specimens from USA and The Gambia at up to 1% frequency. Complete genome comparisons of two STLPyV strains demonstrated 5.2% nucleotide divergence. Alternative splicing of the STLPyV early region yielded a unique form of T antigen, which we named 229T, in addition to the expected large and small T antigens. STLPyV has a mosaic genome and shares an ancestral recombinant origin with MWPyV. The discovery of STLPyV highlights a novel alternative splicing strategy and advances our understanding of the complex evolutionary history of polyomaviruses.

Figure 1. STLPyV is a novel polyomavirus

(A) Diagram of STLPyV genome based on strain GA138. Positions of the two reads obtained from the initial 454 pyrosequencing of DNA generated by MDA of purified virus particles from the index Malawian case are indicated in white. Predicted open reading frames of STLPyV are shown in grey. (B) Diversity plots of nucleotide sequences are shown between STLPyV GA138 and STLPyV WD972 (blue). Their closest relatives MWPyV MA095 (red) and MWPyV WD976 (orange) are included as reference. The proportion of DNA sequence difference is indicated (0.1 = 10%).

Figure 2. Alternative splicing of STLPyV early region

(A) Schematic shows mRNA transcripts expressed from the early region of STLPyV. Exonic regions are indicated by grey boxes, intronic regions by lines. Arrows above the diagram indicate the position of primers. 293T cells were transfected with a plasmid expressing STLPyV LTAg for 48 hr. Cells were harvested and lysed for RT-PCR analysis. RT-PCR reactions were performed with water (Water), STLPyV LTAg plasmid (Control), RNA harvested from mock-transfected (Mock), STLPyV LTAg transfected 293T cells (STLPyV). Data are representative of at least three independent experiments. The three alternatively splice mRNA transcripts were confirmed with a different set of primers (data not shown) and verified in HeLa cells (data not shown). (B) Diagram showing the coding region of T antigens, separated by intron regions in dashed lines. The T’- and truncated T antigens of JCPyV and BKPyV are spliced from LTAg transcripts in a similar manner as SV40 17KT. (C) Sequences of the splice donor and acceptor sites for STLPyV LTAg and 229T transcripts (strain MA138). Exon sequences are highlighted in boxes. The nucleotide sequences of the splice donor and acceptor sites of STLPyV shown are conserved in the three STLPyV strains characterized.

Figure 3. Phylogenetic analysis of STLPyV

Phylogenetic relationships of 28 diverse polyomavirus sequences were inferred from alignment of protein sequences from LTAg (A), VP1 (B) and VP2 (C). Avian polyomaviruses were used as an outgroup to root the phylogenies. STLPyV strains are highlighted in black; MWPyV strains and Wukipolyomavirus whose members show discordant phylogenetic relationships are indicated by boxes. Internal branch labels indicate Bayesian posterior probabilities. The ML method yielded trees with similar topologies.

Figure 4. Prevalance of STLPyV and MWPyV

(A) PCR analysis of STLPyV and MWPyV is shown for control plasmids of the respective LTAg, or representative samples found to be negative,and positive for STLPyV or MWPyV. Bands corresponds to a STLPyV (481 bp) or MWPyV (484 bp) PCR product. PCR products for all positive samples were cloned and sequenced verified.(B) Prevalence of STLPyV and MWPyV in 634 stool specimens collected from a study of children from Saint Louis , as defined by the direct PCR assay described in panel A. Numbers in parenthesis indicate frequency. (C) Results of PCR screening for STLPyV and MWPyV in feces, plasma, nasopharyngeal swabs (NP), and urine specimens collected from a cohort of adult USA kidney transplant recipients. Numbers in parenthesis beside each specimen type indicate the number of specimens screened.(D) Prevalence of STLPyV and MWPyV in a Gambian diarrheal case control study of children is shown, and is based on the PCR screening assay. Odds ratio (OR), 95% confidence intervals (CI) and Fisher exact test indicate that there is no statistically evidence that STLPyV or MWPyV are significantly associated with diarrheal cases (NS = not significant).

Figure 5. STLPyV and MWPyV strains are diverse

Midpoint-rooted neighbor-joining phylogeny inferred from the nucleotide sequences of 9 STLPyV strains and 25 MWPyV strains identified in the three cohorts (Figure 4) and the Malawi index case. Primer sequences were removed to yield a 437 nt alignment. The two genotypes of STLPyV and three genotypes of MWPyV, with strong bootstrap support and > 5% inter-clade sequence difference, are indicated on the right. The ML method yielded a tree with similar topologies.