Complete nucleotide sequence of polyomavirus SA12 - PubMed (original) (raw)

Comparative Study

Complete nucleotide sequence of polyomavirus SA12

Paul Cantalupo et al. J Virol. 2005 Oct.

Abstract

The Polyomaviridae have small icosahedral virions that contain a genome of approximately 5,000 bp of circular double-stranded DNA. Polyomaviruses infect hosts ranging from humans to birds, and some members of this family induce tumors in test animals or in their natural hosts. We report the complete nucleotide sequence of simian agent 12 (SA12), whose natural host is thought to be Papio ursinus, the chacma baboon. The 5,230-bp genome has a genetic organization typical of polyomaviruses. Sequences encoding large T antigen, small t antigen, agnoprotein, and the viral capsid proteins VP1, VP2, and VP3 are present in the expected locations. We show that, like its close relative simian virus 40 (SV40), SA12 expresses microRNAs that are encoded by the late DNA strand overlapping the 3' end of large T antigen coding sequences. Based on sequence comparisons, SA12 is most closely related to BK virus (BKV), a human polyomavirus. We have developed a real-time PCR test that distinguishes SA12 from BKV and the other closely related polyomaviruses JC virus and SV40. The close relationship between SA12 and BKV raises the possibility that these viruses circulate between human and baboon hosts.

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Figures

FIG. 1.

FIG. 1.

SA12 genome. The SA12 genome is represented by the circle, with the numbers indicating base pair positions. The base pairs are numbered in a clockwise direction with base pair 1 being the GC bp of the third GAGGC repeat within the origin of DNA replication. Positions of the coding sequences for large T antigen, small t antigen, agnoprotein, and the viral capsid proteins VP1, VP2, and VP3 are indicated. The positions of the early mRNA introns are indicated. The position of the putative SA12-encoded microRNA is also shown.

FIG. 2.

FIG. 2.

SA12 regulatory region. The 401-bp regulatory region of SA12 extends from nucleotides 5124 to 294. Important regulatory elements are highlighted in color. Light green indicates the consensus pentanucleotide T antigen binding sequence, dark green indicates the nonconsensus pentanucleotide T antigen binding sequence, orange indicates the imperfect palindrome, and blue indicates the A/T-rich region. The enhancer region has been described elsewhere (8).

FIG. 3.

FIG. 3.

SA12 protein coding regions. The cDNA sequence of (A) large T antigen, (B) small t antigen, (C) VP1, (D) VP2, or (E) VP3 is displayed as the bottom sequence, and the translated protein sequence is displayed on top. The lengths of the proteins in numbers of amino acids (aa) are shown. Numbering for both the protein and the DNA sequence is on the left. An * in the protein sequence corresponds to the stop codon.

FIG. 4.

FIG. 4.

Sequence alignments of the variable regions and host range domains. The host range domains from SA12, BKV, and JCV were aligned (A) with and (B) without the host range domain of SV40. The variable regions from BKV, SA12, and JCV were aligned (C) with and (D) without the variable region of SV40. All alignments were done with MultAlin (7). Conserved residues are highlighted in red; partial conservation is indicated with blue, and no conservation is indicated with black. A consensus sequence is shown below each alignment, where conserved residues are represented as capital letters, partially conserved residues are in lowercase, and sites that show no homology are marked with a period.

FIG. 5.

FIG. 5.

Mutation of the SA12 core ori to a consensus sequence does not enhance viral DNA replication or yield. (A) The sequence of the SA12 core ori is shown. Putative T antigen binding sites are underlined. The position of a T10A mutant that changes the left-most binding site to a consensus GAGGC is shown. (B) BSC40 cells were either mock infected or infected with SV40, SA12, or SA12-T10A, and at the indicated times postinfection (days), cells were harvested and lysed. Viral DNA was purified and digested with the appropriate enzyme and resolved through a 0.7% agarose gel. Digested DNAs from SV40, SA12, SA12-T10A, and mock reactions were loaded in lanes 4 to 9, lanes 10 to 15, lanes 16 to 21, and lanes 22 to 23, respectively. As a positive control, pSVB3 was digested with BamHI (lane 2) and pUC119-SA12 was digested with PstI (lane 3). DNA was visualized by staining with GelStar. Replicated viral DNA is indicated by an arrow. (C) Virus growth curve and yield. BSC40 cells were infected with SA12 wild-type wt100 (WT SA12) or SA12-T10A at an MOI of 5. At various times postinfection, the cells were frozen and thawed three times. The virus titer was then determined for each time point by a standard plaque assay.

FIG. 6.

FIG. 6.

Early mRNA splice junctions. (A) Sequence homology of early RNA splice sites. Regions surrounding the early RNA splice sites (5′ large T donor, 5′ small t donor, and 3′ acceptor) from SA12, SV40, JCV, and BKV were aligned using MultAlin (7). Completely conserved residues are represented as capital letters, and partially conserved residues are in lowercase. Sites that show no homology are marked with a period. The consensus 5′ donor and 3′ acceptor splice site sequences are enclosed in a rectangle. (B) Genomic organization of the early region of SA12. Two pairs of PCR primers were designed to flank the putative splice sites, as identified for panel A. The putative splicing sites are marked (DL, putative large T donor site; DS, putative small t donor site; A, putative acceptor site). Sequences shared by large T and small t mRNAs are represented by vertical lines. The unique sequence that is part of the small t mRNA is represented by diamonds. The predicted PCR product sizes for genomic SA12, large T antigen mRNA, and small t antigen mRNA are shown for both primer pairs. (C) PCR analysis of the early region of SA12. Thirty-five cycles of PCR, using primer pair 15/16, were performed on 10 pg of pUC119-SA12 (positive control), 1 μl of the indicated cDNA, or no DNA (negative control). Various amounts of the PCR products were resolved on a 1.5% agarose gel and stained with GelStar. The sizes of the PCR markers are indicated on the right in base pairs. The positions of SA12 and small t and large T PCR products are indicated on the left. (D) PCR analysis of the small t splice junction. Thirty-five cycles of PCR were performed on 1 μl cDNA from the 72-h time point using the indicated primer pairs. PCR products were resolved on a 1.5% agarose gel and stained with GelStar. The sizes of the PCR markers are indicated on the left in base pairs. The positions of SA12 and small t PCR products are indicated on the right. (E) Identification of the early-region splice sites. The early splice junctions of SA12 are shown as in panel B. The SA12 sequence of each splice site (DL, DS, and A) is shown. Large-T antigen mRNA processing is displayed on top, and small t antigen mRNA processing on bottom. The first and last two bases of the intron are underlined. The size of the intron is indicated in base pairs. The exon sequences are highlighted in bold type. Arrows from the exon sequences point to the sequence chromatograph where the exon sequences are boxed.

FIG. 7.

FIG. 7.

Expression of SA12 miRNAs. (A) A secondary-structure prediction of the SA12 hairpin, starting at nucleotide 2785, which is conserved among several polyomaviruses containing the pre-miRNA, is shown. (B) Probe diagram showing the sequence of the hairpin and probes designed to detect miRNAs derived from either arm 5′ or 3′ of the “terminal loop.” (C) Northern blot showing expression of ∼60-nt and ∼22-nt bands in infected cells. Blots were probed with either the 5′ or 3′ probe as indicated. Lanes with RNA from uninfected (U) or 90 h postinfection (90) are indicated. The migration positions of oligonucleotide markers are indicated on the right side. Arrows indicate the locations of the bands corresponding to the pre-miRNA and the miRNA. MW, molecular weight.

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