Splice junction map of simian parvovirus transcripts

Abstract

The transcription map of simian parvovirus (SPV), an Erythrovirus similar to Parvovirus B19, was investigated. RNA was extracted from tissues of experimentally infected cynomolgus macaques and subjected to reverse transcription-PCR with SPV-specific primers. The PCR products were cloned and sequenced to identify splice junctions. A total of 14 distinct sequences were identified as putative partial transcripts. Of these, 13 were spliced; a single unspliced transcript putatively encoded NS1. Sequence analysis revealed that spliced partial transcripts may encode portions of open reading frames for the major capsid proteins VP1 and VP2 and smaller, unknown proteins. These unspliced and spliced transcripts and putative proteins encoded by SPV were similar to those of B19. Initial splice junctions at nucleotides 279 and 333 were analogous to those at nucleotides 406 and 441, respectively, in B19. Seven of the 10 splices identified had typical GT/AG donor/acceptor junctions. The splice sites were confirmed by Northern blotting and autoradiography. In contrast to B19, which has a maximum of two splices per transcript, up to three splices were observed in SPV transcripts. A spliced transcript putatively encoding a truncated version of NS1, as seen with minute virus of mice and adeno-associated virus 2, was also observed. The findings indicate that that the splicing pattern of transcripts of SPV and B19 is similar, but SPV also has coding strategies in common with other parvoviruses.

FIG. 1.

PCR products obtained from the four main primer sets. Lanes 1 and 15, 1-kb and 100-bp markers. Lane 2, mouse liver RNA amplified with mouse liver-specific primers (positive control for PCR of size 361 bp). Lane 3, SPV-infected macaque RNA to which reverse transcriptase was not added during reverse transcription, amplified with the F230 and R2346 primers (negative control). Lane 4, 5 μl of water used for the PCR amplified with the F230 and R2346 primers (negative control). Lane 5, 1 μl of the complete SPV genome amplified with the F230 and R2346 primers. PCR products from amplification of 5 μl of reverse transcription product of infected macaque sample RNA with primers F230 and R2346 (lanes 6 and 7); primers F230 and R2568 (lanes 8 and 9); primers F230 and R3369 (lanes 10 and 11); and primers F230 and R4854 (lanes 12, 13, and 14). PCR products (bands) obtained with the different primer sets (see above) and used in subsequent cloning reactions are numbered 1 through 11.

FIG. 2.

Splice junction map of SPV. Open reading frames (with ATG as the start codon) greater than 40 amino acids are indicated with boxes of different shades. Nt., nucleotide position in simian parvovirus genome (GenBank Accession U26342). ⎧, promoter site (TATAA). ⎫, site for polyadenylation (AATAAA). F, forward primer. R, reverse primer. On the left-hand side are shown the designated numbers and total lengths in nucleotides of cloned partial transcripts. The ends of these transcripts correspond to the forward and reverse primers used. Lines indicate exons, and brackets indicate spliced introns (designated alphabetically in italics). Asterisks indicate stop codons. ORFs and transcripts ending with dotted lines indicate the ends of partial transcripts corresponding to the position of the reverse primer; NS = 307 to 2370, VP1 = 2363 to 4819, and VP2 = 3149 to 4819 (6).

FIG. 3.

Conventional (A) and nonconventional (B) splice donor and acceptor sites. Sequences in the proximity of the splice junctions are shown.

FIG. 4.

Northern blots. Blots A, B, C, E, F, H, I, and J were probed with splice junction-specific 40-mer probes spanning 20 bases on either side of the splices a, b, c, e, f, h, i, and j, respectively (Fig. 2 and Table 3). Lane 1, radiolabeled markers (sizes mentioned on the left). Lane 2, DNase-treated RNA from SPV-infected tissues. Lane 3, negative control RNA, in most instances from tissues of noninfected macaques and occasionally MA104 or CHO cell RNA. Blot Z was probed with radiolabeled antisense strands of the cloned partial transcript of 333 bases. Lane 4, positive control sense strands of RNA transcribed in vitro from the cloned partial transcript of 333 bases. Arrows indicate predicted band sizes.

FIG. 5.

Comparison of the messages encoded by SPV and B19 based on the splice junctions observed. The method of representation of transcripts and ORFs putatively encoded by SPV is the same as in Fig. 2. The transcription map of B19 on the right is based on the data from Luo et al. (17) and Ozawa et al. (23), with all ORFs indicating completely translated proteins (no asterisks).