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.Simian parvovirus (SPV) was first identified in cynomolgus macaques suffering from severe anemia as a consequence of erythroid cell destruction (20). These animals were immunosuppressed due to a concurrent infection with type D simian retrovirus (22). Clinically, this was similar to cases of human infection with parvovirus B19, which is responsible for causing transient aplastic crisis in individuals with underlying hemolysis and red cell aplasia in immunosuppressed individuals, such as AIDS patients (9,22). B19 infection also results in hydrops fetalis and fetal death in pregnant women (9), sequelae also observed with experimental SPV infection in pregnant female cynomolgus macaques (M. G. O'Sullivan, J. C. Veille, W. A. Block, A. R. Turner, N. S. Young, and K. E. Brown, Abstr. 17th Annu. Meet. Am. Soc. Virol., abstr. W22-9, 1998).SPV was classified as a parvovirus considering molecular similarities to other parvoviruses, i.e., adeno-associated virus 2 (prototype dependovirus), the autonomous parvoviruses (the prototype being minute virus of mice), and parvovirus B19 (prototype erythrovirus). In general, members of the subfamily Parvovirinae have a small genome of about 5 kb composed of single-stranded DNA with terminal palindromic sequences that serve as primers for the synthesis of the complementary strand. Only the positive strand of these viruses encodes proteins, suggesting that the positive-sense mRNA is transcribed from the minus-strand genomic DNA (3). To maximize the coding capacity, a number of promoters and all three reading frame...

The Transcription Profile of Aleutian Mink Disease Virus in CRFK Cells Is Generated by Alternative Processing of Pre-mRNAs Produced from a Single Promoter

“…Surprisingly, RNase protection assays could not detect RNAs initiated at the previously identified P36 promoter, following productive AMDV-G infections of CRFK cells at either 37°C or 32°C or following plasmid transfection. Thus, the expression profile of AMDV-G is similar to that of the erythroviruses B19 (24) and simian parvovirus (20,40), which also generate genetic diversity by alternative splicing and polyadenylation of a single pre-mRNA generated from a promoter at the left end of the genome.…”

“…This is often accomplished by transactivation of the capsid gene promoter. In productive AMDV infection of CRFK cells, however, as is also the case for B19 (24) and SPV (20,40), individual mRNAs are generated posttranscriptionally from a single pre-mRNA by a combination of alternative polyadenylation and alternative splicing. There is no evidence that processing of P3-generated pre-mRNAs changes during the course of infection (J. Qiu and D. Pintel, unpublished data), and thus all AMDV RNAs generated during infection increase coordinately with replicating DNA and in concert with increased virus and capsid protein production.…”

Comparison of the Transcription Profile of Simian Parvovirus with That of the Human Erythrovirus B19 Reveals a Number of Unique Features

“…Similar to the organization of B19, SPV has two large open reading frames (ORFs) in either half of the genome. The left ORF encodes the nonstructural NS1 protein, and the right ORF encodes the two capsid proteins VP1 and VP2 (3,31). Expression of the VP2 ORF of SPV results in the generation of virus-like particles and has been useful as a diagnostic reagent for SPV antibody detection (4).…”

“…The splice junctions of SPV RNA isolated from the liver of SPV-infected monkey fetuses have been determined by reverse transcription-PCR (RT-PCR) and Northern blot analysis (31). In this report, we present a detailed characterization of the transcription and protein expression profile of SPV RNA generated following transfection of monkey COS cells.…”

The Abundant R2 mRNA Generated by Aleutian Mink Disease Parvovirus Is Tricistronic, Encoding NS2, VP1, and VP2

“…Strain AMDV-G is a variant of strain Utah 1, which was adapted to grow permissively in Crandell feline kidney cells (CrFK) at 32°C and is nonpathogenic in adult mink (8). It has been demonstrated that AMDV-G, similar to human parvoviruses in the genus of Erythrovirus (20,22,33), uses a single promoter at the left-hand end of the genome (24). This promoter encodes a pre-mRNA that is processed by alternative splicing and alternative polyadenylation to generate six mRNAs (24) (Fig.…”