Internal Polyadenylation of the Parvovirus B19 Precursor mRNA Is Regulated by Alternative Splicing

Abstract

Alternative processing of parvovirus B19 (B19V) pre-mRNA is critical to generating appropriate levels of B19V mRNA transcripts encoding capsid proteins and small nonstructural proteins. Polyadenylation of the B19V pre-mRNA at the proximal polyadenylation site ((pA)p), which prevents generation of fulllength capsid proteins encoding mRNA transcripts, has been suggested as a step that blocks B19V permissiveness. We report here that efficient splicing of the B19V pre-mRNA within the first intron (upstream of the (pA)p site) stimulated the polyadenylation; in contrast, splicing of the B19V pre-mRNA within the second intron (in which the (pA)p site resides) interfered with the polyadenylation, leading to the generation of a sufficient number of B19V mRNA transcripts polyadenylated at the distal polyadenylation site ((pA)d). We also found that splicing within the second intron and polyadenylation at the (pA)p site compete during processing of the B19V pre-mRNA. Furthermore, we discovered that the U1 RNA that binds to the 5 splice donor site of the second intron is fully responsible for inhibiting polyadenylation at the (pA)p site, whereas actual splicing, and perhaps assembly of the functional spliceosome, is not required. Finally, we demonstrated that inhibition of B19V pre-mRNA splicing within the second intron by targeting an intronic splicing enhancer using a Morpholino antisense oligonucleotide prevented B19V mRNA transcripts polyadenylated at the (pA)d site during B19V infection of human erythroid progenitors. Thus, our study reveals the mechanism by which alternative splicing coordinates alternative polyadenylation to generate full-length B19V mRNA transcripts at levels sufficient to support productive B19V infection. Human parvovirus B19 (B19V)2 is a member of the genus Erythrovirus of the family Parvoviridae. B19V causes several diseases in humans (1), the most common of which is erythema infectiosum or Fifth disease. Other diseases include acute and chronic arthropathy, transient aplastic crisis (infection of patients with a high rate of red blood cell turnover), pure red cell aplasia (infection of immunocompromised patients), and hydrops fetalis (infection of pregnant women). Like other parvoviruses, B19V contains a linear single-stranded DNA genome of approximately (ϳ)5.6 kb, which is encapsidated by an icosahedrally symmetric capsid without an envelope (2, 3). In addition to infecting only humans, B19V infection shows a remarkable tropism for human erythroid progenitor cells (4 -7).Like members in the genus Bocavirus (8, 9) and Aleutian mink disease virus (10), also of the Parvoviridae family, the transcription profile of B19V is unusual for a DNA virus in that all of the mRNA transcripts are generated from a single precursor mRNA (pre-mRNA) transcribed from a single promoter at map unit 6 (P6) and feature alternative polyadenylation and splicing (see Fig. 1A) (11, 12). The only unspliced mRNA encoding the large nonstructural protein (NS1) is polyadenylated at the proximal poly(A) site ((pA)p) and retains...

NP1 Protein of the Bocaparvovirus Minute Virus of Canines Controls Access to the Viral Capsid Genes via Its Role in RNA Processing

“…Members of the genera Protoparvovirus and Dependoparvovirus feature transcriptional transactivation of their capsid gene promoters by their nonstructural proteins for this purpose (14,15,39). For parvoviruses that have an internal polyadenylation site, such as AAV5, B19, and GPV, this site lies within an intron whose excision allows extension of the spliced RNA into the capsid gene (16,18,(40)(41)(42). For the Bocaparvoviruses, however, these potent motifs are retained in the capsid protein-encoding cytoplasmic mRNA.…”

“…NP1's effect on MVC internal polyadenylation is different from the effect on polyadenylation in the parvoviruses AAV5 and B19, which feature direct competition between splicing and polyadenylation (16,40). Recently, the HPV-16 E2 protein was reported to regulate the usage of the proximal polyadenylation cisacting elements in the middle of its genome by modulating the recruitment of the polyadenylation machinery to this site (44,45).…”

“…The Dependoparvovirus adeno-associated virus type 2 (AAV2) and Protoparvovirus minute virus of mice (MVM) use transactivation of a capsid gene promoter to generate capsid protein-encoding mRNAs (13)(14)(15). For other parvoviruses that have an internal polyadenylation site, such as adeno-associated virus type 5 (AAV5), Erythroparvovirus B19, and the Dependoparvovirus goose parvovirus (GPV), this site typically lies within an intron whose excision allows extension of the spliced RNA into the capsid gene (6,(16)(17)(18). For the bocaparvoviruses, however, these potent internal polyadenylation motifs are retained in the capsid protein-encoding cytoplasmic mRNA; thus, they must be suppressed to allow processing, export, and accumulation of the spliced capsid-encoding mRNAs in the cytoplas, and hence production of the capsid proteins.…”

Parvovirus B19 Achievements and Challenges

“…Cleavage at pAp1 will generate the most abundant classes of viral mRNAs, cleavage at pAp2 will occur at tenfold lower frequency and generate alternative mRNAs potentially including an additional small ORF for a 9 kDa protein, while readthrough will generate mRNAs extending into the capsid coding region and coding for VP1 protein. Competitive splicing from D2 to A2-1 or A2-2 sites will regulate the production of mRNAs coding for VP2 or 11 kDa proteins, respectively [101]. All of these events appear to be coordinated and in relation to the replicative process involving the DNA template.…”

Characterization of the Nonstructural Proteins of the Bocavirus Minute Virus of Canines

“…The erythroviruses (26) have a single promoter at the left-hand end of the genome and a polyadenylation site within an intron in the center of the genome. Splicing of this intron to remove the polyadenylation signal helps govern access to the capsid gene for these viruses (27). The amdoviruses (28,29) and the bocaviruses (3) have polyadenylation sites that lie within their capsid gene; however, these potent motifs are retained in the capsid-encoding mRNA and must be somehow suppressed to allow export and accumulation of mRNAs that encode capsid protein information.…”