Alternative Polyadenylation of Adeno-Associated Virus Type 5 RNA within an Internal Intron Is Governed by both a Downstream Element within the Intron 3′ Splice Acceptor and an Element Upstream of the P41 Initiation Site

Qiu, Jianming; Nayak, Ramnath; Pintel, David J.

doi:10.1128/jvi.78.1.83-93.2004

Cited by 22 publications

(33 citation statements)

References 43 publications

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“…Although it is similar, to some extent, to that competition during processing of the pre-mRNA generated from AAV5 P41 promoter (34), in AAV5, the DSE of the (pA)p site and the polypyrimidine tract site overlap, and thus polyadenylation factors (e.g. CstF64) compete with splicing factors (i.e.…”

Section: Discussionmentioning

confidence: 99%

“…CstF64) compete with splicing factors (i.e. U2AF65), and this results in the inhibition of polyadenylation at the (pA)p site in ϳ50% of the molecules (34). Binding of U1 snRNP to the central donor site of the AAV5 pre-mRNA inhibits another internal polyadenylation in the other ϳ50% of the molecules.…”

Section: Discussionmentioning

confidence: 99%

“…A functional polyadenylation site within an intron plays a regulatory role in expression of papillomavirus (32), adenovirus (33), and AAV5 (34). In AAV5, different levels of internal polyadenylation are constitutively seen for transcripts generated from different promoters (34). The DSE of the internal polyadenylation site of AAV5 overlaps with the polypyrimidine tract of the 3Ј acceptor site (A2) of the intron (34).…”

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…In AAV5, different levels of internal polyadenylation are constitutively seen for transcripts generated from different promoters (34). The DSE of the internal polyadenylation site of AAV5 overlaps with the polypyrimidine tract of the 3Ј acceptor site (A2) of the intron (34). The B19V system is unique in that all transcripts are processed from a single pre-mRNA transcribed from a single promoter (P6); the core internal (pA)p site is distinct from the 3Ј acceptor of the intron in which the (pA)p site lies (Fig.…”

Section: Discussionmentioning

confidence: 99%

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

Guan

Huang

Cheng

et al. 2011

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 3 more Smart Citations

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

Guan

Huang

Cheng

et al. 2011

Journal of Biological Chemistry

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Parvoviruses

Tattersall¹,

Cotmore²

2010

Topley &Amp; Wilson's Microbiology and Microbial Infections

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Development of a Novel Recombinant Adeno-Associated Virus Production System Using Human Bocavirus 1 Helper Genes

Wang

Cheng

Engelhardt

et al. 2018

Molecular Therapy - Methods & Clinical Development

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Human bocavirus 1 (HBoV1), an autonomous parvovirus, is a helper virus supporting replication of wild-type adeno-associated virus 2 (AAV2). In this study, we compared the helper functions from HBoV1 with those from adenovirus (Ad) for the production of recombinant AAV (rAAV) vector in HEK293 cells. We demonstrated that triple plasmids transfection of (1) a cloned HBoV1 helper minigenome (pBocaHelper) that expresses HBoV1 genes NP1, NS2, and BocaSR, (2) pAAV transfer plasmid, and (3) pAAVRepCap supports rAAV production in HEK293 cells. Despite a production yield of 1–2 log lower than that using pAdHelper (expressing Ad genes E2A, E4, and VA), rAAV vector produced using pBocaHelper transduced cells as efficiently as that produced using pAdHelper. The low vector production is largely due to the inefficient expression of the AAV Rep52 and capsid proteins, as well as reduced rAAV genome replication. When the AAV capsid proteins and Rep52 were ectopically expressed under strong promoters, the enhanced protein expression significantly improved the rAAV production using pBocaHelper, approaching a level of 50%–70% of that produced using pAdHelper. Through further dissection of the helper functions from pAdHelper in a five-plasmid transfection system, we found that the addition of the Ad E2A gene to the above HBoV1 helper system significantly increased rAAV DNA replication, which increased the rAAV vector production to a level of 3–7 times higher than that using pAdHelper. We finally combined HBoV1 NP1 and NS2 genes with Ad helper genes to create a novel dual helper plasmid (pABHelper) for rAAV vector production in the conventional three-plasmid transfection system. The pABHelper facilitated rAAV production at a yield ∼2 times higher than that using the pAdHelper.

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Alternative Polyadenylation of Adeno-Associated Virus Type 5 RNA within an Internal Intron Is Governed by both a Downstream Element within the Intron 3′ Splice Acceptor and an Element Upstream of the P41 Initiation Site

Cited by 22 publications

References 43 publications

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

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

Parvoviruses

Development of a Novel Recombinant Adeno-Associated Virus Production System Using Human Bocavirus 1 Helper Genes

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