Human Parvovirus B19 Induces Cell Cycle Arrest at G
            <sub>2</sub>
            Phase with Accumulation of Mitotic Cyclins

Morita, Eiji; Tada, Kotaro; Chisaka, Hiroshi; Asao, Hironobu; Sato, Hiroyuki; Yaegashi, Nobuo; Sugamura, Kazuo

doi:10.1128/jvi.75.16.7555-7563.2001

Cited by 108 publications

(139 citation statements)

References 32 publications

(36 reference statements)

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“…By using the Isolex 300i magnetic cell selection system (Baxter Healthcare Corporation, Deerfield, IL), we isolated human CD34 ϩ cells from granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSCs) from healthy donors who provided informed consent according to protocols approved by NCI IRB. UT7/Epo-S1 cells (15,25,26) were cultured in Iscove's modified Dulbecco's medium (Mediatech, Herndon, VA) enriched with 10% fetal bovine serum (HyClone, Logan, UT), penicillin, streptomycin, Lglutamine (Invitrogen, Carlsbad, CA), and 2 infectious units per milliliter of recombinant human (rhu) erythropoietin (EPO; Amgen, Thousand Oaks, CA) at 37°C with 5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

“…Only a few permissive cell lines have been described, including erythroleukemia cell lines, such as JK-1 (31,33) and KU812Ep6 (13), and megakaryoblastoid cell lines, such as MB-02 (18), UT7/Epo (25), and UT7/Epo-S1, a subclone of UT7/Epo (15). Recently, a comparative study of various cell lines that are permissive to B19V infection demonstrated that the UT7/Epo-S1 cells provided the greatest sensitivity to B19V replication and expression (38), but even these cells were only semipermissive, with limited viral production.…”

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confidence: 99%

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Ex Vivo-Generated CD36 ⁺ Erythroid Progenitors Are Highly Permissive to Human Parvovirus B19 Replication

Wong

Zhi

Filippone

et al. 2008

J Virol

119

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The pathogenic parvovirus B19 (B19V) has an extreme tropism for human erythroid progenitor cells. In vitro, only a few erythroid leukemic cell lines (JK-1 and KU812Ep6) or megakaryoblastoid cell lines (UT7/Epo and UT7/Epo-S1) with erythroid characteristics support B19V replication, but these cells are only semipermissive. By using recent advances in generating large numbers of human erythroid progenitor cells (EPCs) ex vivo from hematopoietic stem cells (HSCs), we produced a pure population of CD36 ؉ EPCs expanded and differentiated from CD34 ؉ HSCs and assessed the CD36 ؉ EPCs for their permissiveness to B19V infection. Over more than 3 weeks, cells grown in serum-free medium expanded more than 800,000-fold, and 87 to 96% of the CD36 ؉ EPCs were positive for globoside, the cellular receptor for B19V. Immunofluorescence (IF) staining showed that about 77% of the CD36 ؉ EPCs were positive for B19V infection, while about 9% of UT7/Epo-S1 cells were B19V positive. Viral DNA detected by real-time PCR increased by more than 3 logs in CD36 ؉ EPCs; the increase was 1 log in UT7/Epo-S1 cells. Due to the extensive permissivity of CD36 ؉ EPCs, we significantly improved the sensitivity of detection of infectious B19V by real-time reverse transcription-PCR and IF staining 100-and 1,000-fold, respectively, which is greater than the sensitivity of UT7/Epo-S1 cell-based methods. This is the first description of an ex vivo method to produce large numbers of EPCs that are highly permissive to B19V infection and replication, offering a cellular system that mimics in vivo infection with this pathogenic human virus.

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Ex Vivo-Generated CD36 ⁺ Erythroid Progenitors Are Highly Permissive to Human Parvovirus B19 Replication

Wong

Zhi

Filippone

et al. 2008

J Virol

119

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“…NS1 is cytotoxic and plays a crucial role in B19V pathogenesis. NS1 is a multifunctional protein that is involved in regulation of viral p6 promoter activity, DNA replication (15)(16)(17), cell cycle arrest in G 1 and G 2 phases in erythroid lineage cells (18,19), and initiation of apoptosis (20). NS1 also functions as a transcriptional transactivator, regulating a variety of viral and cellular genes, such as TNFA (21) and p21 WAF1/CIP1 (22).…”

Section: Introductionmentioning

confidence: 99%

Human parvovirus B19 causes cell cycle arrest of human erythroid progenitors via deregulation of the E2F family of transcription factors

Wan¹,

Zhi²,

Wong³

et al. 2010

J. Clin. Invest.

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Human parvovirus B19 (B19V) is the only human pathogenic parvovirus. It causes a wide spectrum of human diseases, including fifth disease (erythema infectiosum) in children and pure red cell aplasia in immunocompromised patients. B19V is highly erythrotropic and preferentially replicates in erythroid progenitor cells (EPCs). Current understanding of how B19V interacts with cellular factors to regulate disease progression is limited, due to a lack of permissive cell lines and animal models. Here, we employed a recently developed primary human CD36 + EPC culture system that is highly permissive for B19V infection to identify cellular factors that lead to cell cycle arrest after B19V infection. We found that B19V exploited the E2F family of transcription factors by downregulating activating E2Fs (E2F1 to E2F3a) and upregulating repressive E2Fs (E2F4 to E2F8) in the primary CD36 + EPCs. B19V nonstructural protein 1 (NS1) was a key viral factor responsible for altering E2F1-E2F5 expression, but not E2F6-E2F8 expression. Interaction between NS1 and E2F4 or E2F5 enhanced the nuclear import of these repressive E2Fs and induced stable G 2 arrest. NS1-induced G 2 arrest was independent of p53 activation and increased viral replication. Downstream E2F4/E2F5 targets, which are potentially involved in the progression from G 2 into M phase and erythroid differentiation, were identified by microarray analysis. These findings provide new insight into the molecular pathogenesis of B19V in highly permissive erythroid progenitors.

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“…In this study, we analyzed how alternative polyadenylation of the B19V pre-mRNA is regulated by its alternative splicing in the context of a replication-competent B19V genome in B19V nonpermissive COS-7 and B19V-permisisve UT7/Epo-S1 cells (20,25,26), as well as during B19V infection of ex vivo-expanded primary erythroid progenitor cells. Our results reveal that that alterative splicing at the D2 donor site is a dominate step in processing of the B19V pre-mRNA, attenuating polyadenylation at the (pA)p site and thereby facilitating readthrough of the B19V pre-mRNA such that both the capsid and the 11-kDa proteins are produced.…”

Section: Discussionmentioning

confidence: 99%

“…Cells were transfected with 2 g of DNA per 60-mm plate using Lipofectamine TM and PLUS TM reagent (Invitrogen) according to the manufacturer's instructions. UT7/Epo-S1 cells, which are permissive to B19V infection (20,25,26), were cultured in DMEM with 10% FCS and 2 units/ml of Epo (Epogen; Amgen, Thousand Oaks, CA) at 37°C in 5% CO 2 . Plasmids B19-N8, B19-N8A2-1U2AF, and B19-N8A2-1/ 2KO were digested with XhoI/EcoRI to recover the excised B19V DNA fragments that were used for electroporation.…”

Section: Cells Transfection and Virus Infection-cos-7 Cellsmentioning

confidence: 99%

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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Human Parvovirus B19 Induces Cell Cycle Arrest at G ₂ Phase with Accumulation of Mitotic Cyclins

Cited by 108 publications

References 32 publications

Ex Vivo-Generated CD36 ⁺ Erythroid Progenitors Are Highly Permissive to Human Parvovirus B19 Replication

Ex Vivo-Generated CD36 ⁺ Erythroid Progenitors Are Highly Permissive to Human Parvovirus B19 Replication

Human parvovirus B19 causes cell cycle arrest of human erythroid progenitors via deregulation of the E2F family of transcription factors

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

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Human Parvovirus B19 Induces Cell Cycle Arrest at G 2 Phase with Accumulation of Mitotic Cyclins

Cited by 108 publications

References 32 publications

Ex Vivo-Generated CD36 + Erythroid Progenitors Are Highly Permissive to Human Parvovirus B19 Replication

Ex Vivo-Generated CD36 + Erythroid Progenitors Are Highly Permissive to Human Parvovirus B19 Replication

Human parvovirus B19 causes cell cycle arrest of human erythroid progenitors via deregulation of the E2F family of transcription factors

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

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Human Parvovirus B19 Induces Cell Cycle Arrest at G ₂ Phase with Accumulation of Mitotic Cyclins

Ex Vivo-Generated CD36 ⁺ Erythroid Progenitors Are Highly Permissive to Human Parvovirus B19 Replication

Ex Vivo-Generated CD36 ⁺ Erythroid Progenitors Are Highly Permissive to Human Parvovirus B19 Replication