Parvovirus B19 (B19V) infection is highly restricted to human erythroid progenitor cells. Although previous studies have led to the theory that the basis of this tropism is receptor expression, this has been questioned by more recent observation. In the study reported here, we have investigated the basis of this tropism, and a potential role of erythropoietin (Epo) that EpoR signaling is absolutely required for B19V replication in ex vivo-expanded erythroid progenitor cells after initial virus entry and at least partly accounts for the remarkable tropism of B19V infection for human erythroid progenitors.Parvovirus B19 (B19V) is pathogenic to humans. It replicates autonomously and belongs to the genus Erythrovirus in the family Parvoviridae (14). Clinical manifestations of B19V infection vary among different health conditions. The most common manifestation is erythema infectiosum. However, B19V infection often results in bone marrow failure under the following conditions (9, 10, 62). In patients with increased destruction of erythrocytes and a high turnover of erythrocytes (e.g., sickle cell disease patients), acute B19V infection can cause transient aplastic crisis. In immunocompromised patients, persistent B19V infection may develop manifestations as pure red-cell aplasia, a chronic anemia. Moreover, B19V fetal infection can cause severe anemia in the fetus, resulting in nonimmune hydrops fetalis and fetal death (1,2,16,47,57).Erythropoiesis is the process whereby a fraction of primitive multipotent hematopoietic stem cells (CD34 ϩ ) commit to the erythroid lineage, forming burst-forming units-erythroid (BFU-E; earlier erythroid progenitor) cells, CFU-erythroid (CFU-E; later erythroid progenitor) cells, normoblasts, erythroblasts, reticulocytes, and ultimately, mature erythrocytes. B19V infection shows a remarkable tropism for BFU-E and CFU-E progenitors in human bone marrow and fetal livers. Notably, both cell types express the cell surface marker CD36 (30,39,50,60). The clinical manifestations of B19V infection seen in both aplastic crisis and pure red-cell aplasia are direct outcomes of cell death of the erythroid progenitors that are targets of B19V replication, and this cell death is due to direct cytotoxicity of the virus infection (9, 13). Progressive host cell apoptosis has been observed during B19V infection of erythroid progenitor cells (29,49,60), and this is likely induced during infection of the abundantly expressed 11-kDa nonstructural protein of the virus (12). Apoptosis of erythroid progenitor cells is also characteristic of B19V-induced hydrops fetalis (60).Polyadenylation at the proximal site [(pA)p], which is located in the center of the B19V genome, precludes the inclusion of the capsid-encoding open reading frame (ORF) in transcripts under some conditions (38,61). We have recently shown that replication of the B19V genome enhances readthrough of the (pA)p and, thereafter, the polyadenylation of B19V transcripts at the distal site. Therefore, replication of the B19V genome facilitates the p...

The pre-mRNA processing strategy of the B19 virus is unique among parvoviruses. B19 virus-generated pre-mRNAs are transcribed from a single promoter and are extensively processed by alternative splicing and alternative polyadenylation to generate 12 transcripts. Blockage of the production of full-length B19 virus transcripts at the internal polyadenylation site [(pA)p] was previously reported to be a limiting step in B19 virus permissiveness. We show here that in the absence of genome replication, internal polyadenylation of B19 virus RNAs at (pA)p is favored in cells which are both permissive and nonpermissive for B19 viral replication. The human parvovirus B19 virus (B19V) is a member of the Erythrovirus genus in the subfamily Parvoviridae (8, 41). B19V causes a number of human diseases, such as fifth disease in children, arthropathy, particularly in women, transient aplastic crisis in individuals with a high red cell turnover rate, pure red cell aplasia in immunocompromised patients, and hydrops fetalis following infection of pregnant women (4).The B19V virion is approximately 20 nm in diameter and contains a single-stranded DNA genome of either plus or minus polarity. The left-hand portion of the genome contains the NS-encoding region, which is essential for virus replication, transcription transactivation, and cytotoxicity. The right-hand portion harbors the sequences for the two structural proteins, VP1 and VP2. At least 12 transcripts are generated by alternative splicing and alternative polyadenylation from a single pre-mRNA that is transcribed from the single promoter at map unit 6 (P6) (Fig. 1) (24, 46). B19V transcripts polyadenylated at the proximal polyadenylation site [(pA)p] accumulate as either spliced or unspliced mRNAs. As unspliced mRNAs, they encode the nonstructural NS1 protein, which is essential for virus replication. Spliced RNAs polyadenylated internally at (pA)p encode a 7.5-kDa protein whose function is unknown. All B19V transcripts detected so far that are polyadenylated at the distal polyadenylation site [(pA)d] excise the first intron (D1 to A1-1 or D1 to A1-2) (Fig. 1). Those that are not additionally spliced encode the capsid protein VP1. RNAs in which the second intron (D2 to A2-1) is additionally spliced encode the capsid protein VP2, while those additionally spliced between D2 and A2-2 encode the nonstructural 11-kDa protein that is essential for virus export from the nuclei (38, 48). Two internal polyadenylation sites, (pA)p1 and (pA)p2, have been identified. (pA)p1, which accounts for approximately 90% of the internal polyadenylation events, has a noncanonical cleavage and polyadenylation specificity factor binding motif (AUUAAA) located at nucleotide (nt) 2819. (pA)p2, which accounts for approximately 10% of the internal polyadenylation events, has a canonical signal of AAUAAA located at nt 3115 (46). B19V has been shown to undergo productive replication in erythroid progenitors of human hematopoietic stem cells (26,37). Different disease manifestations are due to the direct...

Human parvovirus B19 (B19V) causes a variety of human diseases. Disease outcomes of bone marrow failure in patients with high turnover of red blood cells and immunocompromised conditions, and fetal hydrops in pregnant women are resulted from the targeting and destruction of specifically erythroid progenitors of the human bone marrow by B19V. Although the ex vivo expanded erythroid progenitor cells recently used for studies of B19V infection are highly permissive, they produce progeny viruses inefficiently. In the current study, we aimed to identify the mechanism that underlies productive B19V infection of erythroid progenitor cells cultured in a physiologically relevant environment. Here, we demonstrate an effective reverse genetic system of B19V, and that B19V infection of ex vivo expanded erythroid progenitor cells at 1% O2 (hypoxia) produces progeny viruses continuously and efficiently at a level of approximately 10 times higher than that seen in the context of normoxia. With regard to mechanism, we show that hypoxia promotes replication of the B19V genome within the nucleus, and that this is independent of the canonical PHD/HIFα pathway, but dependent on STAT5A and MEK/ERK signaling. We further show that simultaneous upregulation of STAT5A signaling and down-regulation of MEK/ERK signaling boosts the level of B19V infection in erythroid progenitor cells under normoxia to that in cells under hypoxia. We conclude that B19V infection of ex vivo expanded erythroid progenitor cells at hypoxia closely mimics native infection of erythroid progenitors in human bone marrow, maintains erythroid progenitors at a stage conducive to efficient production of progeny viruses, and is regulated by the STAT5A and MEK/ERK pathways.

). To further characterize cell cycle arrest during B19V infection of EPCs, we analyzed the cell cycle change using 5-bromo-2=-deoxyuridine (BrdU) pulse-labeling and DAPI (4=,6-diamidino-2-phenylindole) staining, which precisely establishes the cell cycle pattern based on both cellular DNA replication and nuclear DNA content. We found that although both B19V NS1 transduction and infection immediately arrested cells at a status of 4 N DNA content, B19V-infected 4 N cells still incorporated BrdU, indicating active DNA synthesis. Notably, the BrdU incorporation was caused neither by viral DNA replication nor by cellular DNA repair that could be initiated by B19V infection-induced cellular DNA damage. Moreover, several S phase regulators were abundantly expressed and colocalized within the B19V replication centers. More importantly, replication of the B19V wild-type infectious DNA, as well as the M20 mTAD2 mutant, arrested cells at S phase. Taken together, our results confirmed that B19V infection triggers late S phase arrest, which presumably provides cellular S phase factors for viral DNA replication. Human parvovirus B19 (B19V) is a member of the genus Erythrovirus within the family Parvoviridae. Most commonly, it causes a mild disease called "fifth disease" (1); however, under some conditions, B19V infection can be life threatening (2), e.g., hydrops fetalis in pregnant women (3-5), chronic pure red cell aplasia in immunocompromised patients (6, 7), and transient aplastic crisis in sickle cell disease patients (8-10).B19V has a single-stranded DNA genome that is flanked by two identical terminal repeats (11). Under a single p6 promoter, the B19V genome expresses one large nonstructural protein (NS1), two small (11-kDa and 7.5-kDa) nonstructural proteins, and two capsid proteins (VP1 and VP2) (12). B19V infection is restricted to human erythroid progenitor cells (EPCs) of human bone marrow and the fetal liver (5, 13-15). Previously, only a few semipermissive cell lines, such as the human megakaryoblastoid cell line UT7/Epo-S1 (16, 17), were found to support B19V replication. Recently, ex vivo-expanded human primary CD36 ϩ EPCs, which are differentiated from CD34 ϩ hematopoietic stem cells, have been shown to be highly permissive to B19V infection (18)(19)(20). Although B19V replication in UT7/Epo-S1 cells is less efficient (21, 22), the cells can be transfected with a B19V infectious DNA (M20) (23) and produce infectious virions under hypoxic conditions (22).B19V infection of both UT7/Epo-S1 cells and CD36 ϩ EPCs quickly arrests host cells in a tetraploid state (4 N DNA content) (17,24,25), which was previously thought to be "G 2 /M arrest." Expression of B19V NS1 per se in CD36ϩ EPCs was identified as capable of inducing EPCs arrested at a 4 N DNA content through deregulation of the E2F family transcription factors (24). However, it is generally accepted that autonomous parvoviruses rely on host cells at S phase for viral DNA amplification (26-32), because of the simplicity of parvovirus genome structures. In ad...

Human parvovirus B19 (B19V) infection is restricted to erythroid progenitor cells of the human bone marrow. Although the mechanism by which the B19V genome replicates in these cells has not been studied in great detail, accumulating evidence has implicated involvement of the cellular DNA damage machinery in this process. Here, we report that, in ex vivo-expanded human erythroid progenitor cells, B19V infection induces a broad range of DNA damage responses by triggering phosphorylation of all the upstream kinases of each of three repair pathways: ATM (ataxia-telangiectasi mutated), ATR (ATM and Rad3 related), and DNA-PKcs (DNA-dependent protein kinase catalytic subunit). We found that phosphorylated ATM, ATR, and DNA-PKcs, and also their downstream substrates and components (Chk2, Chk1, and Ku70/Ku80 complex, respectively), localized within the B19V replication center. Notably, inhibition of kinase phosphorylation (through treatment with either kinase-specific inhibitors or kinase-specific shRNAs) revealed requirements for signaling of ATR and DNA-PKcs, but not ATM, in virus replication. Inhibition of the ATR substrate Chk1 led to similar levels of decreased virus replication, indicating that signaling via the ATR-Chk1 pathway is critical to B19V replication. Notably, the cell cycle arrest characteristic of B19V infection was not rescued by interference with the activity of any of the three repair pathway kinases.

Human parvovirus B19 (B19V) infection is highly restricted to human erythroid progenitor cells, in which it induces a DNA damage response (DDR). The DDR signaling is mainly mediated by the ATR (ataxia telangiectasia-mutated and Rad3-related) pathway, which promotes replication of the viral genome; however, the exact mechanisms employed by B19V to take advantage of the DDR for virus replication remain unclear. In this study, we focused on the initiators of the DDR and the role of the DDR in cell cycle arrest during B19V infection. We examined the role of individual viral proteins, which were delivered by lentiviruses, in triggering a DDR in ex vivo -expanded primary human erythroid progenitor cells and the role of DNA replication of the B19V double-stranded DNA (dsDNA) genome in a human megakaryoblastoid cell line, UT7/Epo-S1 (S1). All the cells were cultured under hypoxic conditions. The results showed that none of the viral proteins induced phosphorylation of H2AX or replication protein A32 (RPA32), both hallmarks of a DDR. However, replication of the B19V dsDNA genome was capable of inducing the DDR. Moreover, the DDR per se did not arrest the cell cycle at the G 2 /M phase in cells with replicating B19V dsDNA genomes. Instead, the B19V nonstructural 1 (NS1) protein was the key factor in disrupting the cell cycle via a putative transactivation domain operating through a p53-independent pathway. Taken together, the results suggest that the replication of the B19V genome is largely responsible for triggering a DDR, which does not perturb cell cycle progression at G 2 /M significantly, during B19V infection.

Human parvovirus B19 (B19V) infection shows a strong erythroid tropism and drastically destroys erythroid progenitor cells, thus leading to most of the disease outcomes associated with B19V infection. In this study, we systematically examined the 3 B19V nonstructural proteins, 7.5kDa, 11kDa, and NS1, for their function in inducing apoptosis in transfection of primary ex vivo-expanded erythroid progenitor cells, in comparison with apoptosis induced during B19V infection. Our results show that 11kDa is a more significant inducer of apoptosis than NS1, whereas 7.5kDa does not induce apoptosis. Furthermore, we determined that caspase-10, an initiator caspase in death receptor signaling, is the most active caspase in apoptotic erythroid progenitors induced by 11kDa and NS1 as well as during B19V infection. More importantly, cytoplasm-localized 11kDa is expressed at least 100 times more than nucleuslocalized NS1 at the protein level in primary erythroid progenitor cells infected with B19V; and inhibition of 11kDa expression using antisense oligos targeting specifically to the 11kDa-encoding mRNAs reduces apoptosis significantly during B19V infection of erythroid progenitor cells. Taken together, these results demonstrate that the 11kDa protein contributes to erythroid progenitor cell death during B19V infection. (Blood.

Human parvovirus B19 (B19V) infection of primary human erythroid progenitor cells (EPCs) arrests infected cells at both late S-phase and G2-phase, which contain 4N DNA. B19V infection induces a DNA damage response (DDR) that facilitates viral DNA replication but is dispensable for cell cycle arrest at G2-phase; however, a putative C-terminal transactivation domain (TAD2) within NS1 is responsible for G2-phase arrest. To fully understand the mechanism underlying B19V NS1-induced G2-phase arrest, we established two doxycycline-inducible B19V-permissive UT7/Epo-S1 cell lines that express NS1 or NS1mTAD2, and examined the function of the TAD2 domain during G2-phase arrest. The results confirm that the NS1 TAD2 domain plays a pivotal role in NS1-induced G2-phase arrest. Mechanistically, NS1 transactivated cellular gene expression through the TAD2 domain, which was itself responsible for ATR (ataxia-telangiectasia mutated and Rad3-related) activation. Activated ATR phosphorylated CDC25C at serine 216, which in turn inactivated the cyclin B/CDK1 complex without affecting nuclear import of the complex. Importantly, we found that the ATR-CHK1-CDC25C-CDK1 pathway was activated during B19V infection of EPCs, and that ATR activation played an important role in B19V infection-induced G2-phase arrest.