Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication

Abstract

Human parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this report provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication. Human parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red cell aplasia. In fetuses, B19V infection can result in nonimmune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly associated with the replicating single-stranded DNA viral genome and played a critical role in viral DNA replication. In contrast, the DNA damage response-induced phosphorylated forms of RPA32 were dispensable for viral DNA replication.

RNA Binding Motif Protein RBM45 Regulates Expression of the 11-Kilodalton Protein of Parvovirus B19 through Binding to Novel Intron Splicing Enhancers

“…Transfection. Two million UT7/Epo-S1 cells were electroporated with 2 g of SalI-linearized B19V infectious clone pM20 (67) or pM20 ISE3-mut7 in solution V using an Amaxa Nucleofector system (Lonza, Basel, Switzerland), as described previously (37,68). use of the Typhoon FLA 9000 biomolecular imager, and quantification of the bands was carried out using ImageQuant TL.…”

“…Southern blotting. Hirt DNA (lower-molecular-weight DNA) was extracted from either B19V dsDNA genome (pM20)-transfected UT7/Epo-S1 cells or B19V-infected CD36 ϩ EPCs, as reported previously (37,68). B19V RF DNA M20 excised from SalI-digested pM20 was used as a probe.…”

The 11-Kilodalton Nonstructural Protein of Human Parvovirus B19 Facilitates Viral DNA Replication by Interacting with Grb2 through Its Proline-Rich Motifs

“…Particularly, activation of ERK facilitates G 2 /M transition by phosphorylation (activation) of CDC25 (48,49). B19V replication requires S-phase factors to replicate the viral genome (50). B19V infection induces a late S-phase arrest, represented by a prolonged S phase (BrdU ϩ /DNA 4N ) at an early stage followed by a complete G 2 -phase (BrdU Ϫ /DNA 4N ) arrest at a late stage of infection (51,52).…”

Endonuclease Activity Inhibition of the NS1 Protein of Parvovirus B19 as a Novel Target for Antiviral Drug Development

“…B19V replication arrests the cell cycle at late S phase, hijacks cellular DNA replication factors present in the S phase, and replicates its genome following a rolling hairpin model of DNA replication (37)(38)(39). In principle, the B19V ssDNA genome uses the 3=-end hairpin as a self-primer (3=OH) to extend viral ssDNA into the double-stranded DNA (dsDNA) genome by cellular replication proteins (40), a step called first-strand DNA synthesis (39). The extended 3= end is presumably ligated to the 5= end of the genome to form a partial circular DNA genome.…”