Human Parvovirus B19 DNA Replication Induces a DNA Damage Response That Is Dispensable for Cell Cycle Arrest at Phase G ₂ /M

Abstract

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 Infection Causes Cell Cycle Arrest of Human Erythroid Progenitors at Late S Phase That Favors Viral DNA Replication

“…B19V infectious clone pM20 (23), an NS1 endonuclease knockout mutant (pM20 endoϪ ), and an NS1 putative transactivation domain (TAD2) mutant (pM20 mTAD2 ) were described previously (25). Before nucleofection, the B19V DNA (M20 and its mutants) was excised from the clones by SalI digestion and purified.…”

“…Viremic plasma sample P265 (ϳ1 ϫ 10 11 genome copies [gc]/ml) was obtained from ViraCor Laboratories (Lee's Summit, MO). Virus infection was performed at a multiplicity of infection (MOI) of 1,000 gc/cell (ϳ3 fluorescence focus-forming units per cell), as described previously (25,34).…”

“…However, it is generally accepted that autonomous parvoviruses rely on host cells at S phase for viral DNA amplification (26)(27)(28)(29)(30)(31)(32), because of the simplicity of parvovirus genome structures. In addition, we recently identified a mutant B19V infectious clone DNA (M20 mTAD2 ) that bears mutations in a putative transactivation domain (TAD) of NS1 and replicates efficiently in UT7/Epo-S1 cells but without inducing G 2 /M arrest, indicating that G 2 /M arrest is dispensable for B19V DNA replication (25). Therefore, we wondered whether B19V infection creates a "pseudo-G 2 phase" environment, as some other DNA viruses do (33).…”

“…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 CD36…”

“…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 CD36…”