Research Article: Parvovirus B19 NS1 protein induces cell cycle arrest at G2-phase by activating the ATR-CDC25C-CDK1 pathway

Date Published: March 6, 2017

Publisher: Public Library of Science

Author(s): Peng Xu, Zhe Zhou, Min Xiong, Wei Zou, Xuefeng Deng, Safder S. Ganaie, Steve Kleiboeker, Jianxin Peng, Kaiyu Liu, Shengqi Wang, Shui Qing Ye, Jianming Qiu, Colin Parrish.


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.

Partial Text

Human parvovirus B19 (B19V) is a small, non-enveloped, single stranded DNA (ssDNA) virus belonging to the genus Erythroparvovirus within the family Parvoviridae [1]. B19V causes fifth disease or slapped cheek syndrome in children; however, B19V infection can cause hematological disorders [2–6]. B19V infection of immunocompromised patients, such as those with HIV/AIDS, transplant recipients, and infants, leads to a persistent viremia that is associated with chronic anemia and pure red-cell aplasia. Acute B19V infection of patients experiencing increased destruction of erythrocytes, and therefore having a high demand for erythrocyte production (e.g., those with sickle-cell disease), can cause a transient aplastic crisis, whereas B19V infection of pregnant women can cause non-immune hydrops fetalis. B19V infects human erythroid progenitors at the burst-forming unit (BFU)- and colony-forming unit (CFU)-erythroid stages in the bone marrow [7–9] and fetal liver [10,11], which results in the death of infected cells [12–15]. Currently, there are no vaccines or specific antiviral therapeutics that can prevent or treat B19V-induced hematological disorders.

Here, we identified the key mechanism underlying B19V NS1-induced G2-phase arrest in NS1-expressing B19V-permissive cells. We found that the TAD2 domain at the C-terminus of NS1 plays a key role in transactivating cellular gene expression, and that it is essential for NS1-induced G2-phase arrest. We also identified a de novo role for NS1: activation of ATR. NS1 expression results in ATR phosphorylation at threonine 1989; this then inactivates CDC25C via phosphorylation at serine 216. Phosphorylated CDC25C prevents dephosphorylation of CDK1 at tyrosine15; thus, an inactive cyclin B1/CDK1 complex is imported to the nucleus, thereby blocking progression from G2- to M-phase (Fig 11). Finally, we showed that the ATR-CHK1-CDC25C-CDK1 pathway is activated in both NS1(alone)-expressing and B19V-infected CD36+ EPCs.




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