scholarly journals Human Parvovirus B19 Infection Causes Cell Cycle Arrest of Human Erythroid Progenitors at Late S Phase That Favors Viral DNA Replication

2013 ◽  
Vol 87 (23) ◽  
pp. 12766-12775 ◽  
Author(s):  
Yong Luo ◽  
Steve Kleiboeker ◽  
Xuefeng Deng ◽  
Jianming Qiu
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Content ◽  
Parvovirus B19 ◽  
S Phase ◽  
Human Parvovirus B19 ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Cycle Arrest ◽  
Cellular Dna

Human parvovirus B19 (B19V) infection has a unique tropism to human erythroid progenitor cells (EPCs) in human bone marrow and the fetal liver. It has been reported that both B19V infection and expression of the large nonstructural protein NS1 arrested EPCs at a cell cycle status with a 4 N DNA content, which was previously claimed to be “G2/M arrest.” However, a B19V mutant infectious DNA (M20mTAD2) replicated well in B19V-semipermissive UT7/Epo-S1 cells but did not induce G2/M arrest (S. Lou, Y. Luo, F. Cheng, Q. Huang, W. Shen, S. Kleiboeker, J. F. Tisdale, Z. Liu, and J. Qiu, J. Virol.86:10748–10758, 2012). 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 M20mTAD2mutant, 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.

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

2017 ◽  
Vol 92 (5) ◽  
Author(s):  
Wei Zou ◽  
Zekun Wang ◽  
Min Xiong ◽  
Aaron Yun Chen ◽  
Peng Xu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Replication ◽  
Dna Damage Response ◽  
Parvovirus B19 ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Cycle Arrest ◽  
Damage Response ◽  
Cellular Dna

ABSTRACTHuman 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.IMPORTANCEHuman 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.

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

2018 ◽  
Vol 93 (1) ◽  
Author(s):  
Peng Xu ◽  
Aaron Yun Chen ◽  
Safder S. Ganaie ◽  
Fang Cheng ◽  
Weiran Shen ◽  
...  
Keyword(s):  
Dna Replication ◽  
Nonstructural Protein ◽  
Parvovirus B19 ◽  
Growth Factor Receptor ◽  
Cellular Growth ◽  
Human Parvovirus B19 ◽  
Nonstructural Proteins ◽  
Viral Dna ◽  
Viral Dna Replication

ABSTRACTLytic infection of human parvovirus B19 (B19V) takes place exclusively in human erythroid progenitor cells of bone marrow and fetal liver, which disrupts erythropoiesis. During infection, B19V expresses three nonstructural proteins (NS1, 11-kDa, and 7.5-kDa) and two structural proteins (VP1 and VP2). While NS1 is essential for B19V DNA replication, 11-kDa enhances viral DNA replication significantly. In this study, we confirmed the enhancement role of 11-kDa in viral DNA replication and elucidated the underlying mechanism. We found that 11-kDa specially interacts with cellular growth factor receptor-bound protein 2 (Grb2) during virus infection andin vitro. We determined a high affinity interaction between 11-kDa and Grb2 that has an equilibrium dissociation constant (KD) value of 18.13 nM.In vitro, one proline-rich motif was sufficient for 11-kDa to sustain a strong interaction with Grb2. In consistence,in vivoduring infection, one proline-rich motif was enough for 11-kDa to significantly reduce phosphorylation of extracellular signal-regulated kinase (ERK). Mutations of all three proline-rich motifs of 11-kDa abolished its capability to reduce ERK activity and, accordingly, decreased viral DNA replication. Transduction of a lentiviral vector encoding a short hairpin RNA (shRNA) targetingGrb2decreased the expression of Grb2 as well as the level of ERK phosphorylation, which resulted in an increase of B19V replication. These results, in concert, indicate that the B19V 11-kDa protein interacts with cellular Grb2 to downregulate ERK activity, which upregulates viral DNA replication.IMPORTANCEHuman parvovirus B19 (B19V) infection causes hematological disorders and is the leading cause of nonimmunological fetal hydrops during pregnancy. During infection, B19V expresses two structural proteins, VP1 and VP2, and three nonstructural proteins, NS1, 11-kDa, and 7.5-kDa. While NS1 is essential, 11-kDa plays an enhancing role in viral DNA replication. Here, we elucidated a mechanism underlying 11-kDa protein-regulated B19V DNA replication. 11-kDa is tightly associated with cellular growth factor receptor-bound protein 2 (Grb2) during infection.In vitro, 11-kDa interacts with Grb2 with high affinity through three proline-rich motifs, of which at least one is indispensable for the regulation of viral DNA replication. 11-kDa and Grb2 interaction disrupts extracellular signal-regulated kinase (ERK) signaling, which mediates upregulation of B19V replication. Thus, our study reveals a novel mechanism of how a parvoviral small nonstructural protein regulates viral DNA replication by interacting with a host protein that is predominately expressed in the cytoplasm.

scholarly journals Cell Cycle Arrest by Kaposi's Sarcoma-Associated Herpesvirus Replication-Associated Protein Is Mediated at both the Transcriptional and Posttranslational Levels by Binding to CCAAT/Enhancer-Binding Protein α and p21CIP-1

2003 ◽  
Vol 77 (16) ◽  
pp. 8893-8914 ◽  
Author(s):  
Frederick Y. Wu ◽  
Shizhen Emily Wang ◽  
Qi-Qun Tang ◽  
Masahiro Fujimuro ◽  
Chuang-Jiun Chiou ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Arrest ◽  
Lytic Cycle ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Cycle Arrest ◽  
P21 Proteins

ABSTRACT Lytic-cycle replication of Kaposi's sarcoma-associated herpesvirus (KSHV) in PEL cells causes G1 cell cycle arrest mediated by the virus-encoded replication-associated protein (RAP) (or K8 protein), which induces high-level expression of the cellular C/EBPα and p21 proteins. Here we have examined the mechanism of this induction at both the transcriptional and posttranslational levels. RAP proved to bind very efficiently to both C/EBPα and p21 and stabilized them by up to 10-fold from proteasome-mediated degradation in vitro. Cross-linking revealed that RAP itself forms stable dimers and tetramers in solution and forms higher-order complexes but not heterodimers with C/EBPα. Cotransfection of RAP with C/EBPα cooperatively stimulated both the C/EBPα and p21 promoters in luciferase reporter gene assays. Only the basic/leucine zipper region of RAP was needed for interaction with and stabilization of C/EBPα, but both the N-terminal and C-terminal domains were required for transcriptional augmentation. In vitro-translated RAP interfered with DNA binding by C/EBPα in electrophonetic mobility shift assay (EMSA) experiments but did not itself bind to the target C/EBPα sites or form supershifted bands. However, in endogenous chromatin immunoprecipitation (ChIP) assays with tetradecanoyl phorbol acetate-induced PEL cells, RAP proved to specifically associate with the C/EBPα promoter in vivo, but only in a C/EBPα-dependent manner, implying an in vivo piggyback interaction with DNA-bound C/EBPα. Expression of exogenous RAP (Ad-RAP) caused G1/S cell cycle arrest in human dermal microvascular endothelial cells and also induced both the C/EBPα and p21 proteins, which formed punctate nuclear patterns that colocalized with RAP in PML nuclear bodies. In the presence of RAP, C/EBPα was also efficiently recruited into viral DNA replication compartments in both infected and cotransfected cells. In support of a direct role for this interaction in viral DNA replication, three C/EBPα binding sites were identified by in vitro EMSA experiments within a 220-bp core segment of the duplicated KSHV Ori-Lyt region, and although RAP did not bind to Ori-Lyt DNA directly in vitro, both endogenous RAP and C/EBPα were found to be associated with the Ori-Lyt region by ChIP assays in lytically induced PEL cells. Finally, we found that the KSHV lytic cycle could not be triggered by either synchronizing KSHV latently infected PEL cells in G1 phase or inducing p21 in a C/EBPα-independent process.

scholarly journals Human cytomegalovirus RNA2.7 regulates host cell cycle and facilitates viral DNA replication by inhibiting RNA polymerase II phosphorylation

2018 ◽  
Author(s):  
Yujing Huang ◽  
Jing Zhang ◽  
Xin Guo ◽  
Qing Wang ◽  
Zhongyang Liu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Human Cytomegalovirus ◽  
Cell Cycle Control ◽  
S Phase ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Pol Ii

AbstractHuman cytomegalovirus (HCMV) is a ubiquitous pathogen belongs to the beta herpesvirus family. RNA2.7 is a viral long non-coding RNA accounting for more than 20% of total viral transcripts at early time of infection. By construction of RNA2.7 deleted mutant and genome transcriptomic analysis, RNA2.7 is demonstrated to repress host cellular RNA polymerase II (Pol II)-dependent transcription through inhibiting the phosphorylation of RNA polymerase II (Pol II). Co-immunoprecipitation, RNA immunoprecipitation and RNA electrophoretic mobility shift assay are followed to investigate its mechnism. A 145nt-in-length fragment in RNA2.7 is identified to bind to Pol II and block the interaction between Pol II and phosphorylated cyckin-dependent kinase 9 (phospho-CDK9). By inhibiting Pol II phosphorylation, RNA2.7 decreases the transcription and expression levels of chromatin licensing and DNA replication factor 1 (Cdt1) and cell division cycle gene 6 (Cdc6). Through above way, RNA2.7 prevents the entry of cells into S phase and facilitates viral DNA replication. Our results discover the functions of HCMV RNA2.7 in regulation of Pol II phosphorylation and cell cycle control during infection.Author summaryHuman cytomegalovirus (HCMV) RNA2.7 is a viral lncRNA that is most abundant during infection. Here we show that a 145nt-in-length fragment in RNA2.7 binds to RNA polymerase II (Pol II) and blocks the interaction between Pol II and phosphorylated cyckin-dependent kinase 9 (phospho-CDK9). By inhibiting Pol II phosphorylation, RNA2.7 decreases the transcription and expression levels of chromatin licensing and DNA replication factor 1 (Cdt1) and cell division cycle gene 6 (Cdc6), and blocks host cells entering into S phase. RNA2.7 is confirmed to facilitate viral DNA replication through decreasing Cdt1 and Cdc6. Therefore, our results discover the functions of HCMV RNA2.7 in regulation of Pol II phosphorylation and cell cycle control during infection.

scholarly journals Human Parvovirus B19 Nonstructural Protein (NS1) Induces Cell Cycle Arrest at G1 Phase

2003 ◽  
Vol 77 (5) ◽  
pp. 2915-2921 ◽  
Author(s):  
Eiji Morita ◽  
Akitoshi Nakashima ◽  
Hironobu Asao ◽  
Hiroyuki Sato ◽  
Kazuo Sugamura
Keyword(s):  
Cell Cycle ◽  
Virus Infection ◽  
Cell Cycle Arrest ◽  
Nonstructural Protein ◽  
Parvovirus B19 ◽  
Erythroid Cell ◽  
Human Parvovirus B19 ◽  
Cycle Arrest ◽  
Nonstructural Protein 1 ◽  
Ns1 Expression

ABSTRACT Human parvovirus B19 infects predominantly erythroid precursor cells, leading to inhibition of erythropoiesis. This erythroid cell damage is mediated by the viral nonstructural protein 1 (NS1) through an apoptotic mechanism. We previously demonstrated that B19 virus infection induces G2 arrest in erythroid UT7/Epo-S1 cells; however, the role of NS1 in regulating cell cycle arrest is unknown. In this report, by using paclitaxel, a mitotic inhibitor, we show that B19 virus infection induces not only G2 arrest but also G1 arrest. Interestingly, UV-irradiated B19 virus, which has inactivated the expression of NS1, still harbors the ability to induce G2 arrest but not G1 arrest. Furthermore, treatment with caffeine, a G2 checkpoint inhibitor, abrogated the B19 virus-induced G2 arrest despite expression of NS1. These results suggest that the B19 virus-induced G2 arrest is not mediated by NS1 expression. We also found that NS1-transfected UT7/Epo-S1 and 293T cells induced cell cycle arrest at the G1 phase. These results indicate that NS1 expression plays a critical role in G1 arrest induced by B19 virus. Furthermore, NS1 expression significantly increased p21/WAF1 expression, a cyclin-dependent kinase inhibitor that induces G1 arrest. Thus, G1 arrest mediated by NS1 may be a prerequisite for the apoptotic damage of erythroid progenitor cells upon B19 virus infection.

scholarly journals Identification of an Arginine-Rich Motif in Human Papillomavirus Type 1 E1^E4 Protein Necessary for E4-Mediated Inhibition of Cellular DNA Synthesis In Vitro and in Cells

2008 ◽  
Vol 82 (18) ◽  
pp. 9056-9064 ◽  
Author(s):  
Sally Roberts ◽  
Sarah R. Kingsbury ◽  
Kai Stoeber ◽  
Gillian L. Knight ◽  
Phillip H. Gallimore ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Host Cell ◽  
S Phase ◽  
Human Papillomaviruses ◽  
Soluble Form ◽  
Cellular Dna ◽  
In Cells

ABSTRACT Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G2-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1^E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.

scholarly journals Adenovirus E1B55K Region Is Required To Enhance Cyclin E Expression for Efficient Viral DNA Replication

2008 ◽  
Vol 82 (7) ◽  
pp. 3415-3427 ◽  
Author(s):  
Xinyu Zheng ◽  
Xiao-Mei Rao ◽  
Jorge G. Gomez-Gutierrez ◽  
Hongying Hao ◽  
Kelly M. McMasters ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cancer Cells ◽  
Cyclin E ◽  
Serum Starvation ◽  
Viral Dna ◽  
Human Lung Fibroblast ◽  
Viral Dna Replication ◽  
Efficient Replication ◽  
Interfering Rna

ABSTRACT Adenoviruses (Ads) with E1B55K mutations can selectively replicate in and destroy cancer cells. However, the mechanism of Ad-selective replication in tumor cells is not well characterized. We have shown previously that expression of several cell cycle-regulating genes is markedly affected by the Ad E1b gene in WI-38 human lung fibroblast cells (X. Rao, et al., Virology 350:418-428, 2006). In the current study, we show that the Ad E1B55K region is required to enhance cyclin E expression and that the failure to induce cyclin E overexpression due to E1B55K mutations prevents viral DNA from undergoing efficient replication in WI-38 cells, especially when the cells are arrested in the G0 phase of the cell cycle by serum starvation. In contrast, cyclin E induction is less dependent on the function encoded in the E1B55K region in A549 and other cancer cells that are permissive for replication of E1B55K-mutated viruses, whether the cells are in the S phase or G0 phase. The small interfering RNA that specifically inhibits cyclin E expression partially decreased viral replication. Our study provides evidence suggesting that E1B55K may be involved in cell cycle regulation that is important for efficient viral DNA replication and that cyclin E overexpression in cancer cells may be associated with the oncolytic replication of E1B55K-mutated viruses.

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