SMC1-Mediated Intra-S-Phase Arrest Facilitates Bocavirus DNA Replication

Luo, Yong; Deng, Xuefeng; Cheng, Fang; Li, Yang; Qiu, Jianming

doi:10.1128/jvi.03396-12

Cited by 36 publications

(56 citation statements)

References 88 publications

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“…At the indicated times p.i. or p.t., the cells were incubated with BrdU for 1 h, followed by treatment with 1 N HCl (30). The treated cells were costained with anti-B19V NS1 and anti-BrdU antibodies and DAPI for cell cycle analysis by flow cytometry.…”

Section: Cd34mentioning

confidence: 99%

“…Total DNA (both cellular DNA and viral DNA) was extracted using the DNeasy Blood and Tissue Kit (Qiagen). The kit has been optimized for purification of total DNA from virus-infected tissues and had a recovery rate of over 90% for parvoviral DNA (30). The extracted DNA was diluted in 100 l of deionized H 2 O.…”

Section: Cd34mentioning

confidence: 99%

“…Five microliters of the denatured DNA samples was pipetted onto a nitrocellulose membrane. The DNA dots on the membrane were analyzed by a BrdU-based dot blot assay that we recently established for studying DNA replication in minute virus of canines (MVC) (30).…”

Section: Cd34mentioning

confidence: 99%

“…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).…”

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

Luo

Kleiboeker²,

Deng

et al. 2013

J Virol

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). 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...

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Section: Cd34mentioning

confidence: 99%

Section: Cd34mentioning

confidence: 99%

Section: Cd34mentioning

confidence: 99%

mentioning

confidence: 99%

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

Luo

Kleiboeker²,

Deng

et al. 2013

J Virol

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“…This response governs the virusinduced cell cycle arrest and is required for efficient parvovirus replication (26,(33)(34)(35)(36). Parvoviral genomes have unique singlestranded DNA structures with hairpins at both ends.…”

mentioning

confidence: 99%

The ATR Signaling Pathway Is Disabled during Infection with the Parvovirus Minute Virus of Mice

Adeyemi

Pintel

2014

J Virol

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The ATR kinase has essential functions in maintenance of genome integrity in response to replication stress. ATR is recruited to RPA-coated single-stranded DNA at DNA damage sites via its interacting partner, ATRIP, which binds to the large subunit of RPA. ATR activation typically leads to activation of the Chk1 kinase among other substrates. We show here that, together with a number of other DNA repair proteins, both ATR and its associated protein, ATRIP, were recruited to viral nuclear replication compartments (autonomous parvovirus-associated replication [APAR] bodies) during replication of the single-stranded parvovirus minute virus of mice (MVM). Chk1, however, was not activated during MVM infection even though viral genomes bearing bound RPA, normally a potent trigger of ATR activation, accumulate in APAR bodies. Failure to activate Chk1 in response to MVM infection was likely due to our observation that Rad9 failed to associate with chromatin at MVM APAR bodies. Additionally, early in infection, prior to the onset of the virus-induced DNA damage response (DDR), stalling of the replication of MVM genomes with hydroxyurea (HU) resulted in Chk1 phosphorylation in a virus dose-dependent manner. However, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to HU and various other drug treatments. Finally, ATR phosphorylation became undetectable upon MVM infection, and although virus infection induced RPA32 phosphorylation on serine 33, an ATR-associated phosphorylation site, this phosphorylation event could not be prevented by ATR depletion or inhibition. Together our results suggest that MVM infection disables the ATR signaling pathway. IMPORTANCEUpon infection, the parvovirus MVM activates a cellular DNA damage response that governs virus-induced cell cycle arrest and is required for efficient virus replication. ATM and ATR are major cellular kinases that coordinate the DNA damage response to diverse DNA damage stimuli. Although a significant amount has been discovered about ATM activation during parvovirus infection, involvement of the ATR pathway has been less studied. During MVM infection, Chk1, a major downstream target of ATR, is not detectably phosphorylated even though viral genomes bearing the bound cellular single-strand binding protein RPA, normally a potent trigger of ATR activation, accumulate in viral replication centers. ATR phosphorylation also became undetectable. In addition, upon establishment of full viral replication, MVM infection prevented activation of Chk1 in response to hydroxyurea and various other drug treatments. Our results suggest that MVM infection disables this important cellular signaling pathway.

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Effects of SMC1A on immune microenvironment and cancer stem cells in colon adenocarcinoma

Zhou

Liu

et al. 2023

Cancer Medicine

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Background Our previous study suggested that SMC1 has significant functions in colorectal cancer (CRC). However, few reports have shown the effects of structural maintenance of chromosomes 1 (SMC1A) on the immune microenvironment and tumor stem cells. Methods The Cancer Genome Atlas (TCGA) database, CPTAC database, Human Protein Atlas (HPA) database, the Cancer Cell Line Encyclopedia (CCLE) and Tumor Immune Single‐cell Hub were used. Flow cytometry and immunohistochemical analysis were checked for immune infiltration on MC38 mice model. Human CRC tissues were tested with RT‐qPCR. Results The mRNA and protein levels of SMC1A were increased in colon adenocarcinoma (COAD) samples. SMC1A was associated with DNA activity. Interestingly, SMC1A was highly expressed in many types of immune cells at single‐cell levels. Moreover, the high expression of SMC1A was positively correlated with immune infiltration, and immunohistochemical analysis showed that SMC1A was positively associated with CD45 expression in MC38 mice model. Also, the percentage of IL4+CD4+ T cells (Th2) and FoxP3+CD4+ T cells (Tregs) was significantly higher in the SMC1A overexpression group than in control by flow cytometry assay in vivo. SMC1A expression could affect the proliferation of T cells in the mice model. The mutation and somatic cell copy number variation (SCNV) of SMC1A were also associated with immune cell infiltration. In addition to SMC1A in the “hot” T‐cell inflammatory microenvironment of colon cancer, SMC1A also positively correlates with the immune checkpoint genes CD274, CTLA4, and PDCD1 in colon adenocarcinoma (COAD) samples. Furthermore, we also found that SMC1A plays a positive correlation with the induction of cancer stem cells (CSCs). Our results also showed that miR‐23b‐3p binds SMC1A. Conclusion SMC1A may be a bidirectional target switch that simultaneously regulates the immune microenvironment and tumor stem cells. Moreover, SMC1A may be a biomarker for the prediction of immune checkpoint inhibitor (ICI) therapy.

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SMC1-Mediated Intra-S-Phase Arrest Facilitates Bocavirus DNA Replication

Cited by 36 publications

References 88 publications

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

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

The ATR Signaling Pathway Is Disabled during Infection with the Parvovirus Minute Virus of Mice

Effects of SMC1A on immune microenvironment and cancer stem cells in colon adenocarcinoma

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