Murine Norovirus Replication Induces G0/G1Cell Cycle Arrest in Asynchronously Growing Cells

ABSTRACTMany viruses replicate most efficiently in specific phases of the cell cycle, establishing or exploiting favorable conditions for viral replication, although little is known about the relationship between caliciviruses and the cell cycle. Microarray and Western blot analysis of murine norovirus 1 (MNV-1)-infected cells showed changes in cyclin transcript and protein levels indicative of a G1phase arrest. Cell cycle analysis confirmed that MNV-1 infection caused a prolonging of the G1phase and an accumulation of cells in the G0/G1phase. The accumulation in G0/G1phase was caused by a reduction in cell cycle progression through the G1/S restriction point, with MNV-1-infected cells released from a G1arrest showing reduced cell cycle progression compared to mock-infected cells. MNV-1 replication was compared in populations of cells synchronized into specific cell cycle phases and in asynchronously growing cells. Cells actively progressing through the G1phase had a 2-fold or higher increase in virus progeny and capsid protein expression over cells in other phases of the cell cycle or in unsynchronized populations. These findings suggest that MNV-1 infection leads to prolonging of the G1phase and a reduction in S phase entry in host cells, establishing favorable conditions for viral protein production and viral replication. There is limited information on the interactions between noroviruses and the cell cycle, and this observation of increased replication in the G1phase may be representative of other members of theCaliciviridae.IMPORTANCENoroviruses have proven recalcitrant to growth in cell culture, limiting our understanding of the interaction between these viruses and the infected cell. In this study, we used the cell-culturable MNV-1 to show that infection of murine macrophages affects the G1/S cell cycle phase transition, leading to an arrest in cell cycle progression and an accumulation of cells in the G0/G1phase. Furthermore, we show that MNV replication is enhanced in the G1phase compared to other stages of the cell cycle. Manipulating the cell cycle or adapting to cell cycle responses of the host cell is a mechanism to enhance virus replication. To the best of our knowledge, this is the first report of a norovirus interacting with the host cell cycle and exploiting the favorable conditions of the G0/G1phase for RNA virus replication.

Download Full-text

Influenza A Virus Replication Induces Cell Cycle Arrest in G0/G1 Phase

Journal of Virology ◽

10.1128/jvi.01216-10 ◽

2010 ◽

Vol 84 (24) ◽

pp. 12832-12840 ◽

Cited By ~ 85

Author(s):

Yuan He ◽

Ke Xu ◽

Bjoern Keiner ◽

Jianfang Zhou ◽

Volker Czudai ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Influenza A Virus ◽

Influenza A ◽

Cell Cycle Progression ◽

Virus Production ◽

Phase Cell ◽

Cycle Progression ◽

Cycle Arrest ◽

Infected Cells

ABSTRACT Many viruses interact with the host cell division cycle to favor their own growth. In this study, we examined the ability of influenza A virus to manipulate cell cycle progression. Our results show that influenza A virus A/WSN/33 (H1N1) replication results in G0/G1-phase accumulation of infected cells and that this accumulation is caused by the prevention of cell cycle entry from G0/G1 phase into S phase. Consistent with the G0/G1-phase accumulation, the amount of hyperphosphorylated retinoblastoma protein, a necessary active form for cell cycle progression through late G1 into S phase, decreased after infection with A/WSN/33 (H1N1) virus. In addition, other key molecules in the regulation of the cell cycle, such as p21, cyclin E, and cyclin D1, were also changed and showed a pattern of G0/G1-phase cell cycle arrest. It is interesting that increased viral protein expression and progeny virus production in cells synchronized in the G0/G1 phase were observed compared to those in either unsynchronized cells or cells synchronized in the G2/M phase. G0/G1-phase cell cycle arrest is likely a common strategy, since the effect was also observed in other strains, such as H3N2, H9N2, PR8 H1N1, and pandemic swine H1N1 viruses. These findings, in all, suggest that influenza A virus may provide favorable conditions for viral protein accumulation and virus production by inducing a G0/G1-phase cell cycle arrest in infected cells.

Download Full-text

Loss of MCU prevents mitochondrial fusion in G1-S phase and blocks cell cycle progression and proliferation

Science Signaling ◽

10.1126/scisignal.aav1439 ◽

2019 ◽

Vol 12 (579) ◽

pp. eaav1439 ◽

Cited By ~ 19

Author(s):

Olha M. Koval ◽

Emily K. Nguyen ◽

Velarchana Santhana ◽

Trevor P. Fidler ◽

Sara C. Sebag ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Cell Cycle Progression ◽

Mitochondrial Fission ◽

Mitochondrial Fusion ◽

Cycle Phase ◽

Wild Type ◽

Mitochondrial Fragmentation ◽

Cycle Progression ◽

Regulatory Circuit

The role of the mitochondrial Ca2+uniporter (MCU) in physiologic cell proliferation remains to be defined. Here, we demonstrated that the MCU was required to match mitochondrial function to metabolic demands during the cell cycle. During the G1-S transition (the cycle phase with the highest mitochondrial ATP output), mitochondrial fusion, oxygen consumption, and Ca2+uptake increased in wild-type cells but not in cells lacking MCU. In proliferating wild-type control cells, the addition of the growth factors promoted the activation of the Ca2+/calmodulin-dependent kinase II (CaMKII) and the phosphorylation of the mitochondrial fission factor Drp1 at Ser616. The lack of the MCU was associated with baseline activation of CaMKII, mitochondrial fragmentation due to increased Drp1 phosphorylation, and impaired mitochondrial respiration and glycolysis. The mitochondrial fission/fusion ratio and proliferation in MCU-deficient cells recovered after MCU restoration or inhibition of mitochondrial fragmentation or of CaMKII in the cytosol. Our data highlight a key function for the MCU in mitochondrial adaptation to the metabolic demands during cell cycle progression. Cytosolic CaMKII and the MCU participate in a regulatory circuit, whereby mitochondrial Ca2+uptake affects cell proliferation through Drp1.

Download Full-text

DNA damage inhibits proteolysis of the B-type cyclin Clb5 in S. cerevisiae

Journal of Cell Science ◽

10.1242/jcs.110.15.1813 ◽

1997 ◽

Vol 110 (15) ◽

pp. 1813-1820

Author(s):

D. Germain ◽

J. Hendley ◽

B. Futcher

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Tyrosine Phosphorylation ◽

Cell Cycle Progression ◽

Cycle Phase ◽

Cycle Progression ◽

Ubiquitin Pathway ◽

Cyclin Dependent Kinases ◽

Transition Points ◽

Checkpoint Genes

Cell cycle progression is mediated by waves of specific cyclin dependent kinases (CDKs) in all eukaryotes. Cyclins are degraded by the ubiquitin pathway of proteolysis. The recent identification of several components of the cyclin proteolysis machinery has highlighted both the importance of proteolysis at multiple transition points in the cell cycle and the involvement of other substrates degraded by the same machinery. In this study, we have investigated the effects of DNA damage on the cyclin proteolytic machinery in Saccharomyces cerevisiae. We find that the half-life of the B-type cyclin Clb5 is markedly increased following DNA damage while that of G1 cyclins is not. This effect is independent of cell cycle phase. Clb5 turnover requires p34CDC28 activity. Stabilisation of Clb5 correlates with an increase in tyrosine phosphorylation of p34CDC28, but stabilisation does not require this tyrosine phosphorylation. The stabilisation is independent of the checkpoint genes Mec1 and Rad53. These observations establish a new link between the regulation of proteolysis and DNA damage.

Download Full-text

Chloroplast division checkpoint in eukaryotic algae

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1612872113 ◽

2016 ◽

Vol 113 (47) ◽

pp. E7629-E7638 ◽

Cited By ~ 23

Author(s):

Nobuko Sumiya ◽

Takayuki Fujiwara ◽

Atsuko Era ◽

Shin-ya Miyagishima

Keyword(s):

Cell Cycle ◽

Host Cell ◽

Cell Cycle Progression ◽

Chloroplast Division ◽

Dominant Negative ◽

Cyanidioschyzon Merolae ◽

Temperature Sensitive ◽

Specific Expression ◽

Cycle Progression ◽

Division Site

Chloroplasts evolved from a cyanobacterial endosymbiont. It is believed that the synchronization of endosymbiotic and host cell division, as is commonly seen in existing algae, was a critical step in establishing the permanent organelle. Algal cells typically contain one or only a small number of chloroplasts that divide once per host cell cycle. This division is based partly on the S-phase–specific expression of nucleus-encoded proteins that constitute the chloroplast-division machinery. In this study, using the red alga Cyanidioschyzon merolae, we show that cell-cycle progression is arrested at the prophase when chloroplast division is blocked before the formation of the chloroplast-division machinery by the overexpression of Filamenting temperature-sensitive (Fts) Z2-1 (Fts72-1), but the cell cycle progresses when chloroplast division is blocked during division-site constriction by the overexpression of either FtsZ2-1 or a dominant-negative form of dynamin-related protein 5B (DRP5B). In the cells arrested in the prophase, the increase in the cyclin B level and the migration of cyclin-dependent kinase B (CDKB) were blocked. These results suggest that chloroplast division restricts host cell-cycle progression so that the cell cycle progresses to the metaphase only when chloroplast division has commenced. Thus, chloroplast division and host cell-cycle progression are synchronized by an interactive restriction that takes place between the nucleus and the chloroplast. In addition, we observed a similar pattern of cell-cycle arrest upon the blockage of chloroplast division in the glaucophyte alga Cyanophora paradoxa, raising the possibility that the chloroplast division checkpoint contributed to the establishment of the permanent organelle.

Download Full-text

Evidence that the cell cycle is a series of uncoupled, memoryless phases

10.1101/283614 ◽

2018 ◽

Cited By ~ 3

Author(s):

Hui Xiao Chao ◽

Randy I. Fakhreddin ◽

Hristo K. Shimerov ◽

Rashmi J. Kumar ◽

Gaorav P. Gupta ◽

...

Keyword(s):

Cell Cycle ◽

Dna Synthesis ◽

Cell Cycle Progression ◽

Phase 1 ◽

Cell Cycle Phase ◽

Cell Cycle Checkpoints ◽

Cycle Phase ◽

Erlang Distribution ◽

Cycle Progression ◽

Gap Phase

The cell cycle is canonically described as a series of 4 phases: G1 (gap phase 1), S (DNA synthesis), G2 (gap phase 2), and M (mitosis). Various models have been proposed to describe the durations of each phase, including a two-state model with fixed S-G2-M duration and random G1 duration1,2; a “stretched” model in which phase durations are proportional3; and an inheritance model in which sister cells show correlated phase durations2,4. A fundamental challenge is to understand the quantitative laws that govern cell-cycle progression and to reconcile the evidence supporting these different models. Here, we used time-lapse fluorescence microscopy to quantify the durations of G1, S, G2, and M phases for thousands of individual cells from three human cell lines. We found no evidence of correlation between any pair of phase durations. Instead, each phase followed an Erlang distribution with a characteristic rate and number of steps. These observations suggest that each cell cycle phase is memoryless with respect to previous phase durations. We challenged this model by perturbing the durations of specific phases through oncogene activation, inhibition of DNA synthesis, reduced temperature, and DNA damage. Phase durations remained uncoupled in individual cells despite large changes in durations in cell populations. To explain this behavior, we propose a mathematical model in which the independence of cell-cycle phase durations arises from a large number of molecular factors that each exerts a minor influence on the rate of cell-cycle progression. The model predicts that it is possible to force correlations between phases by making large perturbations to a single factor that contributes to more than one phase duration, which we confirmed experimentally by inhibiting cyclin-dependent kinase 2 (CDK2). We further report that phases can show coupling under certain dysfunctional states such as in a transformed cell line with defective cell cycle checkpoints. This quantitative model of cell cycle progression explains the paradoxical observation that phase durations are both inherited and independent and suggests how cell cycle progression may be altered in disease states.

Download Full-text

Characterizing and inferring quantitative cell cycle phase in single-cell RNA-seq data analysis

10.1101/526848 ◽

2019 ◽

Cited By ~ 3

Author(s):

Chiaowen Joyce Hsiao ◽

PoYuan Tung ◽

John D. Blischak ◽

Jonathan E. Burnett ◽

Kenneth A. Barr ◽

...

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Single Cell ◽

Cell Cycle Progression ◽

Cell Types ◽

Cell Cycle Phase ◽

Cellular Heterogeneity ◽

Cycle Phase ◽

Cycle Progression ◽

Cellular Environment

AbstractCellular heterogeneity in gene expression is driven by cellular processes such as cell cycle and cell-type identity, and cellular environment such as spatial location. The cell cycle, in particular, is thought to be a key driver of cell-to-cell heterogeneity in gene expression, even in otherwise homogeneous cell populations. Recent advances in single-cell RNA-sequencing (scRNA-seq) facilitate detailed characterization of gene expression heterogeneity, and can thus shed new light on the processes driving heterogeneity. Here, we combined fluorescence imaging with scRNA-seq to measure cell cycle phase and gene expression levels in human induced pluripotent stem cells (iPSCs). Using these data, we developed a novel approach to characterize cell cycle progression. While standard methods assign cells to discrete cell cycle stages, our method goes beyond this, and quantifies cell cycle progression on a continuum. We found that, on average, scRNA-seq data from only five genes predicted a cell’s position on the cell cycle continuum to within 14% of the entire cycle, and that using more genes did not improve this accuracy. Our data and predictor of cell cycle phase can directly help future studies to account for cell-cycle-related heterogeneity in iPSCs. Our results and methods also provide a foundation for future work to characterize the effects of the cell cycle on expression heterogeneity in other cell types.

Download Full-text

Multiple RNA virus matrix proteins interact with SLD5 to manipulate host cell cycle

Journal of General Virology ◽

10.1099/jgv.0.001697 ◽

2021 ◽

Vol 102 (12) ◽

Author(s):

Li Zhu ◽

Xinyu Li ◽

Henan Xu ◽

Lifeng Fu ◽

George Fu Gao ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Host Cell ◽

Cell Cycle Progression ◽

Matrix Protein ◽

Rna Viruses ◽

Rna Virus ◽

M Protein ◽

Cycle Progression ◽

Cycle Arrest

The matrix protein of many enveloped RNA viruses regulates multiple stages of viral life cycle and has the characteristics of nucleocytoplasmic shuttling. We have previously demonstrated that matrix protein 1 (M1) of an RNA virus, influenza virus, blocks host cell cycle progression by interacting with SLD5, a member of the GINS complex, which is required for normal cell cycle progression. In this study, we found that M protein of several other RNA viruses, including VSV, SeV and HIV, interacted with SLD5. Furthermore, VSV/SeV infection and M protein of VSV/SeV/HIV induced cell cycle arrest at G0/G1 phase. Importantly, overexpression of SLD5 partially rescued the cell cycle arrest by VSV/SeV infection and VSV M protein. In addition, SLD5 suppressed VSV replication in vitro and in vivo, and enhanced type Ⅰ interferon signalling. Taken together, our results suggest that targeting SLD5 by M protein might be a common strategy used by multiple enveloped RNA viruses to block host cell cycle. Our findings provide new mechanistic insights for virus to manipulate cell cycle progression by hijacking host replication factor SLD5 during infection.

Download Full-text

DNA damage checkpoint dynamics drive cell cycle phase transitions

10.1101/137307 ◽

2017 ◽

Cited By ~ 1

Author(s):

Hui Xiao Chao ◽

Cere E. Poovey ◽

Ashley A. Privette ◽

Gavin D. Grant ◽

Hui Yan Chao ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Phase Transitions ◽

Cell Cycle Progression ◽

S Phase ◽

Cell Cycle Phase ◽

Cycle Phase ◽

Dna Damage Checkpoint ◽

Cycle Progression ◽

Dna Damage Checkpoints

ABSTRACTDNA damage checkpoints are cellular mechanisms that protect the integrity of the genome during cell cycle progression. In response to genotoxic stress, these checkpoints halt cell cycle progression until the damage is repaired, allowing cells enough time to recover from damage before resuming normal proliferation. Here, we investigate the temporal dynamics of DNA damage checkpoints in individual proliferating cells by observing cell cycle phase transitions following acute DNA damage. We find that in gap phases (G1 and G2), DNA damage triggers an abrupt halt to cell cycle progression in which the duration of arrest correlates with the severity of damage. However, cells that have already progressed beyond a proposed “commitment point” within a given cell cycle phase readily transition to the next phase, revealing a relaxation of checkpoint stringency during later stages of certain cell cycle phases. In contrast to G1 and G2, cell cycle progression in S phase is significantly less sensitive to DNA damage. Instead of exhibiting a complete halt, we find that increasing DNA damage doses leads to decreased rates of S-phase progression followed by arrest in the subsequent G2. Moreover, these phase-specific differences in DNA damage checkpoint dynamics are associated with corresponding differences in the proportions of irreversibly arrested cells. Thus, the precise timing of DNA damage determines the sensitivity, rate of cell cycle progression, and functional outcomes for damaged cells. These findings should inform our understanding of cell fate decisions after treatment with common cancer therapeutics such as genotoxins or spindle poisons, which often target cells in a specific cell cycle phase.

Download Full-text

Myxoma Virus M-T5 Protects Infected Cells from the Stress of Cell Cycle Arrest through Its Interaction with Host Cell Cullin-1

Journal of Virology ◽

10.1128/jvi.79.16.10750-10763.2005 ◽

2005 ◽

Vol 79 (16) ◽

pp. 10750-10763 ◽

Cited By ~ 66

Author(s):

J. B. Johnston ◽

G. Wang ◽

J. W. Barrett ◽

S. H. Nazarian ◽

K. Colwill ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Arrest ◽

Cell Cycle Progression ◽

Molecular Mechanisms ◽

Regulatory Protein ◽

Myxoma Virus ◽

Cycle Progression ◽

Cycle Arrest ◽

Infected Cells ◽

In Cells

ABSTRACT The myxoma virus (MV) M-T5 gene encodes an ankyrin repeat protein that is important for virus replication in cells from several species. Insight was gained into the molecular mechanisms underlying the role of M-T5 as a host range determinant when the cell cycle regulatory protein cullin-1 (cul-1) was identified as a cellular binding partner of M-T5 and found to colocalize with the protein in both nuclear and cytosolic compartments. Consistent with this interaction, infection with wild-type MV (vMyxlac) or a deletion mutant lacking M-T5 (vMyxT5KO) differentially altered cell cycle progression in a panel of permissive and nonpermissive cells. Cells infected with vMyxlac transitioned rapidly out of the G0/G1 phase and preferentially accumulated at the G2/M checkpoint, whereas infection with vMyxT5KO impeded progression through the cell cycle, resulting in a greater percentage of cells retained at G0/G1. Levels of the cul-1 substrate, p27/Kip-1, were selectively increased in cells infected with vMyxT5KO compared to vMyxlac, concurrent with decreased phosphorylation of p27/Kip-1 at Thr187 and decreased ubiquitination. Compared to cells infected with vMyxlac, cell death was increased in vMyxT5KO-infected cells following treatment with diverse stimuli known to induce cell cycle arrest, including infection itself, serum deprivation, and exposure to proteasome inhibitors or double-stranded RNA. Moreover, infection with vMyxlac, but not vMyxT5KO, was sufficient to overcome the G0/G1 arrest induced by these stimuli. These findings suggest that M-T5 regulates cell cycle progression at the G0/G1 checkpoint, thereby protecting infected cells from diverse innate host antiviral responses normally triggered by G0/G1 cell cycle arrest.

Download Full-text