High Content Image Based Analysis Identifies Cell Cycle Inhibitors as Regulators of Ebola Virus Infection

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Abstract

Viruses modulate a number of host biological responses including the cell cycle to favor their replication. In this study, we developed a high-content imaging (HCI) assay to measure DNA content and identify different phases of the cell cycle. We then investigated the potential effects of cell cycle arrest on Ebola virus (EBOV) infection. Cells arrested in G1 phase by serum starvation or G1/S phase using aphidicolin or G2/M phase using nocodazole showed much reduced EBOV infection compared to the untreated control. Release of cells from serum starvation or aphidicolin block resulted in a time-dependent increase in the percentage of EBOV infected cells. The effect of EBOV infection on cell cycle progression was found to be cell-type dependent. Infection of asynchronous MCF-10A cells with EBOV resulted in a reduced number of cells in G2/M phase with concomitant increase of cells in G1 phase. However, these effects were not observed in HeLa or A549 cells. Together, our studies suggest that EBOV requires actively proliferating cells for efficient replication. Furthermore, multiplexing of HCI based assays to detect viral infection, cell cycle status and other phenotypic changes in a single cell population will provide useful information during screening campaigns using siRNA and small molecule therapeutics.

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Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures.

Jayanta Debnath, Senthil Muthuswamy, Joan S Brugge (2003)

The three-dimensional culture of MCF-10A mammary epithelial cells on a reconstituted basement membrane results in formation of polarized, growth-arrested acini-like spheroids that recapitulate several aspects of glandular architecture in vivo. Oncogenes introduced into MCF-10A cells disrupt this morphogenetic process, and elicit distinct morphological phenotypes. Recent studies analyzing the mechanistic basis for phenotypic heterogeneity observed among different oncogenes (e.g., ErbB2, cyclin D1) have illustrated the utility of this three-dimensional culture system in modeling the biological activities of cancer genes, particularly with regard to their ability to disrupt epithelial architecture during the early aspects of carcinoma formation. Here we provide a collection of protocols to culture MCF-10A cells, to establish stable pools expressing a gene of interest via retroviral infection, as well as to grow and analyze MCF-10A cells in three-dimensional basement membrane culture.

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Influenza A virus replication induces cell cycle arrest in G0/G1 phase.

Y. Shu, Bjoern Keiner, Ze Chen … (2010)

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 G(0)/G(1)-phase accumulation of infected cells and that this accumulation is caused by the prevention of cell cycle entry from G(0)/G(1) phase into S phase. Consistent with the G(0)/G(1)-phase accumulation, the amount of hyperphosphorylated retinoblastoma protein, a necessary active form for cell cycle progression through late G(1) 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 G(0)/G(1)-phase cell cycle arrest. It is interesting that increased viral protein expression and progeny virus production in cells synchronized in the G(0)/G(1) phase were observed compared to those in either unsynchronized cells or cells synchronized in the G(2)/M phase. G(0)/G(1)-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 G(0)/G(1)-phase cell cycle arrest in infected cells.

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Cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication.

David Brooks, Julian A. Hiscox, Brian K. Dove … (2006)

In eukaryotic cells, cell growth and division occur in a stepwise, orderly fashion described by a process known as the cell cycle. The relationship between positive-strand RNA viruses and the cell cycle and the concomitant effects on virus replication are not clearly understood. We have shown that infection of asynchronously replicating and synchronized replicating cells with the avian coronavirus infectious bronchitis virus (IBV), a positive-strand RNA virus, resulted in the accumulation of infected cells in the G2/M phase of the cell cycle. Analysis of various cell cycle-regulatory proteins and cellular morphology indicated that there was a down-regulation of cyclins D1 and D2 (G1 regulatory cyclins) and that a proportion of virus-infected cells underwent aberrant cytokinesis, in which the cells underwent nuclear, but not cytoplasmic, division. We assessed the impact of the perturbations on the cell cycle for virus-infected cells and found that IBV-infected G2/M-phase-synchronized cells exhibited increased viral protein production when released from the block when compared to cells synchronized in the G0 phase or asynchronously replicating cells. Our data suggested that IBV induces a G2/M phase arrest in infected cells to promote favorable conditions for viral replication.

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Author and article information

Journal

Journal ID (nlm-ta): Viruses

Journal ID (iso-abbrev): Viruses

Journal ID (publisher-id): viruses

Title: Viruses

Publisher: MDPI

ISSN (Electronic): 1999-4915

Publication date (Electronic): 25 September 2012

Publication date Collection: October 2012

Volume: 4

Issue: 10

Pages: 1865-1877

Affiliations

[1 ] PerkinElmer, 940 Waltham, MA 02451, USA; Email: krishna.kota@ 123456amedd.army.mil

[2 ] United States Army Medical Research Institute of Infectious Diseases, 1425 Porter st, Fort Detrick, Frederick, MD 21702, USA; Email: jacqueline.g.benko.ctr@ 123456us.army.mil (J.G.B.); rajini.mudhasani@ 123456amedd.army.mil (R.M.); cary.retterer@ 123456us.army.mil (C.R.); julie.tran2@ 123456us.army.mil (J.P.T.); sina.bavari@ 123456amedd.army.mil (S.B.)

Author notes

[† ]

These authors contributed equally to this work.

[* ] Author to whom correspondence should be addressed; Email: rekha.panchal@ 123456amedd.army.mil ; Tel.: +1-301-619-4985; Fax: +1-301-619-1138.

Article

Publisher ID: viruses-04-01865

DOI: 10.3390/v4101865

PMC ID: 3497033

PubMed ID: 23202445

SO-VID: 32dfe35a-ce36-4a41-bc64-140a4a3bd897

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This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/3.0/).

High Content Image Based Analysis Identifies Cell Cycle Inhibitors as Regulators of Ebola Virus Infection

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Abstract

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MDPI: Coronavirus

Most cited references 24

Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures.

Influenza A virus replication induces cell cycle arrest in G0/G1 phase.

Cell cycle perturbations induced by infection with the coronavirus infectious bronchitis virus and their effect on virus replication.

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