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.
Highlights
The filoviruses, marburgviruses and ebolaviruses, are non-segmented single-stranded negative RNA viruses that cause severe hemorrhagic fever with a mortality rate ranging up to 90% in humans [1].The development of efficacious therapeutics against filoviruses is hampered, in part, by our limited knowledge of the mechanisms of virus/host interactions at the molecular level and in part, by lack of suitable tools and methods to screen large libraries of small molecules or siRNAs
Cells were subsequently stained with Hoechst 33342 dye, to measure the DNA content based on the nuclear intensity
We report the development of an high-content imaging (HCI) assay to quantitate different phases of cell cycle
Summary
The filoviruses, marburgviruses and ebolaviruses, are non-segmented single-stranded negative RNA viruses that cause severe hemorrhagic fever with a mortality rate ranging up to 90% in humans [1]. The development of efficacious therapeutics against filoviruses is hampered, in part, by our limited knowledge of the mechanisms of virus/host interactions at the molecular level and in part, by lack of suitable tools and methods to screen large libraries of small molecules or siRNAs. Recently, we reported the development of a high-throughput, high-content image based screening platform to discover novel regulators of EBOV infection [2,3]. Depending on the virus category (DNA, RNA or retroviruses), some viruses cause arrest of cells in particular phase of cell cycle so that they can replicate in the resting cells, while others induce proliferation of arrested cells [7,8]. The advantage of using HCI is that the during image analysis, several hundreds of cellular features can be extracted at the single cell level [14] Phenotypic features such as intensity and localization of fluorescently labeled cellular components such as DNA or protein as well as the size, number, texture.
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