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Molecular characterization of HIV-1 genome in fission yeast Schizosaccharomyces pombe.

Nkeze J, Li L, Benko Z, Li G, Zhao RY - Cell Biosci (2015)

Bottom Line: Three viral proteins, viral protein R (Vpr), protease (PR) and regulator of expression of viral protein (Rev), were found to inhibit cellular proliferation.Mechanistic testing of the Rev effect suggests it triggers transient induction of cellular oxidative stress.Some of the behavioral and functional similarities of Rev between fission yeast and mammalian cells suggest fission yeast might be a useful model system for further studies of molecular functions of Rev and other HIV-1 viral proteins.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Pathology, Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201-1192 USA.

ABSTRACT

Background: The human immunodeficiency virus type 1 (HIV-1) genome (~9 kb RNA) is flanked by two long terminal repeats (LTR) promoter regions with nine open reading frames, which encode Gag, Pol and Env polyproteins, four accessory proteins (Vpu, Vif, Vpr, Nef) and two regulatory proteins (Rev, Tat). In this study, we carried out a genome-wide and functional analysis of the HIV-1 genome in fission yeast (Schizosaccharomyces pombe).

Results: Each one of the HIV-1 genes was cloned and expressed individually in fission yeast. Subcellular localization of each viral protein was first examined. The effect of protein expression on cellular proliferation and colony formations, an indication of cytotoxicity, were observed. Overall, there is a general correlation of subcellular localization of each viral protein between fission yeast and mammalian cells. Three viral proteins, viral protein R (Vpr), protease (PR) and regulator of expression of viral protein (Rev), were found to inhibit cellular proliferation. Rev was chosen for further analysis in fission yeast and mammalian cells. Consistent with the observation in fission yeast, expression of HIV-1 rev gene also caused growth retardation in mammalian cells. However, the observed growth delay was neither due to the cytotoxic effect nor due to alterations in cell cycling. Mechanistic testing of the Rev effect suggests it triggers transient induction of cellular oxidative stress.

Conclusions: Some of the behavioral and functional similarities of Rev between fission yeast and mammalian cells suggest fission yeast might be a useful model system for further studies of molecular functions of Rev and other HIV-1 viral proteins.

No MeSH data available.


Related in: MedlinePlus

Effect of HIV-1 Rev protein production on fission yeast. a Expression of HIV-1 rev delays cellular growth of S. pombe. S. pombe cells containing pYZ1N-rev was grown under gene-repressing (+T) and gene-inducing (−T) conditions in liquid minimal selection EMM at 30°C. The cell density was measured over time and growth curves were plotted. S. pombe cell containing pYZ1N was used as a control. The experiment was repeated three times and the standard errors of each time point were calculated. b Rev delays colony formation in fission yeast cells. A semi-quantitative colony dot dilution assay was use to evaluate the ability of individual rev-expressing S. pombe cells to form colonies on agar plates. The pYZ1N-rev transformed S. pombe cells were grown in liquid EMM medium to log phase with thiamine. Thiamine was then removed from cells by washing and equal number of cells transferred to EMM supplemented plates with (gene-off, left plate) and without (gene-on, right plate) thiamine. The plates were incubated at 30°C for 5 days. Each colony on a plate from left to right (1–6) represents cells plated from approximately 1,000 to 3 cells following threefold dilutions. The pYZ1N plasmid was used as a control. c Rev does not affect cell cycle of fission yeast. Cell cycle profiles were measured in the pYZ1N-rev and pYZ1N control S. pombe cells by flow cytometric analysis. The data show analyses after 24 and 67 h of cell culture in EMM selective media with (gene-off) and without (gene-on) thiamine. The cell cycle stages were monitored after staining with propidium iodide. The up-right numbers are the percentage of cells in G1, S and G2 phases of the cell cycle.
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Fig4: Effect of HIV-1 Rev protein production on fission yeast. a Expression of HIV-1 rev delays cellular growth of S. pombe. S. pombe cells containing pYZ1N-rev was grown under gene-repressing (+T) and gene-inducing (−T) conditions in liquid minimal selection EMM at 30°C. The cell density was measured over time and growth curves were plotted. S. pombe cell containing pYZ1N was used as a control. The experiment was repeated three times and the standard errors of each time point were calculated. b Rev delays colony formation in fission yeast cells. A semi-quantitative colony dot dilution assay was use to evaluate the ability of individual rev-expressing S. pombe cells to form colonies on agar plates. The pYZ1N-rev transformed S. pombe cells were grown in liquid EMM medium to log phase with thiamine. Thiamine was then removed from cells by washing and equal number of cells transferred to EMM supplemented plates with (gene-off, left plate) and without (gene-on, right plate) thiamine. The plates were incubated at 30°C for 5 days. Each colony on a plate from left to right (1–6) represents cells plated from approximately 1,000 to 3 cells following threefold dilutions. The pYZ1N plasmid was used as a control. c Rev does not affect cell cycle of fission yeast. Cell cycle profiles were measured in the pYZ1N-rev and pYZ1N control S. pombe cells by flow cytometric analysis. The data show analyses after 24 and 67 h of cell culture in EMM selective media with (gene-off) and without (gene-on) thiamine. The cell cycle stages were monitored after staining with propidium iodide. The up-right numbers are the percentage of cells in G1, S and G2 phases of the cell cycle.

Mentions: To further differentiate the inhibitory effect of HIV-1 Rev protein on the cellular growth or toxicity of yeast cells, we measured the growth kinetics of S. pombe cells with and without rev gene expression. S. pombe cells containing pYZ1N-rev and pYZ1N control vectors were grown under gene-repressing (+T) and gene-inducing (−T) conditions in liquid minimal and selective EMM medium. Cellular growth was measured by the optical density (OD) over time from 0 to 122 h (5 days). At the first 24 h, both cells grown at the same pace with a doubling time of approx. 24 h. After 24 h of gene induction when the rev gene expression was fully expressed [34, 65], the growth velocity of Rev-producing cells became slower than those without rev gene expression that was grown actively in a logarithmic fashion (Fig. 4a). By 48 h, i.e., 2 days after rev gene induction, the difference in growth between rev-expressing and rev-repressing cells reached to the maximum level. As control, the pYZ1N transformed yeast cells showed no difference in growth of neither gene-repressing nor gene-inducing condition.Fig. 4


Molecular characterization of HIV-1 genome in fission yeast Schizosaccharomyces pombe.

Nkeze J, Li L, Benko Z, Li G, Zhao RY - Cell Biosci (2015)

Effect of HIV-1 Rev protein production on fission yeast. a Expression of HIV-1 rev delays cellular growth of S. pombe. S. pombe cells containing pYZ1N-rev was grown under gene-repressing (+T) and gene-inducing (−T) conditions in liquid minimal selection EMM at 30°C. The cell density was measured over time and growth curves were plotted. S. pombe cell containing pYZ1N was used as a control. The experiment was repeated three times and the standard errors of each time point were calculated. b Rev delays colony formation in fission yeast cells. A semi-quantitative colony dot dilution assay was use to evaluate the ability of individual rev-expressing S. pombe cells to form colonies on agar plates. The pYZ1N-rev transformed S. pombe cells were grown in liquid EMM medium to log phase with thiamine. Thiamine was then removed from cells by washing and equal number of cells transferred to EMM supplemented plates with (gene-off, left plate) and without (gene-on, right plate) thiamine. The plates were incubated at 30°C for 5 days. Each colony on a plate from left to right (1–6) represents cells plated from approximately 1,000 to 3 cells following threefold dilutions. The pYZ1N plasmid was used as a control. c Rev does not affect cell cycle of fission yeast. Cell cycle profiles were measured in the pYZ1N-rev and pYZ1N control S. pombe cells by flow cytometric analysis. The data show analyses after 24 and 67 h of cell culture in EMM selective media with (gene-off) and without (gene-on) thiamine. The cell cycle stages were monitored after staining with propidium iodide. The up-right numbers are the percentage of cells in G1, S and G2 phases of the cell cycle.
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4549081&req=5

Fig4: Effect of HIV-1 Rev protein production on fission yeast. a Expression of HIV-1 rev delays cellular growth of S. pombe. S. pombe cells containing pYZ1N-rev was grown under gene-repressing (+T) and gene-inducing (−T) conditions in liquid minimal selection EMM at 30°C. The cell density was measured over time and growth curves were plotted. S. pombe cell containing pYZ1N was used as a control. The experiment was repeated three times and the standard errors of each time point were calculated. b Rev delays colony formation in fission yeast cells. A semi-quantitative colony dot dilution assay was use to evaluate the ability of individual rev-expressing S. pombe cells to form colonies on agar plates. The pYZ1N-rev transformed S. pombe cells were grown in liquid EMM medium to log phase with thiamine. Thiamine was then removed from cells by washing and equal number of cells transferred to EMM supplemented plates with (gene-off, left plate) and without (gene-on, right plate) thiamine. The plates were incubated at 30°C for 5 days. Each colony on a plate from left to right (1–6) represents cells plated from approximately 1,000 to 3 cells following threefold dilutions. The pYZ1N plasmid was used as a control. c Rev does not affect cell cycle of fission yeast. Cell cycle profiles were measured in the pYZ1N-rev and pYZ1N control S. pombe cells by flow cytometric analysis. The data show analyses after 24 and 67 h of cell culture in EMM selective media with (gene-off) and without (gene-on) thiamine. The cell cycle stages were monitored after staining with propidium iodide. The up-right numbers are the percentage of cells in G1, S and G2 phases of the cell cycle.
Mentions: To further differentiate the inhibitory effect of HIV-1 Rev protein on the cellular growth or toxicity of yeast cells, we measured the growth kinetics of S. pombe cells with and without rev gene expression. S. pombe cells containing pYZ1N-rev and pYZ1N control vectors were grown under gene-repressing (+T) and gene-inducing (−T) conditions in liquid minimal and selective EMM medium. Cellular growth was measured by the optical density (OD) over time from 0 to 122 h (5 days). At the first 24 h, both cells grown at the same pace with a doubling time of approx. 24 h. After 24 h of gene induction when the rev gene expression was fully expressed [34, 65], the growth velocity of Rev-producing cells became slower than those without rev gene expression that was grown actively in a logarithmic fashion (Fig. 4a). By 48 h, i.e., 2 days after rev gene induction, the difference in growth between rev-expressing and rev-repressing cells reached to the maximum level. As control, the pYZ1N transformed yeast cells showed no difference in growth of neither gene-repressing nor gene-inducing condition.Fig. 4

Bottom Line: Three viral proteins, viral protein R (Vpr), protease (PR) and regulator of expression of viral protein (Rev), were found to inhibit cellular proliferation.Mechanistic testing of the Rev effect suggests it triggers transient induction of cellular oxidative stress.Some of the behavioral and functional similarities of Rev between fission yeast and mammalian cells suggest fission yeast might be a useful model system for further studies of molecular functions of Rev and other HIV-1 viral proteins.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Pathology, Department of Pathology, University of Maryland School of Medicine, Baltimore, MD 21201-1192 USA.

ABSTRACT

Background: The human immunodeficiency virus type 1 (HIV-1) genome (~9 kb RNA) is flanked by two long terminal repeats (LTR) promoter regions with nine open reading frames, which encode Gag, Pol and Env polyproteins, four accessory proteins (Vpu, Vif, Vpr, Nef) and two regulatory proteins (Rev, Tat). In this study, we carried out a genome-wide and functional analysis of the HIV-1 genome in fission yeast (Schizosaccharomyces pombe).

Results: Each one of the HIV-1 genes was cloned and expressed individually in fission yeast. Subcellular localization of each viral protein was first examined. The effect of protein expression on cellular proliferation and colony formations, an indication of cytotoxicity, were observed. Overall, there is a general correlation of subcellular localization of each viral protein between fission yeast and mammalian cells. Three viral proteins, viral protein R (Vpr), protease (PR) and regulator of expression of viral protein (Rev), were found to inhibit cellular proliferation. Rev was chosen for further analysis in fission yeast and mammalian cells. Consistent with the observation in fission yeast, expression of HIV-1 rev gene also caused growth retardation in mammalian cells. However, the observed growth delay was neither due to the cytotoxic effect nor due to alterations in cell cycling. Mechanistic testing of the Rev effect suggests it triggers transient induction of cellular oxidative stress.

Conclusions: Some of the behavioral and functional similarities of Rev between fission yeast and mammalian cells suggest fission yeast might be a useful model system for further studies of molecular functions of Rev and other HIV-1 viral proteins.

No MeSH data available.


Related in: MedlinePlus