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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 in mammalian cells. a Stable expression of HIV-1 rev gene in 293VE632 cells. The inducible pZH-1-rev plasmid was transfected into 293VE632 cells. The rev gene was expressed following addition of muristerone A and confirmed by Western blot analysis using a HIV-1 Rev monoclonal antibody. Lane “Off”, rev-repressing 293VE632 cells; lane “On”, rev-expressing 293VE632 cells. Western blot shows a 13 kDa protein band that was reacted to the anti-Rev antibody. β-actin was used as a loading control. b The 293VE632 cells containing the pZH-1-rev plasmid were grown under gene-repressing (rev-Off) and gene-inducing (rev-On) conditions for up to 7 days. The cells were collected every 24 h, and the cell numbers were counted with Tryptan blue staining (Sigma-Aldrich) [69]. The growth curves were generated with an average of three different experiments. c Rev does not affect mammalian cell cycle. The 293VE632 cells transfected with pZH-1 and pZH-1-rev plasmids were cultured without muristerone A (rev-Off) or with muristerone A (rev-On). DNA contents of the cells were determined by flow cytometric analyses after 5 days of culture. The upright numbers are the percentage of cells in the G1, S and G2 phases of the cell cycle. The 293VE632 cells containing pZH-1 plasmid was used as a control (upper row).
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Fig6: Effect of HIV-1 Rev in mammalian cells. a Stable expression of HIV-1 rev gene in 293VE632 cells. The inducible pZH-1-rev plasmid was transfected into 293VE632 cells. The rev gene was expressed following addition of muristerone A and confirmed by Western blot analysis using a HIV-1 Rev monoclonal antibody. Lane “Off”, rev-repressing 293VE632 cells; lane “On”, rev-expressing 293VE632 cells. Western blot shows a 13 kDa protein band that was reacted to the anti-Rev antibody. β-actin was used as a loading control. b The 293VE632 cells containing the pZH-1-rev plasmid were grown under gene-repressing (rev-Off) and gene-inducing (rev-On) conditions for up to 7 days. The cells were collected every 24 h, and the cell numbers were counted with Tryptan blue staining (Sigma-Aldrich) [69]. The growth curves were generated with an average of three different experiments. c Rev does not affect mammalian cell cycle. The 293VE632 cells transfected with pZH-1 and pZH-1-rev plasmids were cultured without muristerone A (rev-Off) or with muristerone A (rev-On). DNA contents of the cells were determined by flow cytometric analyses after 5 days of culture. The upright numbers are the percentage of cells in the G1, S and G2 phases of the cell cycle. The 293VE632 cells containing pZH-1 plasmid was used as a control (upper row).

Mentions: Since HIV-1 Rev caused growth delay in S. pombe, we were interested in whether the same effect could also be observed in mammalian cells. In order to observe the effect of Rev protein on mammalian cellular growth, we created a mammalian gene inducible system to produce Rev. The HIV-1 rev gene was cloned into the mammalian cell expressing vector pZH-1 and transfected into 293VE632 cells [68]. The transfected cells were selected with hygromycin. After exerting drug selection for 2 weeks, Rev protein production was tested by inducing its expression with muristerone A. The cell lysate were collected and monoclonal anti-Rev antibody was used to confirm the expression of Rev protein (Fig. 6a). It was anticipated that if Rev affects mammalian cell growth, there should be a difference of cell numbers between rev-expressing and rev-repressing cells. The cell numbers of rev-containing 293VE632 cells were counted with Trypan blue staining under rev-repressing and rev-inducing conditions, and growth curves were generated. Indeed, there was a diverging growth differences between rev-expressing and rev-repressing cells after 5 days of culture (Fig. 6b). Finally, we tested whether the Rev-induced growth delay was due to, or at least partially due to alteration in cell cycling. To test this, 293VE632 cells containing pZH-1 and pZH-rev were cultured under the rev-repressing and rev-inducing conditions. Following gene induction for 5 days, the cells were harvested and the DNA contents were measured by flow cytometry as described in the materials and methods. No significant differences in cell cycle distributions were seen between rev-expressing and other control 293VE632 cells (Fig. 6c). Therefore, consistent with our observations in fission yeast, Rev also reduces mammalian cellular growth but does not affect its cell cycle distribution.Fig. 6


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 in mammalian cells. a Stable expression of HIV-1 rev gene in 293VE632 cells. The inducible pZH-1-rev plasmid was transfected into 293VE632 cells. The rev gene was expressed following addition of muristerone A and confirmed by Western blot analysis using a HIV-1 Rev monoclonal antibody. Lane “Off”, rev-repressing 293VE632 cells; lane “On”, rev-expressing 293VE632 cells. Western blot shows a 13 kDa protein band that was reacted to the anti-Rev antibody. β-actin was used as a loading control. b The 293VE632 cells containing the pZH-1-rev plasmid were grown under gene-repressing (rev-Off) and gene-inducing (rev-On) conditions for up to 7 days. The cells were collected every 24 h, and the cell numbers were counted with Tryptan blue staining (Sigma-Aldrich) [69]. The growth curves were generated with an average of three different experiments. c Rev does not affect mammalian cell cycle. The 293VE632 cells transfected with pZH-1 and pZH-1-rev plasmids were cultured without muristerone A (rev-Off) or with muristerone A (rev-On). DNA contents of the cells were determined by flow cytometric analyses after 5 days of culture. The upright numbers are the percentage of cells in the G1, S and G2 phases of the cell cycle. The 293VE632 cells containing pZH-1 plasmid was used as a control (upper row).
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Fig6: Effect of HIV-1 Rev in mammalian cells. a Stable expression of HIV-1 rev gene in 293VE632 cells. The inducible pZH-1-rev plasmid was transfected into 293VE632 cells. The rev gene was expressed following addition of muristerone A and confirmed by Western blot analysis using a HIV-1 Rev monoclonal antibody. Lane “Off”, rev-repressing 293VE632 cells; lane “On”, rev-expressing 293VE632 cells. Western blot shows a 13 kDa protein band that was reacted to the anti-Rev antibody. β-actin was used as a loading control. b The 293VE632 cells containing the pZH-1-rev plasmid were grown under gene-repressing (rev-Off) and gene-inducing (rev-On) conditions for up to 7 days. The cells were collected every 24 h, and the cell numbers were counted with Tryptan blue staining (Sigma-Aldrich) [69]. The growth curves were generated with an average of three different experiments. c Rev does not affect mammalian cell cycle. The 293VE632 cells transfected with pZH-1 and pZH-1-rev plasmids were cultured without muristerone A (rev-Off) or with muristerone A (rev-On). DNA contents of the cells were determined by flow cytometric analyses after 5 days of culture. The upright numbers are the percentage of cells in the G1, S and G2 phases of the cell cycle. The 293VE632 cells containing pZH-1 plasmid was used as a control (upper row).
Mentions: Since HIV-1 Rev caused growth delay in S. pombe, we were interested in whether the same effect could also be observed in mammalian cells. In order to observe the effect of Rev protein on mammalian cellular growth, we created a mammalian gene inducible system to produce Rev. The HIV-1 rev gene was cloned into the mammalian cell expressing vector pZH-1 and transfected into 293VE632 cells [68]. The transfected cells were selected with hygromycin. After exerting drug selection for 2 weeks, Rev protein production was tested by inducing its expression with muristerone A. The cell lysate were collected and monoclonal anti-Rev antibody was used to confirm the expression of Rev protein (Fig. 6a). It was anticipated that if Rev affects mammalian cell growth, there should be a difference of cell numbers between rev-expressing and rev-repressing cells. The cell numbers of rev-containing 293VE632 cells were counted with Trypan blue staining under rev-repressing and rev-inducing conditions, and growth curves were generated. Indeed, there was a diverging growth differences between rev-expressing and rev-repressing cells after 5 days of culture (Fig. 6b). Finally, we tested whether the Rev-induced growth delay was due to, or at least partially due to alteration in cell cycling. To test this, 293VE632 cells containing pZH-1 and pZH-rev were cultured under the rev-repressing and rev-inducing conditions. Following gene induction for 5 days, the cells were harvested and the DNA contents were measured by flow cytometry as described in the materials and methods. No significant differences in cell cycle distributions were seen between rev-expressing and other control 293VE632 cells (Fig. 6c). Therefore, consistent with our observations in fission yeast, Rev also reduces mammalian cellular growth but does not affect its cell cycle distribution.Fig. 6

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