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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

Subcellular localization of HIV-1 proteins in fission yeast. Fission yeast strains expressing normal GFP or N-terminally GFP-tagged HIV-1 proteins were grown to a log phase in EMM selective media. The cells were re-inoculated into fresh media without thiamine (to induce gene expression) and grown for 24–30 h. The nuclei were stained with DAPI. The cells were examined using fluorescence microscopy for subcellular localizations of the GFP-tagged proteins with stained cellular nuclei. Each column represents different microscopic views: GFP, for protein subcellular location; DAPI, for localization of the nucleus; Merge, merging images of GFP and DAPI; and the “Contrast” is for the overall view of the cell. The Gag gene products are shown in a, the Pol and Env gene products in b, and the auxiliary and regulatory proteins in c. The scale bar represents 10 μm.
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Fig2: Subcellular localization of HIV-1 proteins in fission yeast. Fission yeast strains expressing normal GFP or N-terminally GFP-tagged HIV-1 proteins were grown to a log phase in EMM selective media. The cells were re-inoculated into fresh media without thiamine (to induce gene expression) and grown for 24–30 h. The nuclei were stained with DAPI. The cells were examined using fluorescence microscopy for subcellular localizations of the GFP-tagged proteins with stained cellular nuclei. Each column represents different microscopic views: GFP, for protein subcellular location; DAPI, for localization of the nucleus; Merge, merging images of GFP and DAPI; and the “Contrast” is for the overall view of the cell. The Gag gene products are shown in a, the Pol and Env gene products in b, and the auxiliary and regulatory proteins in c. The scale bar represents 10 μm.

Mentions: In order to determine the subcellular localization of HIV-1 proteins in fission yeast, SP223 cells were transformed with a fission yeast expression pYZ3N plasmid producing each of the HIV-1 viral protein sequences in fusion with an N-terminal GFP [48]. The fission yeast strains containing different viral proteins expressing plasmids were inoculated into liquid selective medium and the protein expression was induced following the removal of thiamine from the growth medium as described previously [34, 49]. After cultured for 24–30 h (depending on the intensity of green fluorescence), the GFP-viral fusion protein products were observed under a fluorescence microscope. As shown in Fig. 2a, GFP alone disperses throughout the cells indicating no preference of subcellular location. In contrast, the Gag protein aggregated in the cytoplasm of unknown sites. P17 protein was excluded from the nucleus and localized exclusively in the cytoplasm. P24 protein was localized both in the nucleus and cell membrane, while the P7 protein was predominantly localized in the nucleus. P6 was found distributed throughout the cell. The localizations of Pol proteins (P66, P51, IN, PR) and Env proteins (including Gp120 and Gp41) were monitored and shown in Fig. 2b. P66 was found more in the nucleus than in the cytoplasm; whereas P51 proteins were found exclusively in the cytoplasm. Consistent with the role of HIV-1 integrase, IN was indeed found more in the nucleus than in the cytoplasm. PR was distributed evenly throughout the cell, while Gp120 and Gp41 were localized more in the nucleus than in the cytoplasm. The subcellular distribution of HIV-1 accessory proteins (Vpr, Vpu, Vif, Nef, Rev, and Tat) was shown in Fig. 2c. Consistent with previous findings with Vpr [48], it localizes predominantly in the nucleus. Similarly, Vif, Rev, and Tat all accumulated in the nucleus of yeast cells. Nuclear localization of Tat has also been reported in budding yeast previously [50]. Vpu in cytoplasm but Nef was distributed throughout the cell. Finally, the subcellular location patterns observed in fission yeast were compared with that reported from mammalian cell studies. As summarized in Table 1, overall, there is a general correlation of the subcellular localization of each viral protein between fission yeast and that of previously reported in mammalian cells.Fig. 2


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

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

Subcellular localization of HIV-1 proteins in fission yeast. Fission yeast strains expressing normal GFP or N-terminally GFP-tagged HIV-1 proteins were grown to a log phase in EMM selective media. The cells were re-inoculated into fresh media without thiamine (to induce gene expression) and grown for 24–30 h. The nuclei were stained with DAPI. The cells were examined using fluorescence microscopy for subcellular localizations of the GFP-tagged proteins with stained cellular nuclei. Each column represents different microscopic views: GFP, for protein subcellular location; DAPI, for localization of the nucleus; Merge, merging images of GFP and DAPI; and the “Contrast” is for the overall view of the cell. The Gag gene products are shown in a, the Pol and Env gene products in b, and the auxiliary and regulatory proteins in c. The scale bar represents 10 μm.
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Related In: Results  -  Collection

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Fig2: Subcellular localization of HIV-1 proteins in fission yeast. Fission yeast strains expressing normal GFP or N-terminally GFP-tagged HIV-1 proteins were grown to a log phase in EMM selective media. The cells were re-inoculated into fresh media without thiamine (to induce gene expression) and grown for 24–30 h. The nuclei were stained with DAPI. The cells were examined using fluorescence microscopy for subcellular localizations of the GFP-tagged proteins with stained cellular nuclei. Each column represents different microscopic views: GFP, for protein subcellular location; DAPI, for localization of the nucleus; Merge, merging images of GFP and DAPI; and the “Contrast” is for the overall view of the cell. The Gag gene products are shown in a, the Pol and Env gene products in b, and the auxiliary and regulatory proteins in c. The scale bar represents 10 μm.
Mentions: In order to determine the subcellular localization of HIV-1 proteins in fission yeast, SP223 cells were transformed with a fission yeast expression pYZ3N plasmid producing each of the HIV-1 viral protein sequences in fusion with an N-terminal GFP [48]. The fission yeast strains containing different viral proteins expressing plasmids were inoculated into liquid selective medium and the protein expression was induced following the removal of thiamine from the growth medium as described previously [34, 49]. After cultured for 24–30 h (depending on the intensity of green fluorescence), the GFP-viral fusion protein products were observed under a fluorescence microscope. As shown in Fig. 2a, GFP alone disperses throughout the cells indicating no preference of subcellular location. In contrast, the Gag protein aggregated in the cytoplasm of unknown sites. P17 protein was excluded from the nucleus and localized exclusively in the cytoplasm. P24 protein was localized both in the nucleus and cell membrane, while the P7 protein was predominantly localized in the nucleus. P6 was found distributed throughout the cell. The localizations of Pol proteins (P66, P51, IN, PR) and Env proteins (including Gp120 and Gp41) were monitored and shown in Fig. 2b. P66 was found more in the nucleus than in the cytoplasm; whereas P51 proteins were found exclusively in the cytoplasm. Consistent with the role of HIV-1 integrase, IN was indeed found more in the nucleus than in the cytoplasm. PR was distributed evenly throughout the cell, while Gp120 and Gp41 were localized more in the nucleus than in the cytoplasm. The subcellular distribution of HIV-1 accessory proteins (Vpr, Vpu, Vif, Nef, Rev, and Tat) was shown in Fig. 2c. Consistent with previous findings with Vpr [48], it localizes predominantly in the nucleus. Similarly, Vif, Rev, and Tat all accumulated in the nucleus of yeast cells. Nuclear localization of Tat has also been reported in budding yeast previously [50]. Vpu in cytoplasm but Nef was distributed throughout the cell. Finally, the subcellular location patterns observed in fission yeast were compared with that reported from mammalian cell studies. As summarized in Table 1, overall, there is a general correlation of the subcellular localization of each viral protein between fission yeast and that of previously reported in mammalian cells.Fig. 2

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