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Human protein Staufen-2 promotes HIV-1 proliferation by positively regulating RNA export activity of viral protein Rev.

Banerjee A, Benjamin R, Balakrishnan K, Ghosh P, Banerjee S - Retrovirology (2014)

Bottom Line: Our experiments involving transient expressions, siRNA mediated knockdowns and infection assays conclusively established that hStau-2 is a positive regulator of HIV-1 pathogenesis.The Rev-hStau-2 interaction was independent of RNA despite both being RNA binding proteins. hStau-2 mutant, with mutations at Q314R-A318F-K319E, deficient of binding Rev, failed to promote hStau-2 dependent Rev activity and viral production, validating the essentiality of this protein-protein interaction.Altogether, our study provides new insights into the emerging role of the Staufen family of mRNA transporters in host-pathogen interaction and supports the notion that obliterating interactions between viral and host proteins that positively regulate HIV-1 proliferation can significantly contribute to anti-retroviral treatments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, School of Life Sciences, University of Hyderabad, Gachibowli, Hyderabad, Andhra Pradesh 500046, India. sbsl@uohyd.ernet.in.

ABSTRACT

Background: The export of intron containing viral RNAs from the nucleus to the cytoplasm is an essential step in the life cycle of Human Immunodeficiency Virus-1 (HIV-1). As the eukaryotic system does not permit the transport of intron containing RNA out of the nucleus, HIV-1 makes a regulatory protein, Rev, that mediates the transportation of unspliced and partially spliced viral mRNA from the nucleus to the cytoplasm, thereby playing a decisive role in the generation of new infectious virus particles. Therefore, the host factors modulating the RNA export activity of Rev can be major determinants of virus production in an infected cell.

Results: In this study, human Staufen-2 (hStau-2) was identified as a host factor interacting with HIV-1 Rev through affinity chromatography followed by MALDI analyses. Our experiments involving transient expressions, siRNA mediated knockdowns and infection assays conclusively established that hStau-2 is a positive regulator of HIV-1 pathogenesis. We demonstrated that Rev-hStau-2 interactions positively regulated the RNA export activity of Rev and promoted progeny virus synthesis. The Rev-hStau-2 interaction was independent of RNA despite both being RNA binding proteins. hStau-2 mutant, with mutations at Q314R-A318F-K319E, deficient of binding Rev, failed to promote hStau-2 dependent Rev activity and viral production, validating the essentiality of this protein-protein interaction. The expression of this positive regulator was elevated upon HIV-1 infection in both human T-lymphocyte and astrocyte cell lines.

Conclusions: With this study, we establish that human Staufen-2, a host factor which is up-regulated upon HIV-1 infection, interacts with HIV-1 Rev, thereby promoting its RNA export activity and progeny virus formation. Altogether, our study provides new insights into the emerging role of the Staufen family of mRNA transporters in host-pathogen interaction and supports the notion that obliterating interactions between viral and host proteins that positively regulate HIV-1 proliferation can significantly contribute to anti-retroviral treatments.

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Effect of hStau-2 expression on viral p24 levels. A) Over-expression of hStau-2 increased HIV-1 production: hStau-2 over-expressed HEK293T cells or control cells were transfected with pNL4-3 pro-viral DNA and scored after 48 hours for p24 by ELISA in the culture supernatant. The inset shows over-expression of hStau-2 after transfection with increasing concentration of hStau2-59 construct. There was a significant increase in the p24 levels when 0.5 and 1ug of hStau2-59 construct was used. B) The relative quantification of full length (9 kb) viral transcript. Viral transcripts were quantified from hStau-2 overexpressed and control cells by qRT-PCR and normalized to β-actin. C) siRNA mediated knockdown of hStau-2 reduced HIV-1 production: hStau-2 siRNA or scrambled siRNA were transfected one day prior to pNL4-3 transfection in HEK293T cells. Inset: Semiquantitative RT-PCR gel showing a decrease in hStau-2 expression when hStau-2 specific siRNA was used. The viral p24 levels in the culture supernatant were progressively reduced when hStau-2 specific siRNA was used in a dose dependent manner. The experiments were done more than 3 times and error bars represents ± SD. *p value ≤ 0.05 were taken as significant.
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Figure 4: Effect of hStau-2 expression on viral p24 levels. A) Over-expression of hStau-2 increased HIV-1 production: hStau-2 over-expressed HEK293T cells or control cells were transfected with pNL4-3 pro-viral DNA and scored after 48 hours for p24 by ELISA in the culture supernatant. The inset shows over-expression of hStau-2 after transfection with increasing concentration of hStau2-59 construct. There was a significant increase in the p24 levels when 0.5 and 1ug of hStau2-59 construct was used. B) The relative quantification of full length (9 kb) viral transcript. Viral transcripts were quantified from hStau-2 overexpressed and control cells by qRT-PCR and normalized to β-actin. C) siRNA mediated knockdown of hStau-2 reduced HIV-1 production: hStau-2 siRNA or scrambled siRNA were transfected one day prior to pNL4-3 transfection in HEK293T cells. Inset: Semiquantitative RT-PCR gel showing a decrease in hStau-2 expression when hStau-2 specific siRNA was used. The viral p24 levels in the culture supernatant were progressively reduced when hStau-2 specific siRNA was used in a dose dependent manner. The experiments were done more than 3 times and error bars represents ± SD. *p value ≤ 0.05 were taken as significant.

Mentions: With the observation that hStau-2 expression is promoted during HIV-1 infection in human astrocyte and T-lymphocyte cell lines, we studied the influence of hStau-2 overexpression on HIV-1 viral titers. hStau-2 was transiently expressed in HEK293T cells 24 hours prior to transfection with pro-viral DNA pNL4-3. HEK293T cells were transfected with increasing concentration of hStau-2-59 construct and protein expression was checked by Western blot using anti-hStau-2 antibody (Figure 4A, inset). The viral titers were measured by p24 ELISA after 48 hours of pNL4-3 transfection. It was observed that the overexpression of hStau-2 significantly increased the viral titers in a dose dependent manner (Figure 4A). As compared to the control experiment, p24 equivalent of viral titers increased by 56.6 ± 10.28% and 98 ± 3.85% when hStau-2 was overexpressed using 0.5 μg and 1 μg of hStau-2-59 construct respectively (Figure 4A). To check if the over-expression of hStau-2 is influencing the viral transcription and increasing the viral output, the total levels of full length viral transcripts were compared between hStau-2 over-expressed and control experiments. No significant changes at the level of full length transcripts were observed with respect to the control in our experiments (Figure 4B). Hence, the impact of overexpression of hStau-2 on viral transcription was ruled out.


Human protein Staufen-2 promotes HIV-1 proliferation by positively regulating RNA export activity of viral protein Rev.

Banerjee A, Benjamin R, Balakrishnan K, Ghosh P, Banerjee S - Retrovirology (2014)

Effect of hStau-2 expression on viral p24 levels. A) Over-expression of hStau-2 increased HIV-1 production: hStau-2 over-expressed HEK293T cells or control cells were transfected with pNL4-3 pro-viral DNA and scored after 48 hours for p24 by ELISA in the culture supernatant. The inset shows over-expression of hStau-2 after transfection with increasing concentration of hStau2-59 construct. There was a significant increase in the p24 levels when 0.5 and 1ug of hStau2-59 construct was used. B) The relative quantification of full length (9 kb) viral transcript. Viral transcripts were quantified from hStau-2 overexpressed and control cells by qRT-PCR and normalized to β-actin. C) siRNA mediated knockdown of hStau-2 reduced HIV-1 production: hStau-2 siRNA or scrambled siRNA were transfected one day prior to pNL4-3 transfection in HEK293T cells. Inset: Semiquantitative RT-PCR gel showing a decrease in hStau-2 expression when hStau-2 specific siRNA was used. The viral p24 levels in the culture supernatant were progressively reduced when hStau-2 specific siRNA was used in a dose dependent manner. The experiments were done more than 3 times and error bars represents ± SD. *p value ≤ 0.05 were taken as significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4016256&req=5

Figure 4: Effect of hStau-2 expression on viral p24 levels. A) Over-expression of hStau-2 increased HIV-1 production: hStau-2 over-expressed HEK293T cells or control cells were transfected with pNL4-3 pro-viral DNA and scored after 48 hours for p24 by ELISA in the culture supernatant. The inset shows over-expression of hStau-2 after transfection with increasing concentration of hStau2-59 construct. There was a significant increase in the p24 levels when 0.5 and 1ug of hStau2-59 construct was used. B) The relative quantification of full length (9 kb) viral transcript. Viral transcripts were quantified from hStau-2 overexpressed and control cells by qRT-PCR and normalized to β-actin. C) siRNA mediated knockdown of hStau-2 reduced HIV-1 production: hStau-2 siRNA or scrambled siRNA were transfected one day prior to pNL4-3 transfection in HEK293T cells. Inset: Semiquantitative RT-PCR gel showing a decrease in hStau-2 expression when hStau-2 specific siRNA was used. The viral p24 levels in the culture supernatant were progressively reduced when hStau-2 specific siRNA was used in a dose dependent manner. The experiments were done more than 3 times and error bars represents ± SD. *p value ≤ 0.05 were taken as significant.
Mentions: With the observation that hStau-2 expression is promoted during HIV-1 infection in human astrocyte and T-lymphocyte cell lines, we studied the influence of hStau-2 overexpression on HIV-1 viral titers. hStau-2 was transiently expressed in HEK293T cells 24 hours prior to transfection with pro-viral DNA pNL4-3. HEK293T cells were transfected with increasing concentration of hStau-2-59 construct and protein expression was checked by Western blot using anti-hStau-2 antibody (Figure 4A, inset). The viral titers were measured by p24 ELISA after 48 hours of pNL4-3 transfection. It was observed that the overexpression of hStau-2 significantly increased the viral titers in a dose dependent manner (Figure 4A). As compared to the control experiment, p24 equivalent of viral titers increased by 56.6 ± 10.28% and 98 ± 3.85% when hStau-2 was overexpressed using 0.5 μg and 1 μg of hStau-2-59 construct respectively (Figure 4A). To check if the over-expression of hStau-2 is influencing the viral transcription and increasing the viral output, the total levels of full length viral transcripts were compared between hStau-2 over-expressed and control experiments. No significant changes at the level of full length transcripts were observed with respect to the control in our experiments (Figure 4B). Hence, the impact of overexpression of hStau-2 on viral transcription was ruled out.

Bottom Line: Our experiments involving transient expressions, siRNA mediated knockdowns and infection assays conclusively established that hStau-2 is a positive regulator of HIV-1 pathogenesis.The Rev-hStau-2 interaction was independent of RNA despite both being RNA binding proteins. hStau-2 mutant, with mutations at Q314R-A318F-K319E, deficient of binding Rev, failed to promote hStau-2 dependent Rev activity and viral production, validating the essentiality of this protein-protein interaction.Altogether, our study provides new insights into the emerging role of the Staufen family of mRNA transporters in host-pathogen interaction and supports the notion that obliterating interactions between viral and host proteins that positively regulate HIV-1 proliferation can significantly contribute to anti-retroviral treatments.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, School of Life Sciences, University of Hyderabad, Gachibowli, Hyderabad, Andhra Pradesh 500046, India. sbsl@uohyd.ernet.in.

ABSTRACT

Background: The export of intron containing viral RNAs from the nucleus to the cytoplasm is an essential step in the life cycle of Human Immunodeficiency Virus-1 (HIV-1). As the eukaryotic system does not permit the transport of intron containing RNA out of the nucleus, HIV-1 makes a regulatory protein, Rev, that mediates the transportation of unspliced and partially spliced viral mRNA from the nucleus to the cytoplasm, thereby playing a decisive role in the generation of new infectious virus particles. Therefore, the host factors modulating the RNA export activity of Rev can be major determinants of virus production in an infected cell.

Results: In this study, human Staufen-2 (hStau-2) was identified as a host factor interacting with HIV-1 Rev through affinity chromatography followed by MALDI analyses. Our experiments involving transient expressions, siRNA mediated knockdowns and infection assays conclusively established that hStau-2 is a positive regulator of HIV-1 pathogenesis. We demonstrated that Rev-hStau-2 interactions positively regulated the RNA export activity of Rev and promoted progeny virus synthesis. The Rev-hStau-2 interaction was independent of RNA despite both being RNA binding proteins. hStau-2 mutant, with mutations at Q314R-A318F-K319E, deficient of binding Rev, failed to promote hStau-2 dependent Rev activity and viral production, validating the essentiality of this protein-protein interaction. The expression of this positive regulator was elevated upon HIV-1 infection in both human T-lymphocyte and astrocyte cell lines.

Conclusions: With this study, we establish that human Staufen-2, a host factor which is up-regulated upon HIV-1 infection, interacts with HIV-1 Rev, thereby promoting its RNA export activity and progeny virus formation. Altogether, our study provides new insights into the emerging role of the Staufen family of mRNA transporters in host-pathogen interaction and supports the notion that obliterating interactions between viral and host proteins that positively regulate HIV-1 proliferation can significantly contribute to anti-retroviral treatments.

Show MeSH
Related in: MedlinePlus