Limits...
Rev proteins of human and simian immunodeficiency virus enhance RNA encapsidation.

Brandt S, Blissenbach M, Grewe B, Konietzny R, Grunwald T, Uberla K - PLoS Pathog. (2007)

Bottom Line: This restricts expression of structural proteins to the late phase of the lentiviral replication cycle.In contrast, cytoplasmic vector RNA levels were only marginally affected by Rev.In addition to specific interactions of nucleocapsid with the packaging signal at the 5' end of the genome, the Rev/RRE system provides a second mechanism contributing to preferential encapsidation of genomic lentiviral RNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Medical Virology, Ruhr-University Bochum, Germany.

ABSTRACT
The main function attributed to the Rev proteins of immunodeficiency viruses is the shuttling of viral RNAs containing the Rev responsive element (RRE) via the CRM-1 export pathway from the nucleus to the cytoplasm. This restricts expression of structural proteins to the late phase of the lentiviral replication cycle. Using Rev-independent gag-pol expression plasmids of HIV-1 and simian immunodeficiency virus and lentiviral vector constructs, we have observed that HIV-1 and simian immunodeficiency virus Rev enhanced RNA encapsidation 20- to 70-fold, correlating well with the effect of Rev on vector titers. In contrast, cytoplasmic vector RNA levels were only marginally affected by Rev. Binding of Rev to the RRE or to a heterologous RNA element was required for Rev-mediated enhancement of RNA encapsidation. In addition to specific interactions of nucleocapsid with the packaging signal at the 5' end of the genome, the Rev/RRE system provides a second mechanism contributing to preferential encapsidation of genomic lentiviral RNA.

Show MeSH

Related in: MedlinePlus

Effect of Rev on Encapsidation of HIV-1 and SIV Vector RNA(A) The HIV-1 vector plasmid VH was cotransfected with expression plasmids for HIV-1 gag-pol, VSV-G, and HIV-1 tat in the presence (+Rev) or absence (−Rev) of an HIV-1 rev expression plasmid. Cytoplasmic RNA levels, packaging efficiency, and vector titers are presented relative to the values obtained in the same transfection experiment with VH in the presence of Rev. The mean value and standard deviation of three to eight independent transfection experiments are shown.(B) The SIV vector plasmid VS was cotransfected with expression plasmids for SIV gag-pol, VSV-G, and HIV-1 tat in the absence (−Rev) or presence of an SIV (+SIV-Rev) or HIV-1 rev (+Rev) expression plasmid. The mean value and standard deviation of four independent transfection experiments are shown. Numbers above the bars indicate fold induction by Rev.(C) Using a real-time PCR for preGAPDH RNA, threshold cycle numbers (Ct) were determined for four nuclear (N) and cytoplasmic (C) RNA preparations in three independent experiments. Relative amounts of preGAPDH RNA levels were calculated as arbitrary units (AU) per microgram extracted RNA by including 10-fold serial dilutions of standard nuclear RNA preparations. Ct values of a typical experiment are shown in the left panel, and the table summarizes the results.(D) Western blot analysis of total cell lysate (T), and cytoplasmic (C) and nuclear (N) fractions for Lamin B.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1851978&req=5

ppat-0030054-g002: Effect of Rev on Encapsidation of HIV-1 and SIV Vector RNA(A) The HIV-1 vector plasmid VH was cotransfected with expression plasmids for HIV-1 gag-pol, VSV-G, and HIV-1 tat in the presence (+Rev) or absence (−Rev) of an HIV-1 rev expression plasmid. Cytoplasmic RNA levels, packaging efficiency, and vector titers are presented relative to the values obtained in the same transfection experiment with VH in the presence of Rev. The mean value and standard deviation of three to eight independent transfection experiments are shown.(B) The SIV vector plasmid VS was cotransfected with expression plasmids for SIV gag-pol, VSV-G, and HIV-1 tat in the absence (−Rev) or presence of an SIV (+SIV-Rev) or HIV-1 rev (+Rev) expression plasmid. The mean value and standard deviation of four independent transfection experiments are shown. Numbers above the bars indicate fold induction by Rev.(C) Using a real-time PCR for preGAPDH RNA, threshold cycle numbers (Ct) were determined for four nuclear (N) and cytoplasmic (C) RNA preparations in three independent experiments. Relative amounts of preGAPDH RNA levels were calculated as arbitrary units (AU) per microgram extracted RNA by including 10-fold serial dilutions of standard nuclear RNA preparations. Ct values of a typical experiment are shown in the left panel, and the table summarizes the results.(D) Western blot analysis of total cell lysate (T), and cytoplasmic (C) and nuclear (N) fractions for Lamin B.

Mentions: Infectious HIV-1-based vector particles were produced by cotransfection of a codon-optimized HIV-1 gag-pol expression plasmid, a VSV-G expression plasmid, a tat expression plasmid, and the prototypic HIV-1 vector construct VH (Figure 1A) into 293T cells in the presence or absence of a rev expression plasmid [11]. Vector titers in the absence of Rev were only 3% of those obtained with Rev (Figure 2A). To study the role of Rev on packaging efficiency, we determined genomic vector RNA levels in the cytoplasm of transfected cells and in viral particles pelleted from the supernatant of transfected cells through a 30% sucrose cushion. Mean cytoplasmic VH vector RNA copy numbers per microgram extracted RNA were 1.7 × 109 ± 1.1 × 109 and 7.1 × 108 ± 3.5 × 108 in the presence and absence of Rev, respectively, matching well with results obtained in Northern blot analyses (unpublished data). To exclude the possibility that Rev-independent cytoplasmic localization of vector RNA is simply due to contamination of the cytoplasmic RNA fraction with large amounts of nuclear RNA, pre–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA levels in cytoplasmic and nuclear RNA preparations were determined by quantitative real-time PCR. As expected for a nuclear RNA, 16-fold higher concentrations of preGAPDH RNA were observed in the nuclear fraction than in the cytoplasm (Figure 2C). In addition, Western blot analysis revealed the presence of lamin B in the nuclear fraction, but not in the cytoplasmic fraction, confirming the fidelity of the cell fractionation procedure (Figure 2D).


Rev proteins of human and simian immunodeficiency virus enhance RNA encapsidation.

Brandt S, Blissenbach M, Grewe B, Konietzny R, Grunwald T, Uberla K - PLoS Pathog. (2007)

Effect of Rev on Encapsidation of HIV-1 and SIV Vector RNA(A) The HIV-1 vector plasmid VH was cotransfected with expression plasmids for HIV-1 gag-pol, VSV-G, and HIV-1 tat in the presence (+Rev) or absence (−Rev) of an HIV-1 rev expression plasmid. Cytoplasmic RNA levels, packaging efficiency, and vector titers are presented relative to the values obtained in the same transfection experiment with VH in the presence of Rev. The mean value and standard deviation of three to eight independent transfection experiments are shown.(B) The SIV vector plasmid VS was cotransfected with expression plasmids for SIV gag-pol, VSV-G, and HIV-1 tat in the absence (−Rev) or presence of an SIV (+SIV-Rev) or HIV-1 rev (+Rev) expression plasmid. The mean value and standard deviation of four independent transfection experiments are shown. Numbers above the bars indicate fold induction by Rev.(C) Using a real-time PCR for preGAPDH RNA, threshold cycle numbers (Ct) were determined for four nuclear (N) and cytoplasmic (C) RNA preparations in three independent experiments. Relative amounts of preGAPDH RNA levels were calculated as arbitrary units (AU) per microgram extracted RNA by including 10-fold serial dilutions of standard nuclear RNA preparations. Ct values of a typical experiment are shown in the left panel, and the table summarizes the results.(D) Western blot analysis of total cell lysate (T), and cytoplasmic (C) and nuclear (N) fractions for Lamin B.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1851978&req=5

ppat-0030054-g002: Effect of Rev on Encapsidation of HIV-1 and SIV Vector RNA(A) The HIV-1 vector plasmid VH was cotransfected with expression plasmids for HIV-1 gag-pol, VSV-G, and HIV-1 tat in the presence (+Rev) or absence (−Rev) of an HIV-1 rev expression plasmid. Cytoplasmic RNA levels, packaging efficiency, and vector titers are presented relative to the values obtained in the same transfection experiment with VH in the presence of Rev. The mean value and standard deviation of three to eight independent transfection experiments are shown.(B) The SIV vector plasmid VS was cotransfected with expression plasmids for SIV gag-pol, VSV-G, and HIV-1 tat in the absence (−Rev) or presence of an SIV (+SIV-Rev) or HIV-1 rev (+Rev) expression plasmid. The mean value and standard deviation of four independent transfection experiments are shown. Numbers above the bars indicate fold induction by Rev.(C) Using a real-time PCR for preGAPDH RNA, threshold cycle numbers (Ct) were determined for four nuclear (N) and cytoplasmic (C) RNA preparations in three independent experiments. Relative amounts of preGAPDH RNA levels were calculated as arbitrary units (AU) per microgram extracted RNA by including 10-fold serial dilutions of standard nuclear RNA preparations. Ct values of a typical experiment are shown in the left panel, and the table summarizes the results.(D) Western blot analysis of total cell lysate (T), and cytoplasmic (C) and nuclear (N) fractions for Lamin B.
Mentions: Infectious HIV-1-based vector particles were produced by cotransfection of a codon-optimized HIV-1 gag-pol expression plasmid, a VSV-G expression plasmid, a tat expression plasmid, and the prototypic HIV-1 vector construct VH (Figure 1A) into 293T cells in the presence or absence of a rev expression plasmid [11]. Vector titers in the absence of Rev were only 3% of those obtained with Rev (Figure 2A). To study the role of Rev on packaging efficiency, we determined genomic vector RNA levels in the cytoplasm of transfected cells and in viral particles pelleted from the supernatant of transfected cells through a 30% sucrose cushion. Mean cytoplasmic VH vector RNA copy numbers per microgram extracted RNA were 1.7 × 109 ± 1.1 × 109 and 7.1 × 108 ± 3.5 × 108 in the presence and absence of Rev, respectively, matching well with results obtained in Northern blot analyses (unpublished data). To exclude the possibility that Rev-independent cytoplasmic localization of vector RNA is simply due to contamination of the cytoplasmic RNA fraction with large amounts of nuclear RNA, pre–glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA levels in cytoplasmic and nuclear RNA preparations were determined by quantitative real-time PCR. As expected for a nuclear RNA, 16-fold higher concentrations of preGAPDH RNA were observed in the nuclear fraction than in the cytoplasm (Figure 2C). In addition, Western blot analysis revealed the presence of lamin B in the nuclear fraction, but not in the cytoplasmic fraction, confirming the fidelity of the cell fractionation procedure (Figure 2D).

Bottom Line: This restricts expression of structural proteins to the late phase of the lentiviral replication cycle.In contrast, cytoplasmic vector RNA levels were only marginally affected by Rev.In addition to specific interactions of nucleocapsid with the packaging signal at the 5' end of the genome, the Rev/RRE system provides a second mechanism contributing to preferential encapsidation of genomic lentiviral RNA.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Medical Virology, Ruhr-University Bochum, Germany.

ABSTRACT
The main function attributed to the Rev proteins of immunodeficiency viruses is the shuttling of viral RNAs containing the Rev responsive element (RRE) via the CRM-1 export pathway from the nucleus to the cytoplasm. This restricts expression of structural proteins to the late phase of the lentiviral replication cycle. Using Rev-independent gag-pol expression plasmids of HIV-1 and simian immunodeficiency virus and lentiviral vector constructs, we have observed that HIV-1 and simian immunodeficiency virus Rev enhanced RNA encapsidation 20- to 70-fold, correlating well with the effect of Rev on vector titers. In contrast, cytoplasmic vector RNA levels were only marginally affected by Rev. Binding of Rev to the RRE or to a heterologous RNA element was required for Rev-mediated enhancement of RNA encapsidation. In addition to specific interactions of nucleocapsid with the packaging signal at the 5' end of the genome, the Rev/RRE system provides a second mechanism contributing to preferential encapsidation of genomic lentiviral RNA.

Show MeSH
Related in: MedlinePlus