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

Relevance of the Rev–RRE Interaction(A) An HIV-1 vector plasmid in which stem loop II of the RRE was replaced by the MS2 stem loop was cotransfected with expression plasmids for HIV-1 gag-pol, VSV-G, and tat in the absence (−Rev) or presence of a rev (+Rev) expression plasmid, or in the presence of an expression plasmid encoding a fusion protein of Rev and the MS2 coat protein (+Rev-MSC). (B) An SIV vector with a deletion (VSΔSL in Figure 1B) of stem loop II of the RRE was cotransfected with expression plasmids for SIV gag-pol, VSV-G, and tat in the absence (−Rev) or presence of an HIV-1 (+Rev) or SIV rev expression plasmid. Cytoplasmic RNA levels, packaging efficiency, and vector titers were determined as described, and all values are expressed relative to the values obtained in the same transfection experiment for the parental VH (A) and VS (B) vectors in the presence of Rev, which were set as 100%.Mean value and standard deviation of at least three independent transfection experiments are shown. Numbers above the short vertical lines indicate fold induction by Rev.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC1851978&req=5

ppat-0030054-g005: Relevance of the Rev–RRE Interaction(A) An HIV-1 vector plasmid in which stem loop II of the RRE was replaced by the MS2 stem loop was cotransfected with expression plasmids for HIV-1 gag-pol, VSV-G, and tat in the absence (−Rev) or presence of a rev (+Rev) expression plasmid, or in the presence of an expression plasmid encoding a fusion protein of Rev and the MS2 coat protein (+Rev-MSC). (B) An SIV vector with a deletion (VSΔSL in Figure 1B) of stem loop II of the RRE was cotransfected with expression plasmids for SIV gag-pol, VSV-G, and tat in the absence (−Rev) or presence of an HIV-1 (+Rev) or SIV rev expression plasmid. Cytoplasmic RNA levels, packaging efficiency, and vector titers were determined as described, and all values are expressed relative to the values obtained in the same transfection experiment for the parental VH (A) and VS (B) vectors in the presence of Rev, which were set as 100%.Mean value and standard deviation of at least three independent transfection experiments are shown. Numbers above the short vertical lines indicate fold induction by Rev.

Mentions: To determine whether the newly discovered function of Rev was dependent on interaction with the RRE, stem loop II of the RRE, which is essential for Rev binding, was replaced by a heterologous stem loop from the bacteriophage MS2 in the context of the VH vector, resulting in VH-MS2 (Fig 1A). Packaging efficiency and vector titers of VH-MS2 in the presence of Rev were reduced to 9% and 18% of the values obtained with VH, respectively (Figure 5A). Despite deletion of stem loop II of the RRE, Rev enhanced the packaging efficiency and titer of the HIV-1 VH-MS2 vector by 2.5- and 3.2-fold, respectively (Figure 5A). This minor effect of Rev could either be due to residual binding of Rev to the remaining RRE, or to a second Rev-binding site recently identified in the 5′UTR of HIV-1 [15,16]. Using an SIV vector containing a deletion of stem loop II of the SIV RRE (VSΔSL in Figure 1B), HIV-1 and SIV Rev did not enhance packaging efficiency and vector titer either (Figure 5B).


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)

Relevance of the Rev–RRE Interaction(A) An HIV-1 vector plasmid in which stem loop II of the RRE was replaced by the MS2 stem loop was cotransfected with expression plasmids for HIV-1 gag-pol, VSV-G, and tat in the absence (−Rev) or presence of a rev (+Rev) expression plasmid, or in the presence of an expression plasmid encoding a fusion protein of Rev and the MS2 coat protein (+Rev-MSC). (B) An SIV vector with a deletion (VSΔSL in Figure 1B) of stem loop II of the RRE was cotransfected with expression plasmids for SIV gag-pol, VSV-G, and tat in the absence (−Rev) or presence of an HIV-1 (+Rev) or SIV rev expression plasmid. Cytoplasmic RNA levels, packaging efficiency, and vector titers were determined as described, and all values are expressed relative to the values obtained in the same transfection experiment for the parental VH (A) and VS (B) vectors in the presence of Rev, which were set as 100%.Mean value and standard deviation of at least three independent transfection experiments are shown. Numbers above the short vertical lines indicate fold induction by Rev.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-0030054-g005: Relevance of the Rev–RRE Interaction(A) An HIV-1 vector plasmid in which stem loop II of the RRE was replaced by the MS2 stem loop was cotransfected with expression plasmids for HIV-1 gag-pol, VSV-G, and tat in the absence (−Rev) or presence of a rev (+Rev) expression plasmid, or in the presence of an expression plasmid encoding a fusion protein of Rev and the MS2 coat protein (+Rev-MSC). (B) An SIV vector with a deletion (VSΔSL in Figure 1B) of stem loop II of the RRE was cotransfected with expression plasmids for SIV gag-pol, VSV-G, and tat in the absence (−Rev) or presence of an HIV-1 (+Rev) or SIV rev expression plasmid. Cytoplasmic RNA levels, packaging efficiency, and vector titers were determined as described, and all values are expressed relative to the values obtained in the same transfection experiment for the parental VH (A) and VS (B) vectors in the presence of Rev, which were set as 100%.Mean value and standard deviation of at least three independent transfection experiments are shown. Numbers above the short vertical lines indicate fold induction by Rev.
Mentions: To determine whether the newly discovered function of Rev was dependent on interaction with the RRE, stem loop II of the RRE, which is essential for Rev binding, was replaced by a heterologous stem loop from the bacteriophage MS2 in the context of the VH vector, resulting in VH-MS2 (Fig 1A). Packaging efficiency and vector titers of VH-MS2 in the presence of Rev were reduced to 9% and 18% of the values obtained with VH, respectively (Figure 5A). Despite deletion of stem loop II of the RRE, Rev enhanced the packaging efficiency and titer of the HIV-1 VH-MS2 vector by 2.5- and 3.2-fold, respectively (Figure 5A). This minor effect of Rev could either be due to residual binding of Rev to the remaining RRE, or to a second Rev-binding site recently identified in the 5′UTR of HIV-1 [15,16]. Using an SIV vector containing a deletion of stem loop II of the SIV RRE (VSΔSL in Figure 1B), HIV-1 and SIV Rev did not enhance packaging efficiency and vector titer either (Figure 5B).

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