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Suppression of HIV-1 replication by microRNA effectors.

Chable-Bessia C, Meziane O, Latreille D, Triboulet R, Zamborlini A, Wagschal A, Jacquet JM, Reynes J, Levy Y, Saib A, Bennasser Y, Benkirane M - Retrovirology (2009)

Bottom Line: In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression.Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors).RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut de Génétique Humaine CNRS UPR1142, Laboratoire de Virologie Moléculaire, Montpellier, France. christine.chable-bessia@igh.cnrs.fr

ABSTRACT
The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART.

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RCK/p54 restricts HIV-1 mRNA association with polysomes. Cytoplasmic extracts from HeLa cells that were transfected with the indicated siRNA and infected with HIV-1-VSVG-luc were run on glycerol gradient (7% to 47%). Fractions were collected and their RNA contents were monitored by measuring absorbance at 254 nm. HIV-1 mRNA (top panel) and Hdm2 mRNA (lower panel) were quantified in all the fractions by Q-RT-PCR using specific oligonucleotides.
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Figure 2: RCK/p54 restricts HIV-1 mRNA association with polysomes. Cytoplasmic extracts from HeLa cells that were transfected with the indicated siRNA and infected with HIV-1-VSVG-luc were run on glycerol gradient (7% to 47%). Fractions were collected and their RNA contents were monitored by measuring absorbance at 254 nm. HIV-1 mRNA (top panel) and Hdm2 mRNA (lower panel) were quantified in all the fractions by Q-RT-PCR using specific oligonucleotides.

Mentions: To investigate whether RNAi effectors regulate HIV-1 replication, we analyzed virus replication in cells where expression of RNAi effectors was reduced using specific siRNA. HeLa cells were transfected with siRNA specific to RCK/p54, GW182, LSm-1 or XRN1. As controls, HeLa cells were transfected with scrambled siRNA (Scr) or CDK9 specific siRNA and subsequently infected with HIV-1 (Figure 1a). Knockdown of RCK/p54, GW182, LSm-1 and XRN1 enhanced virus replication by up to 10 fold (Figure 1b). As we have previously shown, knockdown of Drosha [21] and DGCR8 (Figure 1b), the two subunits of the microprocessor complex, increased virus production while knockdown of the CDK9 subunit of the PTEFb complex that is required for viral gene expression, reduced HIV-1 production (Figure 1b). Interestingly, analysis of HIV-1 cytoplasmic mRNA distribution on glycerol gradient showed that knockdown of RCK/p54 shifted HIV-1 mRNA from the non-polysomal fraction to polysomes as compared to control siRNA transfected cells (Figure 2, upper panel). As control, we analyzed the distribution of endogenous mRNA expressed from a gene encoding Hdm2. Knockdown of RCK/p54 did not affect Hdm2 mRNA distribution (Figure 2, lower panel). These experiments show that GW182, RCK/p54, LSm-1 and XRN1, factors required for RNAi, are repressors of HIV-1 gene expression that act by preventing HIV-1 mRNA translation.


Suppression of HIV-1 replication by microRNA effectors.

Chable-Bessia C, Meziane O, Latreille D, Triboulet R, Zamborlini A, Wagschal A, Jacquet JM, Reynes J, Levy Y, Saib A, Bennasser Y, Benkirane M - Retrovirology (2009)

RCK/p54 restricts HIV-1 mRNA association with polysomes. Cytoplasmic extracts from HeLa cells that were transfected with the indicated siRNA and infected with HIV-1-VSVG-luc were run on glycerol gradient (7% to 47%). Fractions were collected and their RNA contents were monitored by measuring absorbance at 254 nm. HIV-1 mRNA (top panel) and Hdm2 mRNA (lower panel) were quantified in all the fractions by Q-RT-PCR using specific oligonucleotides.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2657893&req=5

Figure 2: RCK/p54 restricts HIV-1 mRNA association with polysomes. Cytoplasmic extracts from HeLa cells that were transfected with the indicated siRNA and infected with HIV-1-VSVG-luc were run on glycerol gradient (7% to 47%). Fractions were collected and their RNA contents were monitored by measuring absorbance at 254 nm. HIV-1 mRNA (top panel) and Hdm2 mRNA (lower panel) were quantified in all the fractions by Q-RT-PCR using specific oligonucleotides.
Mentions: To investigate whether RNAi effectors regulate HIV-1 replication, we analyzed virus replication in cells where expression of RNAi effectors was reduced using specific siRNA. HeLa cells were transfected with siRNA specific to RCK/p54, GW182, LSm-1 or XRN1. As controls, HeLa cells were transfected with scrambled siRNA (Scr) or CDK9 specific siRNA and subsequently infected with HIV-1 (Figure 1a). Knockdown of RCK/p54, GW182, LSm-1 and XRN1 enhanced virus replication by up to 10 fold (Figure 1b). As we have previously shown, knockdown of Drosha [21] and DGCR8 (Figure 1b), the two subunits of the microprocessor complex, increased virus production while knockdown of the CDK9 subunit of the PTEFb complex that is required for viral gene expression, reduced HIV-1 production (Figure 1b). Interestingly, analysis of HIV-1 cytoplasmic mRNA distribution on glycerol gradient showed that knockdown of RCK/p54 shifted HIV-1 mRNA from the non-polysomal fraction to polysomes as compared to control siRNA transfected cells (Figure 2, upper panel). As control, we analyzed the distribution of endogenous mRNA expressed from a gene encoding Hdm2. Knockdown of RCK/p54 did not affect Hdm2 mRNA distribution (Figure 2, lower panel). These experiments show that GW182, RCK/p54, LSm-1 and XRN1, factors required for RNAi, are repressors of HIV-1 gene expression that act by preventing HIV-1 mRNA translation.

Bottom Line: In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression.Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors).RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut de Génétique Humaine CNRS UPR1142, Laboratoire de Virologie Moléculaire, Montpellier, France. christine.chable-bessia@igh.cnrs.fr

ABSTRACT
The rate of HIV-1 gene expression is a key step that determines the kinetics of virus spread and AIDS progression. Viral entry and gene expression were described to be the key determinants for cell permissiveness to HIV. Recent reports highlighted the involvement of miRNA in regulating HIV-1 replication post-transcriptionally. In this study we explored the role of cellular factors required for miRNA-mediated mRNA translational inhibition in regulating HIV-1 gene expression. Here we show that HIV-1 mRNAs associate and co-localize with components of the RNA Induced Silencing Complex (RISC), and we characterize some of the proteins required for miRNA-mediated silencing (miRNA effectors). RCK/p54, GW182, LSm-1 and XRN1 negatively regulate HIV-1 gene expression by preventing viral mRNA association with polysomes. Interestingly, knockdown of RCK/p54 or DGCR8 resulted in virus reactivation in PBMCs isolated from HIV infected patients treated with suppressive HAART.

Show MeSH
Related in: MedlinePlus