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A re-examination of global suppression of RNA interference by HIV-1.

Sanghvi VR, Steel LF - PLoS ONE (2011)

Bottom Line: The role of Tat as an inhibitor of Dicer has been questioned and our results support and extend the conclusion that Tat does not inhibit RNAi that is mediated by either exogenous or endogenous miRNAs.However, knockdown of Dicer does allow increased viral replication and this occurs at a post-transcriptional level.These results support the idea that although individual miRNAs can act to restrict HIV-1 replication, the virus does not counter these effects through a global suppression of RNAi synthesis or processing.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
The nature of the interaction between replicating HIV-1 and the cellular RNAi pathway has been controversial, but it is clear that it can be complex and multifaceted. It has been proposed that the interaction is bi-directional, whereby cellular silencing pathways can restrict HIV-1 replication, and in turn, HIV-1 can suppress silencing pathways. Overall suppression of RNAi has been suggested to occur via direct binding and inhibition of Dicer by the HIV-1 Tat protein or through sequestration of TRBP, a Dicer co-factor, by the structured TAR element of HIV-1 transcripts. The role of Tat as an inhibitor of Dicer has been questioned and our results support and extend the conclusion that Tat does not inhibit RNAi that is mediated by either exogenous or endogenous miRNAs. Similarly, we find no suppression of silencing pathways in cells with replicating virus, suggesting that viral products such as the TAR RNA elements also do not reduce the efficacy of cellular RNA silencing. However, knockdown of Dicer does allow increased viral replication and this occurs at a post-transcriptional level. These results support the idea that although individual miRNAs can act to restrict HIV-1 replication, the virus does not counter these effects through a global suppression of RNAi synthesis or processing.

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The impact of Dicer depletion on HIV-1 replication in 293T cells.(A) Dicer knockdown was confirmed by immunoblotting total protein extracts prepared 2d post-transfection from 293T cells transfected with indicated amounts of pU6-miDicer. The immunoblot was incubated with antibody specific to Dicer and β-actin that serves as a loading control. (B) 293T cells were transfected with pLAI, together with pmiEGFP or pU6-miDicer, as indicated. 2d post transfection infectious virus released into the supernatant was assayed using P4R5 indicator cells (see Materials and Methods). Error bars represent standard deviation from 6 replicates. (C) Total RNA isolated from cells in (B) was subjected to semi-quantitative RT-PCR to determine the mRNA levels of Dicer, HIV-1 Tat, and β-actin. Following PCR, products were analyzed by electrophoresis in 2% agarose and ethidium bromide staining. PCR products are resolved on the same gel and irrelevant samples are cropped out. D) Total RNA isolated in (C) was treated with DNase, and following cDNA synthesis with a Gag mRNA specific primer, semi-quantitative PCR was performed and products were analyzed as in (C). PCR for β-actin following oligo(dT)-primed cDNA synthesis serves as a loading control.
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pone-0017246-g006: The impact of Dicer depletion on HIV-1 replication in 293T cells.(A) Dicer knockdown was confirmed by immunoblotting total protein extracts prepared 2d post-transfection from 293T cells transfected with indicated amounts of pU6-miDicer. The immunoblot was incubated with antibody specific to Dicer and β-actin that serves as a loading control. (B) 293T cells were transfected with pLAI, together with pmiEGFP or pU6-miDicer, as indicated. 2d post transfection infectious virus released into the supernatant was assayed using P4R5 indicator cells (see Materials and Methods). Error bars represent standard deviation from 6 replicates. (C) Total RNA isolated from cells in (B) was subjected to semi-quantitative RT-PCR to determine the mRNA levels of Dicer, HIV-1 Tat, and β-actin. Following PCR, products were analyzed by electrophoresis in 2% agarose and ethidium bromide staining. PCR products are resolved on the same gel and irrelevant samples are cropped out. D) Total RNA isolated in (C) was treated with DNase, and following cDNA synthesis with a Gag mRNA specific primer, semi-quantitative PCR was performed and products were analyzed as in (C). PCR for β-actin following oligo(dT)-primed cDNA synthesis serves as a loading control.

Mentions: Although HIV-1 does not appear to mount a direct defense against the cellular RNAi machinery, there is considerable evidence that viral replication can be modulated by RNAi and knockdown of Dicer has been shown to up-regulate the production of infectious HIV-1 in primary cells, Jurkat cells, and 293T cells [5], [17], [18]. RNAi has been proposed to restrict HIV-1 replication either through a direct interaction between cellular miRNAs and viral transcripts, or through indirect effects on cellular proteins that act as co-factors in Tat-mediated viral transcription [5], [16]. To begin to investigate at what level Dicer-dependent RNAi acts to restrict HIV-1 replication, we compared viral RNA levels in cells with and without Dicer knockdown. First, we confirmed Dicer knockdown in 293T cells using the plasmid pmiDicer (Figure 6A), and then showed that co-transfection of this plasmid with pLAI led to an approximately 2-fold increase in the production of infectious virus (Figure 6B), consistent with published observations [18]. The release of infectious viral particles by the transfected 293T cells was measured using a P4R5 infection assay, as above. Semi-quantitative RT-PCR of RNA extracted from these cells showed that levels of Tat and Gag mRNAs are similar in cells transfected with pLAI, with or without Dicer knockdown (Figure 6C and 6D), suggesting that the rate limiting restriction to HIV-1 replication imposed by Dicer in 293T cells occurs at a post-transcriptional level. Additionally, we do not find any significant differences in the transcript levels of RNAi-related proteins upon transfection with pLAI (see Figure S4), again arguing against global regulation of the cellular RNAi machinery by products of HIV-1 infection.


A re-examination of global suppression of RNA interference by HIV-1.

Sanghvi VR, Steel LF - PLoS ONE (2011)

The impact of Dicer depletion on HIV-1 replication in 293T cells.(A) Dicer knockdown was confirmed by immunoblotting total protein extracts prepared 2d post-transfection from 293T cells transfected with indicated amounts of pU6-miDicer. The immunoblot was incubated with antibody specific to Dicer and β-actin that serves as a loading control. (B) 293T cells were transfected with pLAI, together with pmiEGFP or pU6-miDicer, as indicated. 2d post transfection infectious virus released into the supernatant was assayed using P4R5 indicator cells (see Materials and Methods). Error bars represent standard deviation from 6 replicates. (C) Total RNA isolated from cells in (B) was subjected to semi-quantitative RT-PCR to determine the mRNA levels of Dicer, HIV-1 Tat, and β-actin. Following PCR, products were analyzed by electrophoresis in 2% agarose and ethidium bromide staining. PCR products are resolved on the same gel and irrelevant samples are cropped out. D) Total RNA isolated in (C) was treated with DNase, and following cDNA synthesis with a Gag mRNA specific primer, semi-quantitative PCR was performed and products were analyzed as in (C). PCR for β-actin following oligo(dT)-primed cDNA synthesis serves as a loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3046114&req=5

pone-0017246-g006: The impact of Dicer depletion on HIV-1 replication in 293T cells.(A) Dicer knockdown was confirmed by immunoblotting total protein extracts prepared 2d post-transfection from 293T cells transfected with indicated amounts of pU6-miDicer. The immunoblot was incubated with antibody specific to Dicer and β-actin that serves as a loading control. (B) 293T cells were transfected with pLAI, together with pmiEGFP or pU6-miDicer, as indicated. 2d post transfection infectious virus released into the supernatant was assayed using P4R5 indicator cells (see Materials and Methods). Error bars represent standard deviation from 6 replicates. (C) Total RNA isolated from cells in (B) was subjected to semi-quantitative RT-PCR to determine the mRNA levels of Dicer, HIV-1 Tat, and β-actin. Following PCR, products were analyzed by electrophoresis in 2% agarose and ethidium bromide staining. PCR products are resolved on the same gel and irrelevant samples are cropped out. D) Total RNA isolated in (C) was treated with DNase, and following cDNA synthesis with a Gag mRNA specific primer, semi-quantitative PCR was performed and products were analyzed as in (C). PCR for β-actin following oligo(dT)-primed cDNA synthesis serves as a loading control.
Mentions: Although HIV-1 does not appear to mount a direct defense against the cellular RNAi machinery, there is considerable evidence that viral replication can be modulated by RNAi and knockdown of Dicer has been shown to up-regulate the production of infectious HIV-1 in primary cells, Jurkat cells, and 293T cells [5], [17], [18]. RNAi has been proposed to restrict HIV-1 replication either through a direct interaction between cellular miRNAs and viral transcripts, or through indirect effects on cellular proteins that act as co-factors in Tat-mediated viral transcription [5], [16]. To begin to investigate at what level Dicer-dependent RNAi acts to restrict HIV-1 replication, we compared viral RNA levels in cells with and without Dicer knockdown. First, we confirmed Dicer knockdown in 293T cells using the plasmid pmiDicer (Figure 6A), and then showed that co-transfection of this plasmid with pLAI led to an approximately 2-fold increase in the production of infectious virus (Figure 6B), consistent with published observations [18]. The release of infectious viral particles by the transfected 293T cells was measured using a P4R5 infection assay, as above. Semi-quantitative RT-PCR of RNA extracted from these cells showed that levels of Tat and Gag mRNAs are similar in cells transfected with pLAI, with or without Dicer knockdown (Figure 6C and 6D), suggesting that the rate limiting restriction to HIV-1 replication imposed by Dicer in 293T cells occurs at a post-transcriptional level. Additionally, we do not find any significant differences in the transcript levels of RNAi-related proteins upon transfection with pLAI (see Figure S4), again arguing against global regulation of the cellular RNAi machinery by products of HIV-1 infection.

Bottom Line: The role of Tat as an inhibitor of Dicer has been questioned and our results support and extend the conclusion that Tat does not inhibit RNAi that is mediated by either exogenous or endogenous miRNAs.However, knockdown of Dicer does allow increased viral replication and this occurs at a post-transcriptional level.These results support the idea that although individual miRNAs can act to restrict HIV-1 replication, the virus does not counter these effects through a global suppression of RNAi synthesis or processing.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
The nature of the interaction between replicating HIV-1 and the cellular RNAi pathway has been controversial, but it is clear that it can be complex and multifaceted. It has been proposed that the interaction is bi-directional, whereby cellular silencing pathways can restrict HIV-1 replication, and in turn, HIV-1 can suppress silencing pathways. Overall suppression of RNAi has been suggested to occur via direct binding and inhibition of Dicer by the HIV-1 Tat protein or through sequestration of TRBP, a Dicer co-factor, by the structured TAR element of HIV-1 transcripts. The role of Tat as an inhibitor of Dicer has been questioned and our results support and extend the conclusion that Tat does not inhibit RNAi that is mediated by either exogenous or endogenous miRNAs. Similarly, we find no suppression of silencing pathways in cells with replicating virus, suggesting that viral products such as the TAR RNA elements also do not reduce the efficacy of cellular RNA silencing. However, knockdown of Dicer does allow increased viral replication and this occurs at a post-transcriptional level. These results support the idea that although individual miRNAs can act to restrict HIV-1 replication, the virus does not counter these effects through a global suppression of RNAi synthesis or processing.

Show MeSH
Related in: MedlinePlus