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The extent of sequence complementarity correlates with the potency of cellular miRNA-mediated restriction of HIV-1.

Houzet L, Klase Z, Yeung ML, Wu A, Le SY, Quiñones M, Jeang KT - Nucleic Acids Res. (2012)

Bottom Line: Using in silico analyses, we predicted target sites for 22 human miRNAs in the HIV genome.Transfection experiments using synthetic miRNAs showed that five of these miRNAs capably decreased HIV replication.Antagomirs to miR-326 that knocked down this cell endogenous miRNA increased HIV-1 replication in cells, suggesting that miR-326 is physiologically functional in moderating HIV-1 replication in human cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Virology Section, Laboratory of Molecular Microbiology National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0460, USA.

ABSTRACT
MicroRNAs (miRNAs) are 22-nt non-coding RNAs involved in the regulation of cellular gene expression and potential cellular defense against viral infection. Using in silico analyses, we predicted target sites for 22 human miRNAs in the HIV genome. Transfection experiments using synthetic miRNAs showed that five of these miRNAs capably decreased HIV replication. Using one of these five miRNAs, human miR-326 as an example, we demonstrated that the degree of complementarity between the predicted viral sequence and cellular miR-326 correlates, in a Dicer-dependent manner, with the potency of miRNA-mediated restriction of viral replication. Antagomirs to miR-326 that knocked down this cell endogenous miRNA increased HIV-1 replication in cells, suggesting that miR-326 is physiologically functional in moderating HIV-1 replication in human cells.

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Increased miRNA–mRNA complementarity correlates withgreater reduction of expression in reporter assays. (A) Schematicrepresentation of CMV promoter-driven reporter constructs engineered to containthree tandem copies of either the Wt or Tgt target sites positioned downstream ofthe luciferase gene. (B) Effect of miR-326 on the Wt and Tgt targetsites. The 293T cells were transfected with Luc-3X Wt or Luc-3X Tgt together with aninternal control pMIR-βgal plasmid. After 24 h post -transfection, cells werelysed and luciferase and βgal activities were measured. The luciferase activityfrom Luc-3X Tgt, after normalization to βgal, was expressed relative to thatfrom the Luc-3X Wt construct. (C) Effect of transfected miR-326 onLuc-3X Wt and Luc-3X Tgt. miR-326 (+) or negative control (−) siRNA(negative control siRNA #1, Ambion) was transfected into 293 T cells with either theLuc-3X Wt or Luc-3X Tgt constructs and the internal control pMIR-βGal.Luciferase and βgal activities were monitored 24 h post-transfection. Values(+) are given as percent of the value from the negative control siRNA (−)transfected cells which was set to 100%. Values are the mean of threeseparate experiments presented with standard deviations.
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gks912-F5: Increased miRNA–mRNA complementarity correlates withgreater reduction of expression in reporter assays. (A) Schematicrepresentation of CMV promoter-driven reporter constructs engineered to containthree tandem copies of either the Wt or Tgt target sites positioned downstream ofthe luciferase gene. (B) Effect of miR-326 on the Wt and Tgt targetsites. The 293T cells were transfected with Luc-3X Wt or Luc-3X Tgt together with aninternal control pMIR-βgal plasmid. After 24 h post -transfection, cells werelysed and luciferase and βgal activities were measured. The luciferase activityfrom Luc-3X Tgt, after normalization to βgal, was expressed relative to thatfrom the Luc-3X Wt construct. (C) Effect of transfected miR-326 onLuc-3X Wt and Luc-3X Tgt. miR-326 (+) or negative control (−) siRNA(negative control siRNA #1, Ambion) was transfected into 293 T cells with either theLuc-3X Wt or Luc-3X Tgt constructs and the internal control pMIR-βGal.Luciferase and βgal activities were monitored 24 h post-transfection. Values(+) are given as percent of the value from the negative control siRNA (−)transfected cells which was set to 100%. Values are the mean of threeseparate experiments presented with standard deviations.

Mentions: To additionally validate that miR-326 more strongly represses the Tgt versus the Wttarget site sequence, we created two Cytomegalovirus (CMV) promoter-driven constructs thatcontain three copies of either the Wt or the Tgt target positioned downstream of aluciferase reporter (Figure 5A). The tworeporters were transfected into 293T cells, and relative luciferase expression wasmeasured. In agreement with the virus replication results in Figures 3 and 4, theTgt-reporter was consistently expressed at a lower magnitude than the Wt-reporter(P < 0.001; Figure 5B)in cell settings where no miR-326 over-expression was performed. To check that thisrepressive effect can also be dose-dependent on miR-326 expression, we repeated theexperiment in the context of miR-326 over-expression. Wt- and Tgt- luciferase reporterswere co-transfected with a synthetic miR-326 or a negative control random RNAoligonucleotide. In the setting of miR-326 over-expression, Tgt-linked luciferaseexpression was decreased by 2-fold more than Wt-linked expression (Figure 5C). Figure5.


The extent of sequence complementarity correlates with the potency of cellular miRNA-mediated restriction of HIV-1.

Houzet L, Klase Z, Yeung ML, Wu A, Le SY, Quiñones M, Jeang KT - Nucleic Acids Res. (2012)

Increased miRNA–mRNA complementarity correlates withgreater reduction of expression in reporter assays. (A) Schematicrepresentation of CMV promoter-driven reporter constructs engineered to containthree tandem copies of either the Wt or Tgt target sites positioned downstream ofthe luciferase gene. (B) Effect of miR-326 on the Wt and Tgt targetsites. The 293T cells were transfected with Luc-3X Wt or Luc-3X Tgt together with aninternal control pMIR-βgal plasmid. After 24 h post -transfection, cells werelysed and luciferase and βgal activities were measured. The luciferase activityfrom Luc-3X Tgt, after normalization to βgal, was expressed relative to thatfrom the Luc-3X Wt construct. (C) Effect of transfected miR-326 onLuc-3X Wt and Luc-3X Tgt. miR-326 (+) or negative control (−) siRNA(negative control siRNA #1, Ambion) was transfected into 293 T cells with either theLuc-3X Wt or Luc-3X Tgt constructs and the internal control pMIR-βGal.Luciferase and βgal activities were monitored 24 h post-transfection. Values(+) are given as percent of the value from the negative control siRNA (−)transfected cells which was set to 100%. Values are the mean of threeseparate experiments presented with standard deviations.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC3526334&req=5

gks912-F5: Increased miRNA–mRNA complementarity correlates withgreater reduction of expression in reporter assays. (A) Schematicrepresentation of CMV promoter-driven reporter constructs engineered to containthree tandem copies of either the Wt or Tgt target sites positioned downstream ofthe luciferase gene. (B) Effect of miR-326 on the Wt and Tgt targetsites. The 293T cells were transfected with Luc-3X Wt or Luc-3X Tgt together with aninternal control pMIR-βgal plasmid. After 24 h post -transfection, cells werelysed and luciferase and βgal activities were measured. The luciferase activityfrom Luc-3X Tgt, after normalization to βgal, was expressed relative to thatfrom the Luc-3X Wt construct. (C) Effect of transfected miR-326 onLuc-3X Wt and Luc-3X Tgt. miR-326 (+) or negative control (−) siRNA(negative control siRNA #1, Ambion) was transfected into 293 T cells with either theLuc-3X Wt or Luc-3X Tgt constructs and the internal control pMIR-βGal.Luciferase and βgal activities were monitored 24 h post-transfection. Values(+) are given as percent of the value from the negative control siRNA (−)transfected cells which was set to 100%. Values are the mean of threeseparate experiments presented with standard deviations.
Mentions: To additionally validate that miR-326 more strongly represses the Tgt versus the Wttarget site sequence, we created two Cytomegalovirus (CMV) promoter-driven constructs thatcontain three copies of either the Wt or the Tgt target positioned downstream of aluciferase reporter (Figure 5A). The tworeporters were transfected into 293T cells, and relative luciferase expression wasmeasured. In agreement with the virus replication results in Figures 3 and 4, theTgt-reporter was consistently expressed at a lower magnitude than the Wt-reporter(P < 0.001; Figure 5B)in cell settings where no miR-326 over-expression was performed. To check that thisrepressive effect can also be dose-dependent on miR-326 expression, we repeated theexperiment in the context of miR-326 over-expression. Wt- and Tgt- luciferase reporterswere co-transfected with a synthetic miR-326 or a negative control random RNAoligonucleotide. In the setting of miR-326 over-expression, Tgt-linked luciferaseexpression was decreased by 2-fold more than Wt-linked expression (Figure 5C). Figure5.

Bottom Line: Using in silico analyses, we predicted target sites for 22 human miRNAs in the HIV genome.Transfection experiments using synthetic miRNAs showed that five of these miRNAs capably decreased HIV replication.Antagomirs to miR-326 that knocked down this cell endogenous miRNA increased HIV-1 replication in cells, suggesting that miR-326 is physiologically functional in moderating HIV-1 replication in human cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Virology Section, Laboratory of Molecular Microbiology National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0460, USA.

ABSTRACT
MicroRNAs (miRNAs) are 22-nt non-coding RNAs involved in the regulation of cellular gene expression and potential cellular defense against viral infection. Using in silico analyses, we predicted target sites for 22 human miRNAs in the HIV genome. Transfection experiments using synthetic miRNAs showed that five of these miRNAs capably decreased HIV replication. Using one of these five miRNAs, human miR-326 as an example, we demonstrated that the degree of complementarity between the predicted viral sequence and cellular miR-326 correlates, in a Dicer-dependent manner, with the potency of miRNA-mediated restriction of viral replication. Antagomirs to miR-326 that knocked down this cell endogenous miRNA increased HIV-1 replication in cells, suggesting that miR-326 is physiologically functional in moderating HIV-1 replication in human cells.

Show MeSH
Related in: MedlinePlus