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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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Increasing the complementarity ofHIV sequence to miR-326 leads to greater restriction of the virus. Co-infectionexperiments in Jurkat cells with mutant and Wt viruses. Each of the seven changedviruses (1.1, 1.2, 1.3, 2.1, 2.2, 2.3 or 3.1) in A was used in head-to-headco-infection experiments with the Wt virus for three successive rounds of infection.The relative amounts of Wt and mutant RNA (in percent of total genomic RNA) atrounds 1, 2 and 3 are given for co-infection of Wt with 1.1, 1.2 or 1.3 virus (leftgraph) or Wt with 2.1, 2.2, 2.3 or 3.1 virus (right graph).
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gks912-F8: Increasing the complementarity ofHIV sequence to miR-326 leads to greater restriction of the virus. Co-infectionexperiments in Jurkat cells with mutant and Wt viruses. Each of the seven changedviruses (1.1, 1.2, 1.3, 2.1, 2.2, 2.3 or 3.1) in A was used in head-to-headco-infection experiments with the Wt virus for three successive rounds of infection.The relative amounts of Wt and mutant RNA (in percent of total genomic RNA) atrounds 1, 2 and 3 are given for co-infection of Wt with 1.1, 1.2 or 1.3 virus (leftgraph) or Wt with 2.1, 2.2, 2.3 or 3.1 virus (right graph).

Mentions: In principle, a strong restriction of viral replication generally selects rapidly for‘escape’ mutations, whereas weak or inconsequential restriction does not(50,51). To ask if miR-326 restriction correlates with better viral RNA targetcomplementarity to miR-326, we created seven new HIV viruses (1.1, 1.2, 1.3, 2.1, 2.2, 2.3and 3.1), all of them fully isogenic, except for 1, 2 or 3 base changes in theirmiR-326-targeted Wt sequence to increase progressively (+1, +2 or +3matches) their base pairing complementarity with miR-326 (Figure 7). These mutant viruses were individually tested in threerounds of co-infection paired with the Wt virus, and viral RNAs were quantified at theconclusion of each round of infection (Figure8A). We noted that all mutant viruses were created to be better base pair-matchedto miR-326 than Wt virus; and indeed all the mutant viruses were more strongly repressedthan Wt virus in head-to-head replication competition (Figure 8), consistent with the extent of sequence complementaritywith miR-326 correlating with the robustness of HIV-1 replication in cells. Figure 7.


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)

Increasing the complementarity ofHIV sequence to miR-326 leads to greater restriction of the virus. Co-infectionexperiments in Jurkat cells with mutant and Wt viruses. Each of the seven changedviruses (1.1, 1.2, 1.3, 2.1, 2.2, 2.3 or 3.1) in A was used in head-to-headco-infection experiments with the Wt virus for three successive rounds of infection.The relative amounts of Wt and mutant RNA (in percent of total genomic RNA) atrounds 1, 2 and 3 are given for co-infection of Wt with 1.1, 1.2 or 1.3 virus (leftgraph) or Wt with 2.1, 2.2, 2.3 or 3.1 virus (right graph).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gks912-F8: Increasing the complementarity ofHIV sequence to miR-326 leads to greater restriction of the virus. Co-infectionexperiments in Jurkat cells with mutant and Wt viruses. Each of the seven changedviruses (1.1, 1.2, 1.3, 2.1, 2.2, 2.3 or 3.1) in A was used in head-to-headco-infection experiments with the Wt virus for three successive rounds of infection.The relative amounts of Wt and mutant RNA (in percent of total genomic RNA) atrounds 1, 2 and 3 are given for co-infection of Wt with 1.1, 1.2 or 1.3 virus (leftgraph) or Wt with 2.1, 2.2, 2.3 or 3.1 virus (right graph).
Mentions: In principle, a strong restriction of viral replication generally selects rapidly for‘escape’ mutations, whereas weak or inconsequential restriction does not(50,51). To ask if miR-326 restriction correlates with better viral RNA targetcomplementarity to miR-326, we created seven new HIV viruses (1.1, 1.2, 1.3, 2.1, 2.2, 2.3and 3.1), all of them fully isogenic, except for 1, 2 or 3 base changes in theirmiR-326-targeted Wt sequence to increase progressively (+1, +2 or +3matches) their base pairing complementarity with miR-326 (Figure 7). These mutant viruses were individually tested in threerounds of co-infection paired with the Wt virus, and viral RNAs were quantified at theconclusion of each round of infection (Figure8A). We noted that all mutant viruses were created to be better base pair-matchedto miR-326 than Wt virus; and indeed all the mutant viruses were more strongly repressedthan Wt virus in head-to-head replication competition (Figure 8), consistent with the extent of sequence complementaritywith miR-326 correlating with the robustness of HIV-1 replication in cells. Figure 7.

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