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Interplay between HIV-1 infection and host microRNAs.

Sun G, Li H, Wu X, Covarrubias M, Scherer L, Meinking K, Luk B, Chomchan P, Alluin J, Gombart AF, Rossi JJ - Nucleic Acids Res. (2011)

Bottom Line: While microRNA-223 levels were significantly enriched in HIV-1-infected CD4(+)CD8(-) PBMCs, microRNA-29a/b, microRNA-155 and microRNA-21 levels were significantly reduced.Among those microRNAs, the microRNA-29 family has seed complementarity in the HIV-1 3'-UTR, but the potential suppressive effect of microRNA-29 on HIV-1 is severely blocked by the secondary structure of the target region.Our data support a possible regulatory circuit at the peak of HIV-1 replication which involves downregulation of microRNA-29, expression of Nef, the apoptosis of host CD4 cells and upregulation of microRNA-223.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biological Science, Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA.

ABSTRACT
Using microRNA array analyses of in vitro HIV-1-infected CD4(+) cells, we find that several host microRNAs are significantly up- or downregulated around the time HIV-1 infection peaks in vitro. While microRNA-223 levels were significantly enriched in HIV-1-infected CD4(+)CD8(-) PBMCs, microRNA-29a/b, microRNA-155 and microRNA-21 levels were significantly reduced. Based on the potential for microRNA binding sites in a conserved sequence of the Nef-3'-LTR, several host microRNAs potentially could affect HIV-1 gene expression. Among those microRNAs, the microRNA-29 family has seed complementarity in the HIV-1 3'-UTR, but the potential suppressive effect of microRNA-29 on HIV-1 is severely blocked by the secondary structure of the target region. Our data support a possible regulatory circuit at the peak of HIV-1 replication which involves downregulation of microRNA-29, expression of Nef, the apoptosis of host CD4 cells and upregulation of microRNA-223.

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Related in: MedlinePlus

Reporter and P24 assays to test repression of HIV-1 NL4-3 by miR-223 and miR-29. (a) Co-transfection of pNL4-3-Luc with miRNAs. The data show miR-223 has weak repression, miR-29 has somewhat better repression. MiRtron-1224 gave mild repression. The y-axis represents the relative Fluc to Rluc signal (as percentages) normalized to the control (fU1-miR) by percentage. The values represent the average from at least three independent experiments and the error bar represents the standard deviation. (b) HIV-1 P24 assays for co-transfection of HIV-1 pNL4-3 with ectopically expressed pri-miRNA-223 or 29 in HeLa-T4 cells. The results indicate strong repression from miR-29 but not miR-223. The y-axis represents the relative p24 values (as percentages) normalized to the control (fU1-miR). The values represent the average from at least three independent experiments and error bar represents the standard deviation. (c) HIV-1 P24 assays for co-transfection of HIV-1 pNL4-3 with chemically synthesized pre-miRNA-223, -29a, -29b and anti-miRs for miR-223, -29a, -29b into HeLa-T4 cells. The results indicate moderate repression mediated by miR-29a, -29b and somewhat milder repression mediated by miR-223. The anti-miR data show an increase in P24 levels for anti-miR-29a and anti-miR-29b, but not for anti-miR-223. The y-axis represents relative p24 values (as percentages) normalized to the control (Negative pre-miR as control for pre-miRNAs and negative anti-miR as control for anti-miRs). The values represent the average from at least three independent experiments and the error bars depict the standard deviation.
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gkr961-F3: Reporter and P24 assays to test repression of HIV-1 NL4-3 by miR-223 and miR-29. (a) Co-transfection of pNL4-3-Luc with miRNAs. The data show miR-223 has weak repression, miR-29 has somewhat better repression. MiRtron-1224 gave mild repression. The y-axis represents the relative Fluc to Rluc signal (as percentages) normalized to the control (fU1-miR) by percentage. The values represent the average from at least three independent experiments and the error bar represents the standard deviation. (b) HIV-1 P24 assays for co-transfection of HIV-1 pNL4-3 with ectopically expressed pri-miRNA-223 or 29 in HeLa-T4 cells. The results indicate strong repression from miR-29 but not miR-223. The y-axis represents the relative p24 values (as percentages) normalized to the control (fU1-miR). The values represent the average from at least three independent experiments and error bar represents the standard deviation. (c) HIV-1 P24 assays for co-transfection of HIV-1 pNL4-3 with chemically synthesized pre-miRNA-223, -29a, -29b and anti-miRs for miR-223, -29a, -29b into HeLa-T4 cells. The results indicate moderate repression mediated by miR-29a, -29b and somewhat milder repression mediated by miR-223. The anti-miR data show an increase in P24 levels for anti-miR-29a and anti-miR-29b, but not for anti-miR-223. The y-axis represents relative p24 values (as percentages) normalized to the control (Negative pre-miR as control for pre-miRNAs and negative anti-miR as control for anti-miRs). The values represent the average from at least three independent experiments and the error bars depict the standard deviation.

Mentions: We first performed reporter assays by co-transfection of the miRNAs with the pNL4-3-Luc vector. This reporter construct containing a Firefly luciferase (Fluc) gene was created by in-frame insertion of the Fluc coding sequence into the pNL4-3 Nef gene. A stop codon was also inserted immediately following the Fluc coding sequence (30,31). The miR-223 and miR-29 target sites are downstream of this stop codon in the 3′-UTR.The co-transfection results showed miRtron-1224, one of the best potential miRNAs predicted by PITA that can target NL4-3 Nef-3′-LTR, which has a 8-mer target seed (position 8979 of NL4-3; Table 3) located ~200 nt upstream of the miR-29 target site in the 3′-LTR, provided repression levels of 20–30%. In contrast, miR-223 only resulted in 5–10% repression. Interestingly, miR-29 triggered a much stronger suppression ranging from 30% to 40% (Figure 3a). We next performed HIV-1 p24 assays following co-transfection of HIV pNL4-3 proviral DNA with ectopically expressed pri-miRNAs 223 or 29 in HeLa-T4 cells. The results of these transfections showed that ectopic expression of miR-29 resulted in ~40–60% reduction of p24 levels (Figure 3b). In order to rule out the possibility that the expressed pri-miRNA may not produce sufficient levels of mature miRNAs for efficient target knockdown, we co-transfected the synthetic Pre-miR™ miRNA precursor molecules with the pNL4-3 proviral DNA in HeLa-T4 cells. We also carried out experiments with HeLa-T4 cells first transfected with the pre-miRs followed by HIV-1 IIIB infection (Figure 3c). The data from these experiments were consistent with the plasmid co-transfection data. Anti-miR™ miRNA inhibitors were used to inactivate the miRNAs. The anti-miR results show that knockdown of endogenous miR-29a/b led to enhanced HIV-1 infection as measured by p24 production. In contrast, knockdown of miR-223 had no effect. The later result is not surprising since miR-223 levels are extremely low in the HeLa-T4 cells and are undetectable in northern blots (Figure 3c).Figure 3.


Interplay between HIV-1 infection and host microRNAs.

Sun G, Li H, Wu X, Covarrubias M, Scherer L, Meinking K, Luk B, Chomchan P, Alluin J, Gombart AF, Rossi JJ - Nucleic Acids Res. (2011)

Reporter and P24 assays to test repression of HIV-1 NL4-3 by miR-223 and miR-29. (a) Co-transfection of pNL4-3-Luc with miRNAs. The data show miR-223 has weak repression, miR-29 has somewhat better repression. MiRtron-1224 gave mild repression. The y-axis represents the relative Fluc to Rluc signal (as percentages) normalized to the control (fU1-miR) by percentage. The values represent the average from at least three independent experiments and the error bar represents the standard deviation. (b) HIV-1 P24 assays for co-transfection of HIV-1 pNL4-3 with ectopically expressed pri-miRNA-223 or 29 in HeLa-T4 cells. The results indicate strong repression from miR-29 but not miR-223. The y-axis represents the relative p24 values (as percentages) normalized to the control (fU1-miR). The values represent the average from at least three independent experiments and error bar represents the standard deviation. (c) HIV-1 P24 assays for co-transfection of HIV-1 pNL4-3 with chemically synthesized pre-miRNA-223, -29a, -29b and anti-miRs for miR-223, -29a, -29b into HeLa-T4 cells. The results indicate moderate repression mediated by miR-29a, -29b and somewhat milder repression mediated by miR-223. The anti-miR data show an increase in P24 levels for anti-miR-29a and anti-miR-29b, but not for anti-miR-223. The y-axis represents relative p24 values (as percentages) normalized to the control (Negative pre-miR as control for pre-miRNAs and negative anti-miR as control for anti-miRs). The values represent the average from at least three independent experiments and the error bars depict the standard deviation.
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gkr961-F3: Reporter and P24 assays to test repression of HIV-1 NL4-3 by miR-223 and miR-29. (a) Co-transfection of pNL4-3-Luc with miRNAs. The data show miR-223 has weak repression, miR-29 has somewhat better repression. MiRtron-1224 gave mild repression. The y-axis represents the relative Fluc to Rluc signal (as percentages) normalized to the control (fU1-miR) by percentage. The values represent the average from at least three independent experiments and the error bar represents the standard deviation. (b) HIV-1 P24 assays for co-transfection of HIV-1 pNL4-3 with ectopically expressed pri-miRNA-223 or 29 in HeLa-T4 cells. The results indicate strong repression from miR-29 but not miR-223. The y-axis represents the relative p24 values (as percentages) normalized to the control (fU1-miR). The values represent the average from at least three independent experiments and error bar represents the standard deviation. (c) HIV-1 P24 assays for co-transfection of HIV-1 pNL4-3 with chemically synthesized pre-miRNA-223, -29a, -29b and anti-miRs for miR-223, -29a, -29b into HeLa-T4 cells. The results indicate moderate repression mediated by miR-29a, -29b and somewhat milder repression mediated by miR-223. The anti-miR data show an increase in P24 levels for anti-miR-29a and anti-miR-29b, but not for anti-miR-223. The y-axis represents relative p24 values (as percentages) normalized to the control (Negative pre-miR as control for pre-miRNAs and negative anti-miR as control for anti-miRs). The values represent the average from at least three independent experiments and the error bars depict the standard deviation.
Mentions: We first performed reporter assays by co-transfection of the miRNAs with the pNL4-3-Luc vector. This reporter construct containing a Firefly luciferase (Fluc) gene was created by in-frame insertion of the Fluc coding sequence into the pNL4-3 Nef gene. A stop codon was also inserted immediately following the Fluc coding sequence (30,31). The miR-223 and miR-29 target sites are downstream of this stop codon in the 3′-UTR.The co-transfection results showed miRtron-1224, one of the best potential miRNAs predicted by PITA that can target NL4-3 Nef-3′-LTR, which has a 8-mer target seed (position 8979 of NL4-3; Table 3) located ~200 nt upstream of the miR-29 target site in the 3′-LTR, provided repression levels of 20–30%. In contrast, miR-223 only resulted in 5–10% repression. Interestingly, miR-29 triggered a much stronger suppression ranging from 30% to 40% (Figure 3a). We next performed HIV-1 p24 assays following co-transfection of HIV pNL4-3 proviral DNA with ectopically expressed pri-miRNAs 223 or 29 in HeLa-T4 cells. The results of these transfections showed that ectopic expression of miR-29 resulted in ~40–60% reduction of p24 levels (Figure 3b). In order to rule out the possibility that the expressed pri-miRNA may not produce sufficient levels of mature miRNAs for efficient target knockdown, we co-transfected the synthetic Pre-miR™ miRNA precursor molecules with the pNL4-3 proviral DNA in HeLa-T4 cells. We also carried out experiments with HeLa-T4 cells first transfected with the pre-miRs followed by HIV-1 IIIB infection (Figure 3c). The data from these experiments were consistent with the plasmid co-transfection data. Anti-miR™ miRNA inhibitors were used to inactivate the miRNAs. The anti-miR results show that knockdown of endogenous miR-29a/b led to enhanced HIV-1 infection as measured by p24 production. In contrast, knockdown of miR-223 had no effect. The later result is not surprising since miR-223 levels are extremely low in the HeLa-T4 cells and are undetectable in northern blots (Figure 3c).Figure 3.

Bottom Line: While microRNA-223 levels were significantly enriched in HIV-1-infected CD4(+)CD8(-) PBMCs, microRNA-29a/b, microRNA-155 and microRNA-21 levels were significantly reduced.Among those microRNAs, the microRNA-29 family has seed complementarity in the HIV-1 3'-UTR, but the potential suppressive effect of microRNA-29 on HIV-1 is severely blocked by the secondary structure of the target region.Our data support a possible regulatory circuit at the peak of HIV-1 replication which involves downregulation of microRNA-29, expression of Nef, the apoptosis of host CD4 cells and upregulation of microRNA-223.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biological Science, Department of Molecular and Cellular Biology, Beckman Research Institute of City of Hope, 1500 E. Duarte Road, Duarte, CA 91010, USA.

ABSTRACT
Using microRNA array analyses of in vitro HIV-1-infected CD4(+) cells, we find that several host microRNAs are significantly up- or downregulated around the time HIV-1 infection peaks in vitro. While microRNA-223 levels were significantly enriched in HIV-1-infected CD4(+)CD8(-) PBMCs, microRNA-29a/b, microRNA-155 and microRNA-21 levels were significantly reduced. Based on the potential for microRNA binding sites in a conserved sequence of the Nef-3'-LTR, several host microRNAs potentially could affect HIV-1 gene expression. Among those microRNAs, the microRNA-29 family has seed complementarity in the HIV-1 3'-UTR, but the potential suppressive effect of microRNA-29 on HIV-1 is severely blocked by the secondary structure of the target region. Our data support a possible regulatory circuit at the peak of HIV-1 replication which involves downregulation of microRNA-29, expression of Nef, the apoptosis of host CD4 cells and upregulation of microRNA-223.

Show MeSH
Related in: MedlinePlus