HIV-1 infects human cells and integrates its DNA into the host genome. HIV-1 infection induces expression of specific sets of cellular miRNAs, including miR-29a. HIV-1 mRNA is transcribed, exported from the nucleus, and translated into viral proteins. Cellular RISC containing specific miRNAs, such as miR-29a, targets HIV-1 mRNA and sequesters the ribonucleoprotein complex in P-bodies. Depending upon cellular stimuli or viral pathogenesis cues, HIV-1 mRNA could be stored in P-bodies and released for subsequent translation of viral proteins. Alternatively, viral mRNA could be degraded in P-bodies.

(A) Inhibiting miR-29a, 29b, or 29c increases HIV-1 production. 293T cells were first transfected with 10 nM miR inhibitor (negative control), miR-29a, b, or c inhibitor, then co-transfected with pNL4-3LucR-E- and VSVG. At 48 h post-transfection, cell supernatants were quantitated for virus production. Data are normalized to data for treatment with anti-miR control (% of control). Error bars represent SD, with significance from t-tests shown by p<0.001 (***). (B) miR-29a, −29b, or −29c decreases HIV-1 production. 293T cells were first transfected with 20 nM control, miR-29a, b, or c mimic, then co-transfected with pNL4-3LucR-E- and VSVG. At 48 h post-transfection, cell supernatants were quantitated for virus production. Data are normalized to data for treatment with miRNA control (% of control). Error bars represent SD, with significance from t-tests shown by p<0.001 (***). (C) Sequence conservation of the miR-29a target nef/LTR region in various HIV-1 subtypes. Sequences of HIV-1 subtypes were downloaded from HIV sequence database (http://www.hiv.lanl.gov/content/sequence/HIV/mainpage.html). Sequences for nef or 3’LTR were aligned using CLC Sequence Viewer 4 software.

(A) miR-29a specifically enhances the association of HIV-1 mRNA with RISC RNP complexes. 293T cells were first transfected with miR-29a or miR-mt29a, then with pNL4-3LucRE(wt) or pNL4-3LucREm29t, VSVG, and Myc-Ago2. At 48 h post-transfection, total cell extracts were immunoprecipitated with antibodies against Myc tag and immunoblotted for Myc-Ago2 (top panel). The mRNA was amplified by RT-PCR for HIV-1 gag and connexin 43 mRNA (middle panels). miRNA expression was monitored (bottom panel) by a commercially available kit (see Experimental Procedures). The mRNA associated with Ago2 was quantified by RT-qPCR (bar graph). Data in the bar graph represent % gag mRNA in the presence of wt and mutant miRNAs normalized to data for transfection with pNL4-3LucR-E- (wt) in the presence of miR-29a and for transfection with pNL4-3LucR-E-m29t in the presence of miR-29a-mt, respectively. (B) miR-29 specifically enhances HIV-1 mRNA co-purification with the immunopurified, endogenous P-body protein, RCK/p54. 293T cells were first transfected with miR-29a or miR-mt29a, then with pNL4-3LucR E(wt) or pNL4-3LucREm29t, and VSVG. At 48 h post-transfection, total cell extracts were immunoprecipitated with antibodies against RCK/p54 and immunoblotted (top panel). The mRNA was amplified by RT-PCR for HIV-1 gag and connexin 43 mRNA (middle panels). miRNA expression was monitored (bottom panel) as in (A). The mRNA associated with RCK/p54 was quantified by RT-qPCR (Figure 4B, bar graph; as described in A). Data in the bar graph represent % gag mRNA in the presence of wt and mutant miRNAs normalized to data for pNL4-3LucR E (wt) with miR-29a and for pNL4-3LucR Em29t with miR-29a-mt, respectively.

(A) Dicer knockdown enhances HIV-1 production. Producer 293T cells were transfected with siRNA mismatched to Dicer mRNA (si-control) or perfectly matched (si-Dicer) and analyzed 48 h post-transfection for virus production (pNL4-3LucR-E-). Efficient knockdown of Dicer is shown by immunoblot analysis of total cell extracts (top panel). Virus production data (lower panel) are normalized to data for cells treated with mismatched siRNA (% of si-control). (B) Dicer knockdown enhances HIV-1 infectivity. Target 293T cells were treated with siRNA mismatched to Dicer mRNA (si-control) or perfectly matched (si-Dicer), and infected for 48 h with the virus equivalent of 50 ng pNL4-3LucR-E-. Virus infectivity was analyzed by measuring luciferase activity of infected cell lysates relative to their protein content. Efficient knockdown of Dicer is shown by immunoblot analysis of total cell extracts (top panel). Virus infectivity data (bottom panel) are normalized to data for cells treated with mismatched siRNA (% of si-control). (C) Drosha knockdown enhances HIV-1 production. Producer 293T cells were transfected with siRNA mismatched to Drosha mRNA (si-control) or perfectly matched (si-Drosha) and analyzed 48 h post-transfection for virus production (bottom panel) as described in Experimental Procedures. Efficient knockdown of Drosha was confirmed by immunoblot analysis of total cell extracts (top panel). (D) Drosha knockdown enhances HIV-1 infectivity. Target 293T cells were treated with siRNA mismatched (si-control) or perfectly matched (si-Drosha) to Drosha mRNA and infected with 50 ng pNL4-3LucR-E-. Virus infectivity was analyzed by measuring luciferase activity in infected cells (bottom panel). Efficient knockdown of Drosha was confirmed by immunoblot analysis of total cell extracts (top panel). Data in all four panels are from at least 3 independent experiments. Error bars represent SD, with significance from t-tests shown by p< 0.05 (*) and 0.001 (***).

(A) HIV-1 infection induces miR-29a expression in 293T cells. 293T cells were infected for 48 h with the virus equivalent of 50 ng pNL4-3LucR-E-. Expression of miR-29a was quantified by RT-qPCR (see Experimental Procedures). The level of miR-29a was about 20% higher in HIV-1 infected 293T cells than in mock-infected 293T cells. (B) Predicted miR-29a target site in the HIV-1 3’-UTR (see Table 1). (C) Inhibiting miR-29a increases HIV-1 production. 293T cells were transfected with 20 nM miR inhibitor (negative control) or miR-29a inhibitor, then co-transfected with pNL4-3LucR-E- and VSVG. At 48 h post-transfection, cell supernatants were quantitated for virus production. Data are normalized to data for cells treated with anti-miR (% of control). (D) Inhibiting miR-29a increases HIV-1 infectivity. Target 293T cells were transfected with 20 nM miR inhibitor (negative control) or miR-29a inhibitor. At 24 h post-transfection, cells were infected with a 50 ng equivalent of pNL4-3LucR-E- virus. At 48 h post-infection, infectivity was measured by luciferase activity in total cell lysates relative to their protein content. Virus infectivity data are normalized to data for cells treated with anti-miR (% of control). (E) miR-29a mimic decreases HIV-1 production. 293T cells were transfected with 10 nM control miRNA or miR-29a mimic, then co-transfected with pNL4-3LucR-E- and VSVG. At 48 h post-transfection, cell supernatants were quantitated for virus production. Virus production data are normalized to data for cells treated with anti-miR (% of control). (F) Inhibiting miR-29a enhances viral infectivity in T lymphocytes. H9 T lymphocytes were transfected with 20 nM miR inhibitor (negative control) or miR-29a inhibitor. At 48 h post-transfection, H9 cells were infected by spinoculation with a virus equivalent of 1 µg pNL4-3LucR-E- virus. At 48 h post-infection, virus infectivity was measured by luciferase activity in total cell lysates relative to their protein content. Virus infectivity data are normalized to data for cells treated with anti-miR (% of control). Data in panels (A), (C), (D), (E), and (F) are from at least 3 independent experiments. Error bars represent SD, with significance from t-tests shown by p< 0.05 (*) and 0.001 (***).

(A) Depleting RCK/p54 disrupts P-bodies. To silence expression of RCK/p54, 293T cells were co-transfected with APOBEC3G–YFP (APO-YFP) and with siRNA against RCK/p54 mRNA (si-RCK/p54) or with mismatched siRNA (si-control). At 24 h post-transfection, cells were fixed and analyzed by confocal microscopy. APO-YFP was detected by direct YFP fluorescence (panels a, d), whereas endogenous RCK/p54 was detected by immunofluorescence with anti-RCK/p54 (panels b, e). Cells were stained to visualize nuclei and images were digitally merged (panels c, f). (B) Depleting Lsm1 disrupts P-bodies. Lsm1 expression was silenced and cells were treated with siRNA against Lsm1 or si-control as in (A). (C) Depleting Ago2 disrupts P-bodies. Ago2 expression was silenced and cells were treated with siRNA against Ago2 or si-control as in (A). (D) Knockdown of RCK/p54, Ago2, or Lsm1 increases HIV-1 production. Producer 293T cells were first transfected with siRNAs mismatched (mm) or perfectly matched (pm) to target P-body protein mRNAs, then co-transfected with pNL4-3LucR-E- and VSVG. Protein knockdown was monitored by immunoblotting (Figure S3). At 48 h post-transfection, viral production was determined by measuring HIV-1 p24 antigen in culture supernatants. Data are normalized to data from mm siRNA-treated cells (si-control). Error bars represent SD, with significance from t-tests shown by p< 0.01 (**) and 0.001 (***). (E) Depleting RCK/p54, Ago2, or Lsm1 increases HIV-1 infectivity. Target 293T cells were transfected with mm or pm siRNA. Protein knockdown was confirmed by immunoblotting (data not shown). Approximately 4 h after the second siRNA transfection, cells were infected for 48 h with the virus equivalent of 50 ng pNL4-3LucR-E. Virus infectivity was determined in total cell lysates from infected cells by measuring luciferase activity determined relative to lysate protein content. Virus infectivity data are normalized to data from mm siRNA-treated cells (% control). Error bars represent SD, with significance from t-tests shown by p< 0.05 (*) and 0.01 (**). (F) Endogenous miRNAs and exogenous HIV-1 mRNAs localize to P-bodies in 293T cells. To detect the localization of miR-18a, 293T cells were transfected with APOBEC3G–YFP (APO-YFP), fixed at 24 h post-transfection, immunostained for APO-YPF or RCK/p54, hybridized in situ for miR-18a with fluorescein-labeled LNA probe, and analyzed by confocal microscopy. APO-YFP and endogenous RCK/p54 were detected by immunofluorescence with anti-YFP and anti-RCK/p54, respectively (panels b, e). Cells were stained to visualize nuclei and images were digitally merged (panels c, f). Arrowheads indicate co-localization of miR-18a and APOBEC3G or RCK/p54 in P-bodies (G) HIV-1 mRNAs co-localize with APOBEC3G. 293T cells were transfected with APO-YFP and pNL4-3, fixed at 36 h post-transfection, immunostained, and hybridized in situ for HIV-1 mRNA as described above. LNA probes complementary to HIV-1 Nef mRNA were labeled with Cy3 and used for FISH.

(A) Schematic representation of HIV-1 reporter constructs. HIV-1 wild-type (pNL4-3Luc wt) and miR-29a target site mutated at the seed sequence (pNL4-3Luc m29t) are shown. The miR-29a target site is indicated by a vertical line (I) and the mutant site by an x (X). Below these construct schema are base-pairing schema of the predicted miR-29a with its target site (pNL4-3Luc wt) and of the seed-mutant miR-29a-mt with its complementary target sequence (pNL4-3Luc m29t). For the mutant sequence, 4 point substitutions (in red) were introduced to disrupt base pairing to miR-29a but to retain pairing to miR-29a-mt. (B) miR-29a specifically represses HIV-1 virus production. 293T cells were co-transfected with HIV-1 construct (pNL4-3Luc wt or pNL4-3Luc m29t) and either miR-29a mimic or seed-mutant mimic (miR-29a-mt). At 48 h post-transfection, virus production in cell supernatants was determined by p24 ELISA. Virus production data are normalized to data for cells treated with miRNA control (% of control). (C) Virus production increases from miR-29a mutant viral constructs and is insensitive to repression by miR-29a. 293 T cells were first transfected with 10 nM miR-29a or control, then co-transfected with pNL4-3LucR E (wt), HIV-1 construct lacking the miR-29a target site pNL4-3LucR E(Δ29t), or pNL4-3LucRE(m29t) and VSVG. At 48 h post-transfection, virus production from cell supernatants was determined by p24 ELISA. Virus production data are normalized to data for cells treated with pNL4-3LucR E (wt) miR (% relative virus production). (D) Mutant viruses pNL4-3LucR E(Δ29t) and pNL4-3LucRE(m29t) are more infectious than pNL4-3LucR E (wt). Target cells were transfected with 10 nM miR-29a or control and infected after 18 h with the virus equivalent of 10 ng pNL4-3LucR E (wt), pNL4-3LucRE(Δ29t), or pNL4-3LucRE(m29t) virus. At 72 h post-infection, virus infectivity was analyzed by measuring luciferase activity in infected cells relative to their protein contents. Virus infectivity data are normalized to data for cells treated with pNL4-3LucR E (wt) (% relative virus infectivity). (E) Mutant virus pNL4-3LucR E(m29t) is significantly more infectious in T lymphocytes than pNL4-3LucR E (wt). H9 T lymphocytes were infected by spinoculation with a virus equivalent of 0.3 µg pNL4-3LucR-E- virus. At 72 h post-infection, virus infectivity was measured by luciferase activity in total cell lysates relative to their protein content. Virus infectivity data are normalized to data for cells treated with pNL4-3LucR E (wt) (% relative virus infectivity). (F) Inhibiting miR-29a increases production of wild-type HIV-1 but not of HIV-1 lacking the miR-29a target site (Δ29t) or HIV-1 containing the mutant miR-29a target site (m29t). 293T cells were first transfected with 20 nM control or miR-29a inhibitor, then co-transfected with pNL4-3LucR-E- (wt), pNL4-3LucR-E-(Δ29t), or pNL4-3LucR-E-(m29t) and VSVG. At 48 h post-transfection, cell supernatants were quantitated for virus production. Virus production data are normalized to data for cells treated with anti-miR (% of control). (G) miR-29a decreases HIV-1 production in Dicer-depleted cells. 293T cells were transfected with 10 nM miRNA control or miR-29a plus mismatched Dicer siRNA (si-control) or perfectly matched Dicer siRNA (si-Dicer). For the second round of transfections, cells were co-transfected with miRNAs, si-RNAs (Figure 1 above), pNL4-3LucR-E-, VSVG, and analyzed 48 h later for virus production. Efficient knockdown of Dicer is indicated by immunoblot analysis of total cell extracts for both miRNA control- and miR-29a-treated cells (top panel). Virus production data (lower panel) are normalized to data for cells treated with miRNA control and si-control (% control). Data in panels (B), (C), (D), (E), (F), and (G) are from at least 3 independent experiments. Error bars represent SD, with significance from t-tests shown by p<0.05 (*), 0.01 (**) and 0.001 (***).