1. Cellular MicroRNA and P Bodies Modulate Host-HIV-1 Interactions
Robin Nathans, Chia-ying Chu, Anna Kristina Serquina, Chih-Chung Lu, Hong Cao, Tariq M. Rana 
Molecular Cell 
Volume 34, Issue 6, Pages (June 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1
Depleting Dicer or Drosha Increases Virus Production and Infectivity
(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 hr posttransfection 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 (percent 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 hr 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 (percent 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 hr posttransfection for virus production (bottom panel) as described in the 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 three independent experiments. Error bars represent SD, with significance from t tests shown by ∗p < 0.05 and ∗∗∗p <0.001.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Predicted Targets of Human miRNAs in HIV-1 3′UTR
To examine miRNA expression after HIV-1 infection, H9 T lymphocytes were infected with HIV-1 and the infections allowed to proceed for five passages. Small RNAs were isolated from HIV-1-infected H9 T lymphocytes, and miRNA expression was quantified by miRNA microarray (Table S1). miRNAs are sorted by their expression signal (log2) in HIV-1-infected H9 T cells, and for comparison, miRNA expression signal (log2) in 293T cells (Lu et al., 2005) is presented: un, undetectable; –, not available. The targets of human miRNAs in the HIV 3′UTR were initially predicted with miRanda ( Default parameters were used (gap open penalty, −8.0; gap extend, −2.0; score threshold, 80; energy threshold, −14 kcal/mol; scaling parameter, −2.0). The HIV-1 3′UTR sequence was obtained from the National Center for Biotechnology Information (NCBI) database with accession number NC_ ( ). miRNA sequences were downloaded from the miRBase ( and 455 human miRNAs were aligned with the HIV 3′UTR sequence. The 11 predicted targets found with perfect seed matches ( are summarized.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 miR-29a Modulates HIV-1 Production and Infectivity
(A) HIV-1 infection induces miR-29a expression in 293T cells. 293T cells were infected for 48 hr with the virus equivalent of 50 ng pNL4-3LucR-E-. Expression of miR-29a was quantified by RT-qPCR (see the 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 Figure 2).
(C) Inhibiting miR-29a increases HIV-1 production. 293T cells were transfected with 20 nM miR inhibitor (negative control) or miR-29a inhibitor, then cotransfected with pNL4-3LucR-E- and VSVG. At 48 hr posttransfection, cell supernatants were quantitated for virus production. Data are normalized to data for cells treated with anti-miR (percent 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 hr posttransfection, cells were infected with a 50 ng equivalent of pNL4-3LucR-E- virus. At 48 hr postinfection, 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 (percent of control).
(E) miR-29a mimic decreases HIV-1 production. 293T cells were transfected with 10 nM control miRNA or miR-29a mimic, then cotransfected with pNL4-3LucR-E- and VSVG. At 48 hr posttransfection, cell supernatants were quantitated for virus production. Virus production data are normalized to data for cells treated with anti-miR (percent 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 hr posttransfection, H9 cells were infected by spinoculation with a virus equivalent of 1 μg pNL4-3LucR-E- virus. At 48 hr postinfection, 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 (percent of control). Data in (A), (C), (D), (E), and (F) are from at least three independent experiments. Error bars represent SD, with significance from t tests shown by ∗p < 0.05 and ∗∗∗p <0.001.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4
miR-29a Specifically Targets HIV-1 mRNA for Translation Repression
(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 and the mutant site by an 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, four 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 cotransfected 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 hr posttransfection, virus production in cell supernatants was determined by p24 ELISA. Virus production data are normalized to data for cells treated with miRNA control (percent 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 cotransfected with pNL4-3LucR-E- (WT), HIV-1 construct lacking the miR-29a target-site pNL4-3LucR-E- (Δ29t), or pNL4-3LucR-E- (m29t) and VSVG. At 48 hr posttransfection, 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) and control miR (percent relative virus production).
(D) Mutant viruses pNL4-3LucR-E- (Δ29t) and pNL4-3LucR-E- (m29t) are more infectious than pNL4-3LucR-E- (WT). Target cells were transfected with 10 nM miR-29a or control and infected after 18 hr with the virus equivalent of 10 ng pNL4-3LucR-E- (WT), pNL4-3LucR-E- (Δ29t), or pNL4-3LucR-E- (m29t) virus. At 72 hr postinfection, 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) (percent 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 hr postinfection, 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) (percent 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 cotransfected with pNL4-3LucR-E- (WT), pNL4-3LucR-E- (Δ29t), or pNL4-3LucR-E- (m29t) and VSVG. At 48 hr posttransfection, cell supernatants were quantitated for virus production. Virus production data are normalized to data for cells treated with anti-miR (percent 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 cotransfected with miRNAs, si-RNAs (Figure 1, above), pNL4-3LucR-E-, and VSVG and analyzed 48 hr 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 (percent control). Data in (B), (C), (D), (E), (F), and (G) are from at least three independent experiments. Error bars represent SD, with significance from t tests shown by ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p <
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5
miR-29a Directly Targets HIV-1 mRNA for Its Association with RISC and P Body RNP Assemblies
(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 pNL4-3LucR-E- (WT) or pNL4-3LucR-E-m29t, VSVG, and Myc-Ago2. At 48 hr posttransfection, 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 the Experimental Procedures). The mRNA associated with Ago2 was quantified by RT-qPCR (bar graph). Data in the bar graph represent percent 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 copurification with the immunopurified, endogenous P body protein, RCK/p T cells were first transfected with miR-29a or miR-mt29a, then with pNL4-3LucR-E- (WT) or pNL4-3LucR-E-m29t, and VSVG. At 48 hr posttransfection, 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 (bar graph; as described in A). Data in the bar graph represent percent 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-E-m29t with miR-29a-mt, respectively.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

7. Figure 6
Depleting P Body Proteins Disrupts P Body Formation and Increases HIV-1 Production and Infectivity
(A) Depleting RCK/p54 disrupts P bodies. To silence expression of RCK/p54, 293T cells were cotransfected with APOBEC3G-YFP (A3G-YFP) and with siRNA against RCK/p54 mRNA (si-RCK/p54) or with mismatched siRNA (si-control). At 24 hr posttransfection, cells were fixed and analyzed by confocal microscopy. A3G-YFP was detected by direct YFP fluorescence (Aa and Ad), whereas endogenous RCK/p54 was detected by immunofluorescence with anti-RCK/p54 (Ab and Ae). Cells were stained to visualize nuclei, and images were digitally merged (Ac and Af).
(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 cotransfected with pNL4-3LucR-E- and VSVG. Protein knockdown was monitored by immunoblotting (Figure S3). At 48 hr posttransfection, 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 ∗∗∗p <
(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 hr after the second siRNA transfection, cells were infected for 48 hr 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 (percent control). Error bars represent SD, with significance from t tests shown by ∗p < 0.05 and ∗∗p < 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 (A3G-YFP), fixed at 24 hr posttransfection, immunostained for A3G-YPF or RCK/p54, hybridized in situ for miR-18a with fluorescein-labeled LNA probe, and analyzed by confocal microscopy. A3G-YFP and endogenous RCK/p54 were detected by immunofluorescence with anti-YFP and anti-RCK/p54, respectively (Fb and Fe). Cells were stained to visualize nuclei, and images were digitally merged (Fc and Ff). Arrowheads indicate colocalization of miR-18a and APOBEC3G or RCK/p54 in P bodies.
(G) HIV-1 mRNAs colocalize with APOBEC3G. 293T cells were transfected with A3G-YFP and pNL4-3, fixed at 36 hr posttransfection, 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.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
A Highly Conserved miR-29 Family Modulates HIV-1 Production and Infectivity
(A) Inhibiting miR-29a, miR-29b, or miR-29c increases HIV-1 production. 293T cells were first transfected with 10 nM miR inhibitor (negative control), miR-29a, miR-29b, or miR-29c inhibitor, then cotransfected with pNL4-3LucR-E- and VSVG. At 48 hr posttransfection, cell supernatants were quantitated for virus production. Data are normalized to data for treatment with anti-miR control (percent of control). Error bars represent SD, with significance from t tests shown by ∗∗∗p < 0.001.
(B) miR-29a, miR-29b, or miR-29c decreases HIV-1 production. 293T cells were first transfected with 20 nM control or miR-29a, miR-29b, or miR-29c mimic, then cotransfected with pNL4-3LucR-E- and VSVG. At 48 hr posttransfection, cell supernatants were quantitated for virus production. Data are normalized to data for treatment with miRNA control (percent of control). Error bars represent SD, with significance from t tests shown by ∗∗∗p <
(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 ( Sequences for nef or 3′LTR were aligned using CLC Sequence Viewer 4 software.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 8 Model for Cellular miRNAs Modulating Host-Virus Interactions
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 RNP 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.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions