MicroRNA-155 Reinforces HIV Latency^*

qRT-PCR

Total J-Lat cell RNA was purified using TRIzol reagent (Invitrogen), and the RNA concentration was determined with an ND-1000 spectrophotometer (NanoDrop). TaqMan low density array (TLDA) human miRNA assays (versions 2.1 and 3.0; Applied Biosystems) were used to calculate changes in miRNA expression between latent cells and reactivated cells collected from four independent experiments. Briefly, cDNA was generated using the TaqMan reverse transcription kit and Megaplex primer pools A (v2.1) and B (v3.0). TLDA cards A and B were loaded with reverse transcription products and PCR was performed on a 7900HT Fast Real-Time PCR system (Applied Biosystems). Fold changes in the expression of each miRNA were calculated (ΔΔCt method), and differences in expression between latent and reactivated cells were analyzed using moderated t statistics. Linear contrasts were used to make all pairwise comparisons between groups. Follow-up analyses of specific miRNAs were performed using TaqMan microRNA assays. RNU6 was used as an endogenous control. TaqMan gene expression assays (Applied Biosystems) were used to quantify the expression of mRNA transcripts. The following primers and probes were used in gene expression assays: DGCR8 (Hs00256062_m1), Dicer (Hs00229023_m1), and TRIM32 (Hs00705875_s1). GAPDH or β-actin was used as an endogenous control for ΔΔCt calculations.

Lentiviral Infection

Lentiviral particles were produced as described (17). For J-Lat infections, 100,000 cells were incubated with 4 μg/ml Polybrene (Sigma), RPMI, and viral suspension for ∼2 h at 37 °C. After 24 h, the cells were washed and cultured in RPMI.

Lentiviral Vectors

shRNAs were cloned into the pSicoR lentiviral vector, which encodes an mCherry reporter driven by an EF-1α promoter (pSicoR-MS1). shRNAs against human DGCR8, Dicer, TRIM32, and negative control scramble were cloned into pSicoR-MS1 using the following oligonucleotide sequences: shScramble forward (TGT CAA GTC TCA CTT GCG TCT TCA AGA GAG ACG CAA GTG AGA CTT GAC TTT TTT C), shScramble reverse (TCG AGA AAA AAG TCA AGT CTC ACT TGC GTC TCT CTT GAA GAC GCA AGT GAG ACT TGA CA); shDGCR8 forward (TGA AAG AGT TTG TTA TTA ACT TCA AGA GAG TTA ATA ACA AAC TCT TTC TTT TTT C), shDGCR8 reverse (TCG AGA AAA AAG AAA GAG TTT GTT ATT AAC TCT CTT GAA GTT AAT AAC AAA CTC TTT CA); shDicer forward (TGC AGC TCT GGA TCA TAA TAT TCA AGA GAT ATT ATG ATC CAG AGC TGC TTT TTT C), shDicer reverse (TCG AGA AAA AAG CAG CTC TGG ATC ATA ATA TCT CTT GAA TAT TAT GAT CCA GAG CTG CA); and shTRIM32 forward (TGC AAA CAA ATG CTG ATA TAT TCA AGA GAT ATA TCA GCA TTT GTT TGC TTT TTT C), shTRIM32 reverse (TCG AGA AAA AAG CAA ACA AAT GCT GAT ATA TCT CTT GAA TAT ATC AGC ATT TGT TTG CA). The pCDH-TRIM32 lentiviral expression vector was constructed by performing PCR on the MGC human TRIM32 cDNA clone (accession number: BC003154, CloneID: 2906024; Thermo Scientific) using the following primer sequences: TRIM32 (EcoRI) forward (GTT TCT GAA TTC GAT GGC TGC AGC AGC AGC TTC TC), TRIM32 (BamHI) reverse (GTT TCT GGA TCC CTA TGG GGT GGA ATA TCT TCT CAG AT). PCR products were digested with EcoRI and BamHI and ligated into the pCDH-EF1α-MCS-IRES-Puro lentiviral vector.

Infection of CD4 T Cells with Dual Reporter Virus

Primary CD4 T cells and peripheral blood mononuclear cells were purified from healthy donor blood (Blood Centers of the Pacific, San Francisco, CA, and Stanford Blood Center). CD4 T cells were isolated by negative selection using the RosetteSep human CD4 T cell enrichment cocktail (StemCell Technologies). Peripheral blood mononuclear cells were purified by Histopaque-1077 density gradient. Purified CD4 T cells from peripheral blood were cultured in RPMI 1640 medium supplemented with 10% FBS, l-glutamine (2 mm), penicillin (50 units/ml), and streptomycin (50 mg/ml). Purified CD4 T cells isolated from peripheral blood were stimulated with αCD3/αCD28 activating beads (Life Technologies) at a concentration of 1 bead/cell in the presence of 30 units/ml IL-2 (PeproTech) for 3 days. All cells were spinoculated with HIV Duo-Fluo I at a concentration of 100 ng of p24 per 1 × 10⁶ cells for 2 h at 1,200 × g at 37 °C. After spinoculation, all cells were returned to culture in the presence of 30 units/ml IL-2. Infected CD4 T cells were sorted based on GFP and mCherry fluorescence 4 days after infection using a FACSAria II (BD Biosciences), and RNA was extracted immediately using the Ambion PARIS kit (Life Technologies).

Antibodies

Protein levels were analyzed by immunoblotting using the following antibodies: DGCR8 (ab37272, Abcam), Dicer (ab13502, Abcam), TRIM32 (ab96612, Abcam), IκBα (sc-371, Santa Cruz Biotechnology), RelA (sc-109, Santa Cruz Biotechnology), β-actin (A5316, Sigma), Sp1 (sc-59, Santa Cruz Biotechnology), GAPDH (ab8245, Abcam), HSP90 (sc7947, Santa Cruz Biotechnology), and His (sc-803, Santa Cruz Biotechnology). Immunoprecipitations were carried out using protein G Dynabeads (BD Biosciences protocol) and the following antibodies: FLAG (F1804, Sigma) and HA (sc-7392, Santa Cruz Biotechnology). For ubiquitin assays, 5A8 cells were transfected, pre-treated with 1.5 μm β-lactone overnight, and lysed in NP-40 buffer supplemented with protease inhibitor and N-ethylmaleimide.

Transfections

J-Lat 5A8 cells were infected with lentivirus expressing shRNA targeting Dicer and sorted by the mCherry-positive population. These sorted shDicer-infected J-Lat cells were cultured for ∼1 week and were nucleofected (Lonza) using Solution R and the program O-028 with 2 μm miRIDIAN microRNA mimics (Dharmacon), unless otherwise noted. After 24 h, cells were stimulated with α-CD3 and α-CD28 or with TNFα overnight or for 24 h, and analyzed by flow cytometry. For immunoprecipitation experiments, 4 million 293T cells were transfected with 3.5 μg of TRIM32, IκBα, pCMV constructs, and 0.7 μg of His-ubiquitin using FuGENE HD for 24 h. For Dual-Luciferase assays, the indicated luciferase plasmid (150 ng) was transfected into HeLa cells in a 24-well plate with or without miR-155 mimic (10 pmol) using Lipofectamine 2000 (Invitrogen). The Dual-Luciferase reporter assay system (Promega) was used to measure the levels of both firefly and Renilla luciferase 24 h after transfection.

Plasmid for Luciferase Assay

Luciferase reporter construct was generated using the psiCHECK2 vector (Promega). The TRIM32 3′-UTR was ligated into the XhoI/NotI sites of psiCHECK2. Sequencing was performed to confirm that construct contained the expected DNA sequence.

Plasmids in 293T Experiments

TRIM32 plasmids were a kind gift of Dr. Ki-Sun Kwon (18). IκBα plasmids were constructed as described previously (19).

In Vitro Kinase Assay

5A8 cells were unstimulated or treated (30 min) with PMA (20 nm) and ionomycin (2 μm). A total of 15 million cells were lysed in whole cell lysis buffer, and the assay was performed as described previously (4).

EMSA

Nuclear extracts were prepared (4 μg/sample) and were incubated with poly(dI-dC) and salmon sperm DNA for 15 min, followed by the addition of [γ-³²]ATP-labeled consensus κB or Oct-1 oligonucleotides (Promega). For cold-probe competition, 100× cold κB probes were added 10 min prior to labeled probes. Samples were separated on non-denaturing gels and analyzed by autoradiography.

Cloning, Expression, and Purification of TRIM32 and TRIM25

TRIM cDNA was cloned between the KasI and XhoI restriction sites of pPROEX-HTa. The TRIM32 I22E mutant was cloned by site-directed mutagenesis. A saturated culture of ArcticExpress (DE3) cells (Agilent) harboring pPROEX-HTa-TRIM32 was diluted to an A₆₀₀ of ∼0.2 and grown at 30 °C for 3 h. Cells were subsequently induced with 0.4 mm isopropyl-1-thio-β-d-galactopyranoside (supplemented with 0.1 mm ZnSO₄) and grown for an additional 48 h at 12 °C. Cells were resuspended in lysis buffer (50 mm Tris 8.0, 150 mm NaCl, 0.1 mm ZnSO₄, 10 mm imidazole, 10% glycerol, and 0.5 mm PMSF) and lysed by sonication. Recombinant TRIM proteins were batch-purified using nickel-nitrilotriacetic acid resin, eluted with elution buffer (50 mm Tris 8.0, 150 mm NaCl, 0.1 mm ZnSO₄, 200 mm imidazole, 10% glycerol), dialyzed overnight in dialysis buffer (50 mm Tris 8.0, 150 mm NaCl, 0.1 mm ZnSO₄, 10% glycerol, 1 mm DTT), and concentrated.

Cloning and in Vitro Synthesis of IκBα (NFKBIA)

IκBα cDNA was cloned between the FseI and AscI restriction sites of pCS2. 40 μl of rabbit reticulocyte lysate (Promega), 12 μl of ³⁵S-labeled amino acids (MP Biomedicals, catalogue number 0151006), and 12 μl of pCS2-IκBα (at a stock concentration of 500 ng/μl) were mixed and incubated at 30 °C for 2 h. In vitro transcribed/translated IκBα substrate was purified with anti-HA-agarose resin, eluted with 0.2 ml of 0.4 mg/ml 3×HA peptide, and concentrated 5-fold in volume.

Ubiquitylation Assay

In a 15-μl reaction volume, 0.3 μl of 10 μm E1 (200 nm final), 0.3 μl of 235 μm UbcH5 (5 μm final), 1.5 μl of 10 mg/ml ubiquitin (1 mg/ml final), 1.5 μl of 10× ubiquitylation assay buffer (250 mm Tris 7.5, 500 mm NaCl, and 100 mm MgCl₂), 2.3 μl of energy mix (150 mm creatine phosphate, 20 mm ATP, 20 mm MgCl₂, 2 mm EGTA, pH to 7.5 with KOH), and 2 μl of ³⁵S-labeled IκBα was mixed with 1.5 μl of 20 μm TRIM25, TRIM32, or TRIM32 I22E (2 μm final) at 30 °C for 1 h. Reactions were stopped by the addition of SDS sample loading buffer, resolved on a 10% SDS-acrylamide gel, and exposed on an autoradiography screen.

Knockdown of Enzymes Involved in miRNA Biogenesis Increases Reactivation of Latent HIV

Because various miRNAs block HIV replication, we hypothesized that select miRNAs promote HIV latency in infected CD4 T cells. To test this possibility, miRNA-deficient J-Lat 5A8 cells were prepared by knocking down the expression of two enzymes required for miRNA biogenesis, DGCR8 and Dicer. Knockdown was achieved by introduction of an shRNA lentiviral vector (pSicoR-MS1) containing an mCherry reporter into the J-Lat 5A8 cells. The mCherry-positive cells, which were successfully infected with the lentivirus, were isolated by fluorescent cell sorting followed by qRT-PCR to assess target gene expression. Knockdown of both DGCR8 and Dicer exceeded 90% in the mCherry-positive 5A8 cells as compared with levels obtained in cells receiving a negative control (5A8 shScramble) (Fig. 1A). Additionally, DGCR8 and Dicer levels were much lower in the knock-out cells than in cells receiving no shRNA, and further each knockdown appeared specific as expression of the counterpart microRNA biogenic enzyme was not affected, nor was the expression of GAPDH altered (Fig. 1B). To assess the effects of diminished miRNAs on HIV reactivation, the infected cells were cultured alone or stimulated with α-CD3/CD28 or TNFα. Both DGCR8 and Dicer knockdown cells exhibited significantly greater increases in HIV-1 reactivation with both agonists as demonstrated by increased GFP expression as compared with the shRNA scramble control knockdown (Fig. 1C). GFP expression in the absence of stimulation was not markedly affected by knockdown of either enzyme. These findings suggest that one or more miRNAs naturally function as inhibitors of latent HIV reactivation.

To identify the specific miRNAs that contribute to this inhibition, we used a TLDA to profile 754 unique human miRNAs (supplemental Table 1). We identified miRNAs that were differentially expressed between reactivated and latent cells (Table 1), validating top candidates in individual TaqMan assays (data not shown).

Introducing miR-155 into Dicer-deficient Cells Decreases the Level of HIV Reactivation

To determine whether candidate miRNAs inhibited HIV-1 reactivation, each candidate miRNA was reintroduced into Dicer-deficient cells. Although knocking down Dicer nearly doubled the level of reactivation in stimulated cells (Fig. 1C), introducing the top four candidate miRNAs from both GFP-positive and GFP-negative cells countered this effect, restoring a lower level of reactivation as compared with cells receiving a negative control miRNA mimic (Fig. 2A). Introducing miR-155 produced the greatest decline in HIV-1 reactivation (78.3% rescue) (Fig. 2A), occurring in a dose-related manner (Fig. 2B). Two of the miRNAs tested, miR-1290 and miR-505* (asterisk indicates passenger strand), did not significantly affect reactivation levels, indicating that this effect is specific and not associated with the introduction of every miRNA. To further evaluate miR-155 levels when cells were stimulated, individual TaqMan assays were performed. miR-155 was expressed at 50-fold higher levels in GFP-positive cells (∼50-fold more) as compared with unstimulated cells. miR-155 levels were also increased in GFP-negative cells following stimulation, albeit to a lesser extent (Fig. 2C). Together, these findings raise the possibility that specific miRNAs abundantly expressed in reactivated cells serve to counter viral activation and perhaps promote a return to latency in cells undergoing transient virus production.

To determine the levels of miR-155 in a model of HIV-1 latency formed with primary CD4 T cells, we employed a dual-fluorescence virus, HIV-DuoFluo, to identify latent and productively infected primary CD4 T cells occurring after HIV infection. This model has been recently described (20). In this model, HIV-DuoFluo (R7GEmC) expresses GFP under control of the HIV promoter and expresses mCherry under control of a constitutive EF1α promoter. Following integration of this virus into primary CD4 T cells, mCherry is constitutively expressed and marks all successfully infected cells. Active viral expression occurs in a subset of these cells indicated by the expression of GFP. Latently infected cells are identified by the expression of mCherry in the absence of GFP expression. CD4 T cells from two blood donors (Donor 9600 and Donor 9601) were infected with R7GEmC and sorted for uninfected cells, latently infected cells (mCherry-positive, GFP-negative), and actively infected cells (mCherry-positive, GFP-positive). Both donors exhibited high levels of miR-155 in cells that were TCR-stimulated but uninfected. This finding is consistent with prior studies describing up-regulation of miR-155 in activated lymphoid and myeloid cells (21, 22). Levels of miR-155 were much higher in virus-producing cells (mCherry-positive, GFP-positive) as compared with latently infected cells (mCherry-positive, GFP-negative) (Fig. 2D). These findings suggest that miR-155 may principally act within an activated cellular environment. Interestingly, miR-29b levels did not change in any of the tested cell populations. This microRNA has been implicated in the regulation of cyclin T1, a participant in the PTEF-b complex that functions as a key cofactor for HIV Tat (9).

MicroRNA-155 Targets TRIM32

Next, we sought to identify host genes whose action was suppressed by miR-155 using the TargetScan prediction algorithms. These analyses identified TRIM32 as a high-value hit because it had a favorable aggregate P_CT and context+ score. In addition, the 3′-UTR of TRIM32 contains an 8-mer site that precisely matches the seed region (positions 1–8) of miR-155 (Fig. 3A). Furthermore, TRIM32 represented an intriguing potential target because of a prior report of its direct binding to the activation domain of HIV Tat (21). To study whether miR-155 directly binds to the 3′-UTR of TRIM32, the full 3′-UTR of TRIM32 including the predicted target site was cloned downstream of a Renilla luciferase reporter construct. In this assay, a decrease in luciferase activity in the presence of miR-155 demonstrates binding of the miRNA and subsequent inhibition of luciferase expression. (Fig. 3A). Indeed, miR-155 inhibited luciferase expression of the reporter containing the full 3′-UTR of TRIM32 (Fig. 3B). These results confirm that TRIM32 is a target of miR-155.

Studies were next performed to assess miR-155 effects on endogenous TRIM32 levels in J-Lat 5A8 cells. The introduction of miR-155 decreased levels of TRIM32 mRNA in these cells by ∼70% as compared with a negative control miRNA (Fig. 3C). Recent studies in the Rudensky laboratory (22) have analyzed endogenous targets of miR-155 within the entire murine transcriptome using AGO differential HITS-CLIP (dCLIP) and mRNA changes in CD4 T cells isolated from wild type or miR-155 knock out mice. Binding maps were made accessible online for all 3′-UTRs on CLIP Base. We searched for TRIM32 in CLIP Base and found that the putative miR-155 binding site in the TRIM32 3′-UTR is in fact bound by the miR-155-AGO complex in wild type CD4 T cells, but not in CD4 T cells lacking miR-155 due to gene knock-out (Fig. 3D, image obtained from CLIP Base). Together, these findings provide strong evidence supporting TRIM32 as a bona fide cellular target of miR-155.

TRIM32 Regulates HIV-1 Latency

Next, we assessed the effects of shRNA knockdown of TRIM32 in latently infected J-Lat 5A8 cells. After infection with a TRIM32 shRNA, TRIM32 mRNA and protein levels were both reduced by ∼40% (Fig. 4, A and B). Conversely, TRIM32 mRNA and protein levels were not reduced in cells infected with virus encoding a scrambled control shRNA (Fig. 4, A and B). In agreement with one or more miRNAs posttranscriptionally regulating TRIM32 expression, TRIM32 protein, but not mRNA, was increased when Dicer was knocked down (Fig. 4, A and B). TCR activation of J-Lat 5A8 cells with anti-CD3 and anti-CD28 antibodies decreased GFP expression when TRIM32 expression was knocked down but increased expression following Dicer knockdown (Fig. 4C). These findings suggest that TRIM32 promotes reactivation of latent HIV-1 and that its activity is regulated by miRNAs, notably miR-155.

Next, we examined the effects of lentivirus-mediated overexpression of TRIM32 (pCDH-TRIM32) in J-Lat 5A8 cells. By both qRT-PCR and immunoblotting TRIM32 levels were significantly higher in pCDH-TRIM32 cells as compared with cells infected with the empty lentiviral vector (pCDH) (Fig. 4, D and E). TRIM32 alone promoted HIV-1 reactivation in the absence of stimulation. Treatment with anti-CD3/CD28 and TNFα resulted in increased levels of latent virus reactivation in cells expressing TRIM32 (Fig. 4F). Together, these findings confirm TRIM32 as an HIV activator and effective host antagonist of HIV latency.

TRIM32 Promotes Reactivation of Latent HIV-1 by Stimulating NF-κB Signaling

TRIM32 has been reported to interact with the activation domain of HIV-1, HIV-2, and equine infectious anemia virus Tat proteins (21). To assess whether the activating effects of TRIM32 on latent HIV required the presence of Tat, TRIM32 was expressed in J-Lat A72 cells that lack Tat (23). TRIM32 effectively induced latent provirus expression alone or in combination with anti-CD3/CD28 antibodies or TNFα (Fig. 5A). These results indicate that the TRIM32 is able to activate the HIV LTR in a Tat-independent manner. Recent studies have shown that TRIM32 induces NF-κB in 293 cells (14, 16). We examined whether TRIM32 similarly activates NF-κB using Jurkat κB-dsRed cells, which contain a basal promoter and five κB enhancer elements controlling expression of a dsRed reporter (6). When TRIM32 was expressed in these cells, the level of dsRed expression increased as compared with the empty vector control in both the absence and the presence of stimulation (Fig. 5B). These findings confirm the NF-κB-inducing activity of TRIM32.

Next, nuclear and cytoplasmic extracts from TRIM32-expressing cells were immunoblotted. In the absence of concomitant PMA and ionomycin stimulation, TRIM32 expression was associated with an increase in nuclear RelA expression and a decrease in cytoplasmic IκBα levels (Fig. 5C). Based on the pattern of nuclear Sp1 and cytoplasmic IκBα, nuclear and cytoplasmic fractions were of high quality (Fig. 5C). To confirm TRIM32-induced nuclear translocation of NF-κB, EMSA were performed with nuclear extracts and ³²P-labeled κB and Oct1 DNA probes. Unstimulated cells expressing TRIM32 displayed increased binding activity in the form of a more slowly migrating complex (p50/RelA heterodimer) that was fully competed with unlabeled wild type κB probe but not by a mutant κB probe. (Fig. 5D). In contrast, Oct1 DNA binding did not change in the presence or absence of TRIM32. Together, these results indicate that TRIM32 expression induces NF-κB activation in CD4 T cells.

To further investigate the mechanism through which TRIM32 induces NF-κB, an IκB kinase (IKK) assay was performed (Fig. 5E). Unexpectedly, the same level of IKK activity was found in cells expressing TRIM32 versus the negative control. However, brief stimulation with PMA and ionomycin increased IKK activity. Furthermore, there was no evidence of phosphorylated IκBα in the lysate of cells expressing TRIM32 (Fig. 5E). These findings suggest that TRIM32 acts downstream of the IKKs in the NF-κB pathway. Because our prior experiments indicated that TRIM32 expression promotes degradation of IκBα, we hypothesized that TRIM32 directly binds to and ubiquitinates IκBα. To test this possibility, TRIM32 and IκBα interaction was tested in cells expressing HA-tagged IκBα and FLAG-tagged full-length TRIM32 or FLAG-tagged mutant TRIM32 containing a deletion in the RING domain (TRIM32ΔRING), thereby removing its E3 ubiquitin ligase activity. When anti-TRIM32 antibodies were used in the immunoprecipitations, both IκBα and the IκBα super-repressor containing S32A/S36A mutations corresponding to the key IKK phosphoacceptor sites (24) were coimmunoprecipitated. However, when anti-IκBα antibodies were used, lower amounts of the TRIM32ΔRING protein were coimmunoprecipitated as compared with wild type TRIM32 (Fig. 5F). The ability of TRIM32 to ubiquitinate IκBα was next tested. In the presence of the β-lactone proteasome inhibitor, full-length TRIM32 promoted ubiquitination of IκBα, whereas TRIM32ΔRING did not. In addition, TRIM32 also promoted ubiquitination of the IκBα super-repressor, but at slightly lower levels as compared with wild type IκBα (Fig. 5G). To confirm these findings, we assayed the ability of recombinant TRIM32 (wild type versus catalytic-dead mutant) to ubiquitinate radiolabeled IκBα in vitro. Wild type TRIM32 mediated ubiquitination of IκBα, whereas the I22E mutant, lacking ubiquitin transfer activity, did not (Fig. 5H). Furthermore, we tested the ability of another NF-κB-activating TRIM protein, TRIM25, to directly ubiquitinate IκBα. We found that recombinant TRIM25 ubiquitinated IκBα in vitro at levels similar to that of TRIM32 (Fig. 5I). Together, these results suggest that both TRIM32 and TRIM25 bypass many of the usual upstream NF-κB signaling steps by directly binding to and ubiquitinating IκBα and that this mechanism may be conserved in other NF-κB-activating TRIM E3 ligases.