Long Noncoding RNA uc002yug.2 Activates HIV-1 Latency through Regulation of mRNA Levels of Various RUNX1 Isoforms and Increased Tat Expression

Abstract

The HIV-1 reservoir is a major obstacle to complete eradication of the virus. Although many proteins and RNAs have been characterized as regulators in HIV-1/AIDS pathogenesis and latency, only a few long noncoding RNAs (lncRNAs) have been shown to be closely associated with HIV-1 replication and latency. In this study, we demonstrated that lncRNA uc002yug.2 plays a key role in HIV-1 replication and latency. uc002yug.2 potentially enhances HIV-1 replication, long terminal repeat (LTR) activity, and the activation of latent HIV-1 in both cell lines and CD4⁺ T cells from patients. Further investigation revealed that uc002yug.2 activates latent HIV-1 through downregulating RUNX1b and -1c and upregulating Tat protein expression. The accumulated evidence supports our model that the Tat protein has the key role in the uc002yug.2-mediated regulatory effect on HIV-1 reactivation. Moreover, uc002yug.2 showed an ability to activate HIV-1 similar to that of suberoylanilide hydroxamic acid or phorbol 12-myristate 13-acetate using latently infected cell models. These findings improve our understanding of lncRNA regulation of HIV-1 replication and latency, providing new insights into potential targeted therapeutic interventions.IMPORTANCE The latent viral reservoir is the primary obstacle to curing HIV-1 disease. To date, only a few lncRNAs, which play major roles in various biological processes, including viral infection, have been identified as regulators in HIV-1 latency. In this study, we demonstrated that lncRNA uc002yug.2 is important for both HIV-1 replication and activation of latent viruses. Moreover, uc002yug.2 was shown to activate latent HIV-1 through regulating alternative splicing of RUNX1 and increasing the expression of Tat protein. These findings highlight the potential merit of targeting lncRNA uc002yug.2 as an activating agent for latent HIV-1.

Keywords: HIV-1 latency; RUNX1; Tat; activation; lncRNA uc002yug.2.

FIG 1

uc002yug.2 enhances HIV-1 replication. (A to D) uc002yug.2 overexpression enhances HIV-1 replication in HeLa cells. (A) The pNL4-3 viral vector with increasing amounts of uc002yug.2 (0, 100, 300, and 900 ng) was cotransfected into HeLa cells as indicated. After 48 h, cells and supernatants were harvested. The RNA level of uc002yug.2 was detected with the qRT-PCR assay. (B) p55 levels in cell lysate and CAp24 levels in supernatants were determined by immunoblotting, and the densities of bands were analyzed with ImageJ software to calculate the values relative to that for β-actin. (C) The infectivity of HIV-1 was measured by detecting the luciferase activity after TZM-bl cells were infected with the supernatant for another 48 h. (D) The mRNA levels of RUNX1a and RUNX1b and -1c were detected by qRT-PCR in the same cells as for panel A. (E to H) Knockdown of uc002yug.2 decreases HIV-1 replication in HEK293T cells. The endogenous uc002yug.2 in HEK293T cells was knocked down by using uc002yug.2-specific shRNA. (E) The knockdown of uc002yug.2 and corresponding RUNX1a and RUNX1b and -1c mRNA levels were detected by qRT-PCR. (F) Both control and uc002yug.2sh HEK293T cells were cotransfected with pNL4-3 viral vector plus uc002yug.2 or control vector as indicated. At 48 h after transfection, the cells and supernatants were harvested. p55 levels in cell lysate and CAp24 levels in supernatants were determined by immunoblotting, and the densities of bands were analyzed with ImageJ software to calculate the values relative to that for β-actin. (G) Virus production was measured by detecting the luciferase activity after TZM-bl cells were infected with the supernatant for another 48 h. The infectivity of 293T-pLKO.1 cells transected with pNL4-3 plus control vector was set as 100%. (H) The RNA level of uc002yug.2 in the same cells as described for panel F was detected by qRT-PCR. Results are representative of those from three independent repeats, and the corresponding value of the mock control was set as 1 or 100%. Data are presented as means ± SDs.

FIG 2

RUNX1b- and -1c-induced suppression of HIV-1 partially contributes to the effect of uc002yug.2 on HIV-1. (A and B) Overexpression of RUNX1b and RUNX1c inhibits HIV-1 replication in HEK293T cells. (A) The pNL4-3 vector and plasmids expressing RUNX1 isoforms, with or without CBF-β, were cotransfected into HEK293T cells as indicated. At 48 h after transfection, the levels of expression of RUNX1 isoforms, CBF-β, p55, and CAp24 in cells were determined by immunoblotting. (B) HIV-1 NL4-3 infectivity in the supernatant was measured by detecting luciferase activity after TZM-bl cells were infected with the supernatant for another 48 h. The infectivity of 293T cells transfected with pNL4-3 plus control vector was set as 100%. (C to E) RUNX1 is involved in the function of uc002yug.2 to regulate HIV-1 replication. (C) siRNA targeting RUNX1b and -1c was cotransfected with pNL4-3 into uc002yug.2 knockdown and control HEK293T cells. After 48 h, cells and supernatants were harvested. The qRT-PCR assay was used to detect the mRNA level of RUNX1b and -1c. (D) p55 levels in cell lysate and CAp24 levels in supernatants were determined by immunoblotting, and the densities of bands were analyzed with ImageJ software to calculate the values relative to that for β-actin. (E) The infectivity of HIV-1 was measured as described for panel B. (F to I) Overexpression of uc002yug.2 increases HIV-1 replication in Jurkat cells. (F) Jurkat cells were transduced with lentiviruses containing uc002yug.2 or a scramble control, and at 48 h postinfection, puromycin (1 μg/ml) was added to the medium for selection. The RNA level of uc002yug.2 was detected by qRT-PCR. (G) The uc002yug.2-overexpressing or control Jurkat cells were infected with HIV-1 pNL4-3-deltaE-EGFP virus, and GFP-positive cells were measured by flow cytometry at 48 h postinfection. (H) The relative ratio of GFP-positive cells in uc002yug.2-overexpressing or control Jurkat cells was calculated. The ratio of GFP-positive cells in control Jurkat cells infected with pNL4-3-deltaE-EGFP virus was set as 100%. (I) mRNA levels of RUNX1a and RUNX1b and -1c in uc002yug.2-overexpressing or control Jurkat cells was detected with qRT-PCR. Results are representative of those from three independent repeats, and the corresponding value of the mock control was set as 1 or 100%. Data are presented as means ± SDs.

FIG 3

uc002yug.2 expression is upregulated after HIV-1 infection or reactivation. (A and B) HIV-1 infection upregulates uc002yug.2 expression in Jurkat cells. (A) Differences in expression levels of uc002yug.2 between the mock- and HIV-1 NL4-3-infected Jurkat cells were detected by qRT-PCR. (B) Levels of CAp24 from HIV-1 NL4-3 were measured by immunoblotting. (C to F) Reactivation of HIV-1 increases uc002yug.2. HIV-1 latently infected J-Lat 6.3 and ACH-2 cells were utilized for measuring the change of uc002yug.2 during virus reactivation. (C) J-Lat 6.3 cells were treated or not with PMA (1 μM) for 48 h. Differences in expression levels of uc002yug.2, RUNX1a, and RUNX1b and -1c between the mock-infected and reactivated cells were detected by qRT-PCR. (D) HIV-1 reactivation of J-Lat 6.3 cells was measured by detecting GFP-positive cells by flow cytometry. (E) ACH-2 cells were treated or not with PMA (1 μM) for 48 h. Differences in expression levels of uc002yug.2, RUNX1a, and RUNX1b and -1c between the mock-infected and reactivated cells were detected by qRT-PCR. (F) HIV-1 reactivation of ACH-2 cells was measured by immunoblotting with anti-CAp24. (G and H) Low uc002yug.2 level maintains HIV-1 latency. (G) ACH-2 cells are HIV-1 latently infected CEM cells. The uc002yug.2 expression in acutely or latently HIV-1-infected CEM cells was detected using a qRT-PCR assay. (H) J-Lat 6.3 cells are HIV-1 latently infected Jurkat cells. The uc002yug.2 expression in acutely or latently HIV-1-infected Jurkat cells was detected using a qRT-PCR assay. Results are representative of those from three independent repeats. The qRT-PCR results in panels A, C, E, G, and H are represented with corresponding values, with the mock-infected cells set at 100%. Data are presented as means ± SDs.

FIG 4

uc002yug.2 affects HIV-1 LTR activity in HEK293T cells. (A) Knockdown of uc002yug.2 decreases HIV-1 LTR activity in HEK293T cells. The endogenous uc002yug.2 in HEK293T cells was knocked down as described for Fig. 1E. (A) HIV-1 LTR-luciferase and pRenilla plasmids were cotransfected into control or uc002yug.2 knockdown HEK293T cells. HIV-1 LTR activity was determined by a dual-luciferase reporter assay at 48 h after transfection. (B and C) Overexpression of RUNX1b and RUNX1c inhibits LTR activity in HEK293T cells. (B) The HIV-1 LTR-luciferase, pRenilla, and RUNX1 isoforms, with or without CBF-β plasmids, were cotransfected into HEK293T cells as indicated. At 48 h after transfection, RUNX1a or RUNX1b and -1c and CBF-β expression levels in cells were determined by immunoblotting. (C) HIV-1 LTR activity was determined by a dual-luciferase reporter assay. The luciferase activity of HEK293T cells transfected with HIV-1 LTR-luciferase was determined; pRenilla plus control vector (lane 2) was set as 100%. (D and E) Overexpression of RUNX1b and RUNX1c inhibits HIV-1 LTR activity in TZM-bl cells. (D) In TZM-bl cells, which contain an integrated LTR driving expression of firefly luciferase, Tat and RUNX1 isoforms were cotransfected as indicated. (E) HIV-1 LTR activity was determined at 48 h posttransfection, and the luciferase activity of TZM-bl cells transfected with Tat and control vector (lane 2) was set as 100% (E). Results are representative of those from three independent repeats. Data are presented as means ± SDs.

FIG 5

Expression of uc002yug.2 activates latent HIV-1 replication. (A) ACH-2 cells were infected with lentiviruses containing uc002yug.2 or a scramble control, and at 48 h postinfection, puromycin (1 μg/ml) was added to the medium for selection. The expression of uc002yug.2 was determined by qRT-PCR. (B) uc002yug.2-overexpressing or control ACH-2 cells were treated or not with PMA (1 μM), and CAp24 levels were detected by immunoblotting 48 h posttreatment. The densities of bands were analyzed with ImageJ software to calculate the values relative to that for β-actin. (C) Expression levels of RUNX1a and RUNX1b and -1c were determined by qRT-PCR when uc002yug.2 was overexpressed in ACH-2 cells. (D and E) CAp24 levels were detected by immunoblotting 48 h after uc002yug.2-overexpressing or control ACH-2 cells were treated or not with different concentrations of SAHA (0, 1, and 10 mM) (D) or TNF-α (1 μM) (E) for 48 h. The densities of bands were analyzed with ImageJ software to calculate the values relative to that for β-actin. (F) uc002yug.2-overexpressing or control ACH-2 cells at a concentration of 1 × 10³ per well were seeded in a 96-well plate. Cell growth was measured using Cell Counting Kit-8 (Transgen, China) according to the manufacturer's instructions at 24 h, 48 h, 72 h, and 96 h. Cell proliferation curves were plotted based on the absorbance at 450 nm using an iMark microplate reader (Bio-Rad) at each time point. The absorbance value at 0 h was set as 100%. Results are representative of those from three independent repeats. Data are presented as means ± SDs.

FIG 6

uc002yug.2 upregulates Tat protein. (A to C) uc002yug.2 increases HIV-1 LTR activity through upregulating Tat protein. HIV-1 LTR-luciferase reporter, pRenilla, and increasing amounts of uc002yug.2 (0, 100, 300, and 900 ng) with or without Tat plasmids were cotransfected into HeLa cells as indicated. The cells were harvested at 48 h posttransfection. (A) The expression of uc002yug.2 was detected by qRT-PCR. (B) Tat protein levels in cells were determined by immunoblotting, and the densities of bands were analyzed with ImageJ software to calculate the values relative to that for β-actin. (C) The LTR activity was determined by a dual-luciferase reporter assay, and the luciferase activity of HeLa cells transfected with an LTR-luciferase reporter, pRenilla without Tat plus control vector, was set as 100%. (D) mRNA levels of CUL4B and PSMD11 were negatively correlated with increased uc002yug.2 expression. (E) Latent cell line ACH2 stably overexpressing uc002yug.2 also showed downregulated mRNA levels of CUL4B and PSMD11. Results are representative of those from three independent repeats. Data are presented as means ± SDs.

FIG 7

uc002yug.2 affects HIV-1 transcription through altering RUNX1 and Tat expression. (A) Schematic of HIV-1 LTR mutants. HIV-1 LTR mutants that had previously been shown to lack RUNX binding ability (middle row) or have a deletion in the TAR element (bottom row) were constructed. (B) Schematic of the uc002yug.2 mutant. Compared with the wild-type uc002yug.2 (uc002yug.2-WT), uc002yug.2-d275nt was constructed to delete 275 nt at its 3′ terminus, which is responsible for the alternative splicing of RUNX1 via binding with RUNX1 pre-mRNA (lower diagram). (C) Effect of uc002yug.2 and its mutants on the HIV-1 LTR as well as its mutants. Wild-type or mutant HIV-1 LTR, pRenilla plasmid plus control vector, or uc002yug.2-WT or uc002yug.2-d275nt was cotransfected into HEK293T cells. After 48 h, cells were harvested. The LTR activity was determined by a dual-luciferase reporter assay, and the luciferase activity of HEK293T cells transfected with wild-type HIV-1 LTR, Tat plus control vector, was set as 100%. (D) Tat protein levels in cells were determined by immunoblotting. (E) The mRNA levels of RUNX1a and RUNX1b and -1c in uc002yug.2-WT-, uc002yug.2-d275nt-, and mock-transfected HEK293T cells were detected by qRT-PCR, and the corresponding value of the mock control was set as 100%. Results are representative of those from three independent repeats. Data are presented as means ± SDs.

FIG 8

uc002yug.2 increases viral replication and reactivation in CD4⁺ T cells. (A and B) uc002yug.2 was correlated with HIV-1 loads in plasma of HIV-1-infected patients. (A) Expression levels of uc002yug.2 in CD4⁺ T cells isolated from HIV-1 patients who underwent HAART (n = 12) and HIV-1 patients who had not received HAART (n = 12) were detected by qRT-PCR. The geometric means of the β-actin, GAPDH, and HMBS genes were used for normalization. (B) The HIV-1 loads and uc002yug.2 RNA levels of HIV-1-infected patients who had not received HAART were plotted, and linear regression analysis was performed. The geometric means of the β-actin, GAPDH, and HMBS genes were used for normalization. (C) uc002yug.2 increases viral replication. Primary CD4⁺ T lymphocytes from healthy donors were nucleofected with uc002yug.2 or control vector and then were infected with HIV-1 NL4-3. HIV-1 production in the supernatant was quantified with p24 ELISA at indicated time points postinfection. (D) uc002yug.2 increases HIV-1 reactivation in primary resting CD4⁺ T cells from patients. Resting CD4⁺ T cells were isolated from HAART-treated patients and nucleofected with uc002yug.2 or control vector. P1 to P3 represent three patients. HIV-1 reactivation in CD4⁺ T cells upon PHA-M (5 ng/ml) stimulation was detected by measuring p24 levels in the supernatants by ELISA.

FIG 9

Model of uc002yug.2 functional reactivation of HIV-1 uc002yug.2 may promote HIV-1 replication and reactivation through two mechanisms, which are the downregulation of RUNX1b and -1c and upregulation of Tat, leading to the activation of HIV-1 LTR. When uc002yug.2 is increased, RUNX1b and -1c would be downregulated due to alternative splicing of RUNX1 pre-mRNA, thereby reducing the suppression of RUNX1b and -1c on HIV-LTR activity (1); meanwhile, Tat protein would be upregulated and exert its transactivator function to promote HIV-1 LTR activity (2); however, whether uc002yug.2 upregulates Tat indirectly through RUNX1 needs to be further explored (3). Solid arrow, pathway with experimental data; dashed arrow, hypothetical potential pathways.