Fig 1.
NOP2 inhibits HIV-1 replication.
(A) RNAi gene enrichment ranking (RIGER) method was applied to analyze screens performed using multiple orthologous RNAi reagents (MORRs). Genes were ranked in order of their RIGER scores (lowest → highest), from host dependency factors to host restriction factors. RIGER analysis of these screens recognized several known host restriction factors (CCNK, BRD4) as well as new ones, such as NOP2. (B) MAGI-HeLa cells were transiently transfected with the indicated siRNAs (siNT or siNOP2), and NOP2 knockdown was analyzed by immunoblotting. (C) MAGI-HeLa cells transfected with the indicated siRNAs were infected with HIV-1 IIIB viruses, followed by the immunostaining of p24 (green). Nuclei were stained with Hoechst 33342 (blue). The infection rate is calculated by dividing p24-expressing cells by total cells, and normalized to that of non-targeting siRNA (siNT). (D) MAGI-HeLa cells transfected with the indicated siRNAs were infected with HIV-1 NL4–3-Luc (dEnv) viruses. The relative luminometer units (RLU) of luciferase was measured and normalized total proteins, and normalized to that of non-targeting siRNA (siNT). (E) Jurkat cells were stably transduced with indicated shRNAs (shNT or shNOP2) in pAPM vector, and NOP2 knockdown was analyzed by immunoblotting. (F) Jurkat cells stably expressing shNOP2 or shNT were infected with HIV IIIB viruses. A portion of supernatant was harvested every 2 days until 12 days post-of-infection (dpi), and titrated using the TZM-bl cells. The RLU was measured, and normalized to that of non-targeting shRNA (shNT). (G) MAGI-HeLa cells were stably transduced with the indicated lentiviral vectors expressing V5-tagged FLAG peptide or NOP2 ORF (pLEX-FLAG or pLEX-NOP2), and protein expression of V5-NOP2 was analyzed by immunoblotting. (H) MAGI-HeLa cells stably transduced with pLEX-FLAG or pLEX-NOP2 were infected with HIV-1 NL4–3-Luc (dEnv) viruses. The RLU was measured, and normalized to that of pLEX-FLAG. (I) Jurkat cells were stably transduced with the indicated vectors (pLEX-FLAG or pLEX-NOP2), and protein expression of V5-NOP2 was analyzed by immunoblotting. (J) Jurkat cells stably transduced with pLEX-FLAG or pLEX-NOP2 were infected with HIV-1 IIIB viruses. A portion of supernatant was harvested every 2 days until 14 dpi, and titrated using the TZM-bl cells. The RLU was measured, and normalized to that of pLEX-FLAG. Results were based on n = 3 experiments and presented as mean ± S.D., * p < 0.05; ** p < 0.01; *** p < 0.001, ANOVA.
Fig 2.
NOP2 suppresses HIV-1 transcription.
(A) TZM-bl cells stably expressing FLAG-tagged HIV-1 Tat protein in the retroviral vector pQXCIP (pQCXIP-Tat) were transiently transduced with the indicated shRNA (shNT or shNOP2) in pAPM vector, and NOP2 knockdown was analyzed by reverse transcription coupled qPCR (RT-qPCR). (B) The RLU of cells in (A) was measured, and normalized to that of shNT. (C) HEK293 cells were stably transduced with the indicated vectors (pLEX-FLAG or pLEX-NOP2), and protein expression of V5-NOP2 was analyzed by immunoblotting. (D) The cells in (C) were transiently co-transfected with pcDNA-Tat, HIV-LTR-Luciferase, and pTK-Renilla vectors, followed by the dual-luciferase reporter assay. The RLU (luciferase/renilla) was measured, and normalized to that of pLEX-FLAG. (E) Jurkat cells were stably transduced with the indicated shRNA (shNT, shNOP2, or shNSUN2) in pAPM vector, and NOP2 or NSUN2 knockdown was analyzed by RT-qPCR. (F) The cells in (E) were infected with HIV-1 IIIB viruses, followed by the RNA extraction and RT-qPCR to measure HIV-1 initiated or elongated (proximal [Pro], intermediate [Int], and distal [Dis]) transcripts. The level of each HIV-1 transcript was normalized to that of shNT. Results were based on n = 3 experiments and presented as mean ± S.D., * p < 0.05; ** p < 0.01; *** p < 0.001, ANOVA.
Fig 3.
Knockdown of NOP2 benefits HIV-1 reactivation in multiple latency cell lines.
(A, C, E) Jurkat-based HIV-1 latency cell lines, J-Lat 10.6 (A) and EF7 (C), as well as a monocytic one, U1/HIV (E), were stably transduced with the indicated shRNA (shNT or shNOP2) in pAPM vector, and NOP2 knockdown was analyzed by RT-qPCR. (B, D) Jurkat-based J-Lat 10.6 (B) and EF7 (D) cells stably expressing shNT or shNOP2 were treated with DMSO, SAHA (1 uM), JQ1 (0.5 uM), or Prostratin (0.5 uM), to reactivate latent HIV-1. Percentage of GFP-expressing cells was determined by flow cytometry. (E) U1/HIV cells stably expressing shNT or shNOP2 were stimulated with DMSO, TNF-α (10 ng/ml), JQ1 (0.5 uM), or Prostratin (0.5 uM), to reactivate latent HIV-1. Total RNA was extracted, followed by RT-qPCR to measure the mRNA level of HIV-1 Gag, which was normalized to that of shNT. Results were based on n = 3 experiments and presented as mean ± S.D., * p < 0.05; ** p < 0.01; *** p < 0.001, ANOVA.
Fig 4.
Validation of NOP2’s effect on HIV-1 latency using primary CD4+ T cell models.
(A) A primary cell model of HIV-1 latency based on the use of activated CD4+ T cells. (B, C) Primary CD4+ T cells isolated from three donors (donors 1–3) were activated and infected with VSV-G pseudo-typed dHIV-nef. Once cells returned back to quiescent stage and HIV-1 latency was established, these cells were transiently transfected with the indicated siRNA (siNT or siNOP2) through electroporation, followed by the total RNA extraction and RT-qPCR to measure the knockdown of NOP2 (B) and expression of HIV-1 Gag (C), which was normalized to that of siNT. (D) Primary CD4+ T cells isolated from the additional donor (donor 4) was subjected to the same treatment as in (B, C), except that HIV-1 reactivation was analyzed by measuring Gag mRNA level at both the basal level with mock treatment and the stimulation condition using anti-CD3/28 antibodies on Dynabeads. (E) A primary cell model of HIV-1 latency based on the use of resting CD4+ T cells. (F, G) Resting CD4+ T cells isolated from three donors (donors 1–3) were spinoculated with VSV-G pseudotyped dHIV-nef. Cells were transiently transfected with the indicated siRNA (siNT or siNOP2) through electroporation. Total RNAs were extracted from these cells and analyzed by RT-qPCR to measure the mRNA level for NOP2 (F) or HIV-1 Gag (G), which was normalized to that of siNT. (H) Primary CD4+ T cells isolated from the additional donor (donor 4) was subjected to the same treatment as in (F, G), except that HIV-1 reactivation was analyzed by measuring Gag mRNA level at both the basal level with mock treatment and the stimulation condition using anti-CD3/28 antibodies on Dynabeads. Results were based on n = 3 replicates and presented as mean ± S.E.M., * p < 0.05; ** p < 0.01; *** p < 0.001, ANOVA.
Fig 5.
NOP2 binds with HIV-1 TAR RNA and contributes to its m5C methylation.
(A) TZM-bl cells were transiently transfected with either pQCXIP-NOP2 (HA-tagged) or PQCXP-Tat (FLAG-tagged). Cells were cross-linked using formaldehyde. Cell lysates were prepared and split to halves for incubation with mouse anti-HA/FLAG antibody or mouse IgG (mIgG). Co-precipitated DNA samples were analyzed by semi-quantitative PCR using primer sets that amplify HIV-1 5’ LTR region (Nuc0, Nuc1, PPR). (B) J-Lat A2 cells were treated with or without TNF-α (10 ng/ml). Lysates from cross-linked cells were prepared and split to halves for incubation with mouse anti-NOP2 antibody or mIgG. Co-precipitated DNA samples were analyzed by qPCR using primer sets that amplify Nuc0, Nuc1, or the negative control GAPDH DNA sequence. The DNA level of Nuc0, Nuc1 and GAPDH amplicon was determined as the percentage of input (1% of lysate). (C) HEK293 cells stably expressing the indicated shRNA (shNT or shNOP2) were transiently transfected with the pU16TAR plasmid for expression of HIV-1 TAR RNA. Cell lysates were prepared and treated with DNase I, followed by total RNA extraction. Extracted RNAs were incubated with an anti-m5C antibody or control IgG. Immuno-precipitated RNA samples were analyzed by RT-qPCR using the primer set that amplifies HIV-1 TAR. The DNA amplicon of TAR was determined as the percentage of input (1% of lysate). Results in (B) and (C) were based on n = 3 experiments and presented as mean ± S.D., * p < 0.05; ** p < 0.01; *** p < 0.001, ANOVA. (D) Recombinant NOP2 or Tat protein (6xHis-tagged) was purified from bacteria, and incubated with the equal amount of synthesized biotinylated TAR RNA (bio-TAR), its scrambled RNA (bio-scram), or the free biotin in vitro. The protein-RNA complex was affinity-precipitated using streptavidin magnetic beads. The input or precipitated Tat or NOP2 protein was analyzed by immunoblotting. (E) Recombinant Tat protein was incubated with bio-TAR in the presence (1:1 molar ratio) or absence of recombinant NOP2 protein, or with free biotin in vitro. Vise versa, recombinant NOP2 protein was incubated with bio-TAR in the presence (1:1 molar ratio) or absence of recombinant Tat protein, or with free biotin in vitro. In both cases, the protein-RNA complex was affinity-precipitated using streptavidin magnetic beads. The input or precipitated Tat or NOP2 protein was analyzed by immunoblotting, and the relative intensity of pulled down Tat or NOP2 was calculated. (F) TZM-bl cells stably expressing FLAG-tagged HIV-1 Tat protein in the retroviral vector pQXCIP (pQCXIP-Tat) were transiently transduced with pQCXIP-NOP2 (HA-tagged) or empty vector. Protein expression of HA-NOP2 and FLAG-Tat was analyzed by immunoblotting. (G) Cells in (F) were either without (left panel) or with (right panel) cross-linking. Cell lysates were prepared and incubated with bio-TAR or free biotin in vitro. The protein-RNA complex was affinity-precipitated using streptavidin magnetic beads. The input or precipitated Tat protein was analyzed by immunoblotting, and the relative intensity of pulled down Tat was calculated. * A non-specific protein band beneath FLAG-Tat at the “without cross-linking” condition was noted.
Fig 6.
The MTD domain of NOP2 competes with Tat for TAR binding.
(A) Recombinant NOP2 protein with deletion of 1–57 amino acids (6xHis-tagged) was purified from bacteria, and incubated with bio-TAR or free biotin in vitro. The protein-RNA complex was affinity-precipitated using streptavidin magnetic beads. The input or precipitated NOP2 (1-57aa deleted) protein was analyzed by immunoblotting. (B) Recombinant NOP2 protein domains, NTD (1-200aa), MTD (201-620aa), and CTD (621-845aa), were purified from bacteria, and incubated with bio-TAR or free biotin in vitro, followed by the affinity precipitation using streptavidin magnetic beads. The input or precipitated NOP2 protein domains (NTD, MTD, CTD) were analyzed by immunoblotting. (C) Recombinant Tat protein was incubated with bio-TAR in the absence or presence of NOP2 MTD domain at the increased doses, or with free biotin in vitro, followed by the affinity precipitation using streptavidin magnetic beads. The input or precipitated Tat or NOP2 MTD domain was analyzed by immunoblotting. The relative intensity of pulled down Tat was calculated. (D) NOP2 MTD domain was further divided into five smaller domains (MTD-1 to 5), which were ~140aa with 70aa overlap. The positions of two catalytic cysteine residues (496aa, 550aa) in the MTD were indicated by asterisks (*). (E) Recombinant NOP2 MTD smaller domains, MTD-1 to 5, were purified from bacteria, and incubated with bio-TAR or free biotin in vitro, followed by the affinity precipitation using streptavidin magnetic beads. The input or precipitated smaller MTD domains of NOP2 were analyzed by immunoblotting. (F, G) Recombinant Tat protein was incubated with bio-TAR in the absence or presence of NOP2 MTD-3 (F) or MTD-5 (G) domain at the increased amount, or with free biotin in vitro, followed by the affinity precipitation using streptavidin magnetic beads. The input or precipitated Tat or smaller MTD domain of NOP2 was analyzed by immunoblotting. The relative intensity of pulled down Tat was calculated.
Fig 7.
A tentative model illustrates NOP2’s silencing effect on HIV-1 proviral expression.
At the latent phase of HIV-1 infection, NOP2 occupies at the 5′ LTR of HIV-1 proviruses, binds with HIV-1 TAR RNA through its methylation domain and leads to its m5C methylation. HIV-1 Tat protein is a critical viral factor that recruits the host positive transcription elongation complex b (P-TEFb), composed of cyclin T1 (CCNT1) and CDK9, to activate RNA polymerase II (Pol II) for HIV-1 transcriptional elongation. The binding of NOP2 with TAR competes with Tat-TAR interaction, preventing the recruitment of Tat and P-TEFb as well as the activation of Pol II. Overall, the association of NOP2 at 5’ LTR suppresses HIV-1 transcriptional elongation. Once latent HIV-1 proviruses are reactivated, for example, by TNFα, NOP2 dissociates from the 5′ LTR of HIV-1 proviruses. Thus, Tat binds with TAR and recruits P-TEFb for activation of Pol II, promoting HIV-1 transcriptional elongation.