Nucleolar protein NOP2/NSUN1 suppresses HIV-1 transcription and promotes viral latency by competing with Tat for TAR binding and methylation
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.