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. 2014 May 21;9(5):e98304.
doi: 10.1371/journal.pone.0098304. eCollection 2014.

V体育官网 - Interplay of Rad51 with NF-κB pathway stimulates expression of HIV-1

Rafal Kaminski  1 , Hassen S Wollebo  1 , Prasun K Datta  1 , Martyn K White  1 , Shohreh Amini  2 , Kamel Khalili  1
Affiliations

Interplay of Rad51 with NF-κB pathway stimulates expression of HIV-1

Rafal Kaminski et al. PLoS One. .

Abstract

Transcription from the HIV-1 promoter is controlled by a series of ubiquitous and inducible cellular proteins with the ability to enter the nucleus and interact with the promoter. A DNA sequence spanning nucleotides -120 to -80, which supports the association of the inducible NF-κB transcription factor, has received much attention. Here we demonstrate that the interplay between Rad51, a key regulator of the homologous recombination pathway of DNA repair and whose level is induced upon HIV-1 infection, with the NF-κB pathway, augments transcription of the viral promoter VSports手机版. Evidently, stimulation of the NF-κB pathway by PMA and/or TSA promotes association of Rad51 with the LTR DNA sequence and that the p65 subunit of NF-κB is important for this event. Our results also demonstrate that, similar to p65, Rad51 utilizes the NF-κB pathway to position itself in the nucleus as ectopic expression of an IκB mutant impedes its nuclear appearance and transcriptional activity upon the HIV-1 LTR. Treatment of peripheral blood mononuclear cells with small molecules that inhibit Rad51 activity results in greater than 50% decrease in the HIV-1 infection of cells. These observations provide evidence for the involvement of DNA repair factors in control of HIV-1 gene activation and offer a new avenue for the development of anti-viral therapeutics that affect viral gene transcription in latently infected cells. .

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Activation of the HIV-1 LTR by Rad51 and NF-κB p65 requires the κB motif.
(A) Primary cultures of human astrocytes were transfected with luciferase reporter plasmids pGL3-LTR encompassing various segments of the LTR (as indicated), either alone or together with plasmids expressing NF-κB p65 and/or Rad51. The total amount of DNA was equalized with relevant plasmid vector DNA. (B) Western blot of protein extracts from the cells shown in Panel A showing NF-κB p65 and Rad51 levels in each sample. α-Tubulin served as a protein loading control. (C) Primary cultures of human astrocytes were transfected with NF-κB p65 siRNA and Rad51 siRNA alone or together. The total amount of siRNA was equalized with non-target siRNA. After 48 hours, cells were lysed and luciferase assays performed. (D) Western blot analysis illustrating the levels of NF-κB p65 and Rad51 in cells treated with various siRNAs as indicated. Transcriptional activity represents fold effect in which controls (NF-κB p65 and Rad51) were set at 1.0.
Figure 2
Figure 2. Rad51 stimulates expression of integrated copies of HIV-1.
(A) TZM-bl cells multiple endogenous integrated copies of LTR-luciferase were transfected with 0, 3, 10 and 30 ng of pCMV (lanes 1–4 or 5–8, respectively). After 24 hours, cells either remained unchanged or infected with HIV-1 and 48 hours later cells were harvested for luciferase assay for promoter activity and Western blot for measuring Rad51 level. (B) TZM-bl cells were treated as above except that plasmid expressing siRad51 was used to decrease expression of endogenous Rad51. Luciferase activity and Rad51 Western blots are shown. (C) Primary human fetal astrocytes were transfected with luciferase reporter plasmid treated with RI-1 or B02 in various concentrations after 24 hours and luciferase activity was determined after 48 hours. (D) MTT assay showing cell viability upon treatment under the same conditions used in Panel C.
Figure 3
Figure 3. Recruitment of Rad51 to an integrated copy of the HIV-1 LTR and activation of its transcription.
(A) TZM-bl cells were treated with TSA or PMA and the level of promoter activity was determined after 36 hours by luciferase assay. (B) ChIP analysis of extracts from TZM-bl cells treated with TSA and PMA illustrating the presence of LTR DNA corresponding to the κB motif in immunocomplexes pulled down by anti-Rad51 and anti-p65 antibodies. The bar graphs illustrate quantitative analyses of the intensity of bands corresponding to LTR DNA detected in the complex pulled down by anti-Rad51 and anti-p65 antibodies. The intensity of the input band was used for normalizing the data of each. (C) ChIP analysis for detection of Rad51 association with endogenous LTR DNA in TZM-bl cells, untreated or treated with PMA, after transfection with plasmids expressing non-target (NT) siRNA or NF-κB p65-specific siRNA. The bar graph illustrates quantitative analysis of the intensity of bands corresponding to LTR DNA detected in the complex pulled down by anti-Rad51 antibody. The intensity of the input band was used for normalizing the data. (D) ChIP analysis demonstrating the interaction of NF-κB p65 with integrated copies of the LTR under various conditions as indicated. ADU (Absorption density units) were set at the arbitrary unit of one. Ten represents the intensity of the bands obtained from ChIP assay.
Figure 4
Figure 4. Impact of NF-κB pathway on Rad51 activation of the LTR.
(A) Primary cultures of astrocytes were transfected with LTR-luciferase reporter constructs along with plasmids expressing Rad51, NF-κB p65 and IκBmut.in various combinations as indicated. Luciferase activity was determined after 36 hours and fold changes are illustrated. (B) Cytoplasmic or nuclear protein fractions from extracts of TZM-bl cells transfected with plasmids expressing NF-κB p65, Rad51 and IκBmut. were analyzed for p65 and Rad51 by Western blot. Lamin and α-tubulin were used to measure the integrity of the protein extracts. (C) Subcellular localization of p65 in TZM-bl cells transfected with pCMV-p65, pCMV-myc-Rad51 and either with pCMV (control) plasmid or a plasmid expressing IκBmut and treated with PMA. (D) Subcellular localization of myc-Rad51 in TZM-bl cells transfected with pCMV-p65, pCMV-myc-Rad51 and either control or IκBmut, and treated with PMA. (E) Quantification of the image shown in Panel C was performed by scoring 50 cells. (F) Quantification of the image shown in Panel D was performed by scoring 50 cells.
Figure 5
Figure 5. Inhibition of Rad51 function represses HIV-1 infection.
(A) Top: PBMCs isolated from 3 different donors were infected with HIV-1 SF162 and incubated in the presence of 10 or 50 µM of Rad51 inhibitor (RI-1) for 3 days. Infection was quantified by examining the level of virus in the supernatants from the infected cells using p24 Gag ELISA. (B) Western blot analysis of protein extracts from the infected cells using anti-Rad51 and anti-α-tubulin antibody. The ratio of the intensity of the Rad51 and α-tubulin in each sample is shown. (C) MTT assay showing cell viability upon treatment under the same conditions used in Panel A.
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