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. 2021 Jun 30:9:693706.
doi: 10.3389/fcell.2021.693706. eCollection 2021.

HIV-1 Transactivator of Transcription (Tat) Co-operates With AP-1 Factors to Enhance c-MYC Transcription

Leonardo Alves de Souza Rios  1 , Lungile Mapekula  1 , Nontlantla Mdletshe  1 , Dharshnee Chetty  2 , Shaheen Mowla  1
Affiliations

HIV-1 Transactivator of Transcription (Tat) Co-operates With AP-1 Factors to Enhance c-MYC Transcription

Leonardo Alves de Souza Rios et al. Front Cell Dev Biol. .

Abstract

HIV-1 infection often leads to the development of co-morbidities including cancer. Burkitt lymphoma (BL) is one of the most over-represented non-Hodgkin lymphoma among HIV-infected individuals, and displays a highly aggressive phenotype in this population group, with comparatively poorer outcomes, despite these patients being on anti-retroviral therapy. Accumulating evidence indicates that the molecular pathogenesis of HIV-associated malignancies is unique, with components of the virus playing an active role in driving oncogenesis, and in order to improve patient prognosis and treatment, a better understanding of disease pathobiology and progression is needed. In this study, we found HIV-1 Tat to be localized within the tumor cells of BL patients, and enhanced expression of oncogenic c-MYC in these cells. Using luciferase reporter assays we show that HIV-1 Tat enhances the c-MYC gene promoter activity and that this is partially mediated via two AP-1 binding elements located at positions -1128 and -1375 bp, as revealed by mutagenesis experiments. We further demonstrate, using pull-down assays, that Tat can exist within a protein complex with the AP-1 factor JunB, and that this complex can bind these AP-1 sites within the c-MYC promoter, as shown by in vivo chromatin immunoprecipitation assays. Therefore, these findings show that in HIV-infected individuals, Tat infiltrates B-cells, where it can enhance the expression of oncogenic factors, which contributes toward the more aggressive disease phenotype observed in these patients.

Keywords: AP-1; HIV-1; c-MYC; non-Hodgkin lymphoma; transactivator of transcription.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
HIV-1 Tat is detected in BL tumor tissue and elevates c-MYC expression at the transcriptional and translational levels. (A–E) Immunohistochemical detection of Tat protein in FFPE tumor samples from lymphoma patients. An HIV positive BL incubated without primary antibody (replaced with buffer only) (A) and an HIV negative DLBCL incubated with primary Tat antibody (ab43014, Abcam, United Kingdom) as used for the HIV positive BL tumor samples (B), were used as controls. (C) IHC for Tat protein expression in HIV positive BL tumor tissue. Positive staining is indicated by deposition of DAB (brown) and Hematoxylin was used as counterstain (blue/purple). Distinct nuclear Tat expression (marked by black arrows in E). Images (A–C) were taken at 400X magnification and (D,E) at 1000X. Scale bar in (A–C) represents 50 and 20 μm in (D,E). (F) Western blotting of total proteins isolated from Ramos (Left) and BL41 (Right) cells electroporated with pcDNA-Tat expression vector or pcDNA-Empty control using anti-c-MYC (SC-764, Santa Cruz Biotechnology, United States) antibody. Anti-p38 (M0800, Sigma-Aldrich, United States) was used as a loading control. Bar graphs represent densitometric analysis of western blots (ImageJ) and error bars represent standard deviation (SD). (G) Dual-Luciferase Reporter assay shows a significant dose-dependent increase in c-MYC promoter activity with increasing HIV-1 Tat concentration, relative to the empty-vector control which has been set at 1. The following fold increases were obtained for the indicated pcDNA-Tat concentrations: 50 ng: 1.5 ± 0.15; 100 ng: 2 ± 0.03; 200 ng: 2.86 ± 0.2; 400 ng: 3.5 ± 0.2 and 500 ng: 5.3 ± 0.02. Error bars represent SD. (H) Western blot showing increasing expression of HIV- 1 Tat in transfected HT1080 cells in G, with p38 as a loading control. Statistical analysis was done using GraphPad PRISM 8 with Student’s t-test and one-way ANOVA, significance indicates ∗(p < 0.05), ∗∗(p < 0.01) ∗∗∗(p < 0.001), ****(p < 0.0001). The results are representative of at least three separate repeats.
FIGURE 2
FIGURE 2
The AP-1 sites located at positions -1128 and -1375 bp partially mediate the effect of HIV-1 Tat on the c-MYC promoter. (A) The WT c-MYC promoter (-2324 to + 537 bp) construct was analyzed for TFBS using PROMO. Sites are represented as different colored shapes which are labeled in the key and the transcription start site (TSS) is denoted by a black arrow. Shortened promoter constructs are labeled accordingly on the right side of the diagram (pGL3-cMYC-DF1, pGL3-cMYC-DF2 and pGL3-cMYC-DF3). (B) Luciferase reporter assays comparing the fold activation of the c-MYC WT promoter to the deletion constructs (DF1-DF3) in the presence of HIV-1 Tat. (C) Luciferase assays comparing fold activation in the presence of HIV-1 Tat between the WT promoter, DF2 and AP-1 MT1, AP-1 MT2 and AP-1 MT1 + 2 constructs. Fold activation was obtained by comparing the relative luciferase units (RLU) of the pcDNA-Tat transfected groups to their empty transfected controls, which were set to 1. Statistical analysis was done using one-way ANOVA in GraphPad PRISM 8. Significance ∗(p < 0.05) and ns (not significant). Error bars represent SD. The results are representative of at least three separate repeats.
FIGURE 3
FIGURE 3
HIV-1 Tat and JunB form protein to protein associations and occupy the c-MYC promoter at two AP-1 binding sites. HT1080 cells were transfected with both 500 ng HIV-1 Tat expression vector (pcDNA-Tat) and 500 ng JunB expression vector (pCMV-JunB). (A) The cell lysate was incubated with anti-HIV-1 Tat antibody (ab43014, Abcam, United Kingdom) and pulled down using protein A agarose beads (Roche, Germany). Western blot was carried out using an anti-JunB antibody (Santa Cruz, United States) The goat-anti-rabbit secondary antibody (Bio-Rad, United States) was used as a negative control (lane 3). (B) Diagrammatic representation of the c-MYC promoter depicting the location of the two AP-1 sites in square blocks and the positions of the forward and reverse primers used for the ChIP assay are denoted by black arrows. (C–F) Bar graphs show qRT-PCR data using immunoprecipitated DNA obtained from ChIP with either anti-JunB, anti-HIV-1 Tat or anti-IgG (negative control) antibodies and amplified with the primers depicted in the diagram, error bars represent SD. Statistical significance determined using Student’s t-test in GraphPad PRISM 8, ∗∗∗(p < 0.001). The graphs are representative of at least three separate repeats.
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References

    1. Abayomi E. A., Somers A., Grewal R., Sissolak G., Bassa F., Maartens D., et al. (2011). Impact of the HIV epidemic and anti-retroviral treatment policy on lymphoma incidence and subtypes seen in the western cape of South Africa, 2002–2009: preliminary findings of the tygerberg lymphoma study group. Transfus. Apher. Sci. 44 161–166. 10.1016/j.transci.2011.01.007 - DOI - PMC - PubMed
    1. Abudulai L. N., Fernandez S., Corscadden K., Hunter M., Kirkham L.-A. S., Post J. J., et al. (2016). Chronic HIV-1 infection induces B-cell dysfunction that is incompletely resolved by long-term antiretroviral therapy. J. Acquir. Immune Defic. Syndr. 71 381–389. 10.1097/QAI.0000000000000869 - DOI - PubMed
    1. Anand A. R., Rachel G., Parthasarathy D. (2018). HIV proteins and endothelial dysfunction: implications in cardiovascular disease. Front. Cardiovasc. Med. 5:185. 10.3389/fcvm.2018.00185 - DOI - PMC - PubMed
    1. Blanco A., Álvarez S., Fresno M., Muñoz-Fernández M. Á. (2008). Extracellular HIV-tat induces cyclooxygenase-2 in glial cells through activation of nuclear factor of activated T cells. J. Immunol. 180 530–540. 10.4049/jimmunol.180.1.530 - DOI - PubMed
    1. Carroll V. A., Lafferty M. K., Marchionni L., Bryant J. L., Gallo R. C., Garzino-Demo A. (2016). Expression of HIV-1 matrix protein p17 and association with B-cell lymphoma in HIV-1 transgenic mice. Proc. Natl. Acad. Sci. U.S.A. 113 13168–13173. 10.1073/pnas.1615258113 - DOI - PMC - PubMed

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