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. 2020 Feb 1:147:102-113.
doi: 10.1016/j.freeradbiomed.2019.12.015. Epub 2019 Dec 19.

The HIV-Tat protein interacts with Sp3 transcription factor and inhibits its binding to a distal site of the sod2 promoter in human pulmonary artery endothelial cells

Terrin L Manes  1 , Ari Simenauer  1 , Jason L Geohring  1 , Juliana Flemming  1 , Michael Brehm  2 , Adela Cota-Gomez  3
Affiliations

The HIV-Tat protein interacts with Sp3 transcription factor and inhibits its binding to a distal site of the sod2 promoter in human pulmonary artery endothelial cells

Terrin L Manes et al. Free Radic Biol Med. .

Abstract

Redox imbalance results in damage to cellular macromolecules and interferes with signaling pathways, leading to an inflammatory cellular and tissue environment. As such, the cellular oxidative environment is tightly regulated by several redox-modulating pathways. Many viruses have evolved intricate mechanisms to manipulate these pathways for their benefit, including HIV-1, which requires a pro-oxidant cellular environment for optimal replication. One such virulence factor responsible for modulating the redox environment is the HIV Transactivator of transcription (Tat). Tat is of particular interest as it is actively secreted by infected cells and internalized by uninfected bystander cells where it can elicit pro-oxidant effects resulting in inflammation and damage. Previously, we demonstrated that Tat regulates basal expression of Superoxide Dismutase 2 (sod2) by altering the binding of the Sp-transcription factors at regions relatively near (approx. -210 nucleotides) upstream of the transcriptional start site. Now, using in silico analysis and a series of sod2 promoter reporter constructs, we have identified putative clusters of Sp-binding sites located further upstream of the proximal sod2 promoter, between nucleotides -3400 to -210, and tested their effect on basal transcription and for their sensitivity to HIV-1 Tat. In this report, we demonstrate that under basal conditions, maximal transcription requires a cluster of Sp-binding sites in the -584 nucleotide region, which is extremely sensitive to Tat. Using chromatin immunoprecipitation (ChIP) we demonstrate that Tat results in altered occupancy of Sp1 and Sp3 at this distal Tat-sensitive regulatory element and strongly stimulated endogenous expression of SOD2 in human pulmonary artery endothelial cells (HPAEC). We also report altered expression of Sp1 and Sp3 in Tat-expressing HPAEC as well as in the lungs of HIV-1 infected humanized mice. Lastly, Tat co-immunoprecipitated with endogenous Sp3 but not Sp1 and did not alter the acetylation state of Sp3. Thus, here, we have defined a novel and important cis-acting factor in HIV-1 Tat-mediated regulation of SOD2, demonstrated that modulation of Sp1 and Sp3 activity by Tat promotes SOD2 expression in primary human pulmonary artery endothelial cells and determined that pulmonary levels of Sp3 as well as SOD2 are increased in the lungs of a mouse model of HIV infection.

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Figures

Figure 1.
Figure 1.. Schematics of predicted Sp binding clusters and Sod2 promoter constructs.
A) Sp-binding clusters in sequences upstream of the sod2 transcriptional start site as predicted by MatInspector and PhysBinder algorithmns: MatInspector predictions = pink/red, PhysBinder = green. Darker shades signify stronger predicted Sp binding affinity. Black arrows indicate locations of ChIP qPCR primers. B) Sod2-promoter-reporter constructs (sod2-prom) utilized in this study: Constructs of −210, −1240, −1605, −2987 and −3406 sod2-prom plasmids were a kind gift from Dr. Daret St. Clair. Construct of −584 sod2prom was created in house.
Figure 2.
Figure 2.. Tat represses expression from sod2 promoter reporters.
A) Expression from sod2- promoter-reporter constructs in HeLa-WT and stable HeLa transfectant that constitutively expresses the HIV Tat protein, HeLa-TatIII. B) Expression from the sod2-promoter reporter constructs in HPAEC with (Tat(+)) and without (Tat(−)) co-transfection with pCP2-Tat. Firefly luciferase values were normalized to Renilla luciferase from co-transfected pRL-CMV plasmid. Shown is the Mean ± SEM of biological replicates, n ≥ 6, (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 by un-paired student’s T-test).
Figure 3.
Figure 3.. Chromatin immunoprecipitation (ChIP) analysis of Sp1 and Sp3 bound to the −584 region of the sod2 promoter in un-transfected (Tat(−)) or pCP2-Tat101 transfected (Tat(+)) HPAEC.
Following IP with either Sp1 or Sp3 antibodies, the number of copies of the −584-nucleotide region of the endogenous sod2 promoter was determined by absolute qPCR via a standard curve generated utilizing a plasmid containing −3406sod2-promoter sequences. n = 3. (*p ≤ 0.05 by un-paired student’s T-test). DNA that was immunoprecipitated using mouse IgG1 (serotype control) did not amplify.
Figure 4.
Figure 4.. SOD2 expression in HPAEC in the presence and absence of Tat.
A) sod2 transcription is strongly stimulated by HIV-Tat. Relative fold change calculated by the ΔΔCT method and normalized to GAPDH. Mean ± SEM of biological replicates, n= 6, p < 0.0001 by unpaired student’s t-test. B and C) SOD2 protein levels are increased in the presence of Tat. Mean ± SEM of biological replicates, n= 4, (p ≤ 0.0001 by unpaired student’s t-test). SOD2 Densitometry values normalized to β-actin. L = MW ladder.
Figure 5.
Figure 5.. Sp1 and Sp3 Binding activity in HPAEC in the absence and presence of Tat.
Using an Sp-specific oligonucleotide-binding ELISA (TransAm, Active Motif), we measured the levels of active Sp1 (Panel A) and Sp3 (Panel B) in the nucleus of mock transfected (Tat(−)) or pCP2-Tat101 transfected (Tat(+)) HPAEC. The signal was normalized to μg of nuclear protein. Mean ± SEM of biological replicates, n = 3, (***p ≤ 0.001 by unpaired student’s T-test).
Figure 6.
Figure 6.. Tat Co-Immunoprecipitates with Sp3 but does not alter Sp3 acetylation State:
A) Sp3 and Tat Co-immunoprecipitate: Sp3 was immunoprecipitated from Tat (−) or Tat (+) HPAEC whole cell lysate. Total input lysate and immunoprecipitate were analyzed via immunoblot for successful Sp3 IP and potential Tat CoIP. B) Sp3 acetylation state is not altered by interaction with Tat: The total input lysate and Sp3 immunoprecipitate from Panel A was also analyzed via immunoblot with anti-acetyl-lysine antibodies. C) Acetyl lysine detection in the total protein lysates of Untreated or Trichostatin A (TSA) treated HPAEC.
Figure 7.
Figure 7.. Transcription of Sp1 and Sp3 in the presence and absence of Tat in HPAEC.
A) Transcript levels of Sp1 and Sp3 in Tat (−) (mock transfected) and Tat (+) (pCP2-Tat101-tranfected) HPAEC: Relative levels of transcript calculated by the ΔΔCT method and normalized to GAPDH (n = 6). Mean ± SEM of biological replicates (**p ≤ 0.01, ***p ≤ 0.001 by unpaired student’s T-test).
Figure 8.
Figure 8.. Sp1 and Sp3 protein levels in the absence and presence of Tat in HPAEC.
A-B) Sp3 and Sp1 expression is increased in Tat (+) HPAEC. Whole cell extract of mock transfected (Tat (−)) and pCP2-Tat101-transfected (Tat (+)) HPAEC were analyzed via immunoblot for total levels of Sp3/Sp1. C) Fold increase of Sp3 (top band) and Sp1 in Tat (+) and Tat (−) HPAEC. Fold change in Sp expression was calculated as actin normalized Tat (+) densitometry values over actin normalized Tat (−) densitometry values (n = 4). Mean ± SEM of biological replicates L = MW ladder.
Figure 9.
Figure 9.. Sp3 expression increased in the Lungs of HIV infected mice while SOD2 did not change.
A) Immunoblot analysis of Sp3 and Sod2 levels in total lung tissue: Protein was extracted from whole lung tissue of un-infected, mock-infected, or HIV-1 infected humanized mice for immunoblot analysis. B) Sp3 densitometry: Values shown as normalized to β-actin. (un-infected n = 3, mock infected n = 4, HIV-1 infected n = 3). C) Sod2 densitometry: Values shown as normalized to β-actin. (un-infected n = 3, mock infected n = 4, HIV-1 infected n = 3). Mean ± SEM of biological replicates (*p ≤ 0.05, **p ≤ 0.01 by un-paired student’s T-test).
Figure 10.
Figure 10.. Sp1 expression in the Lungs of HIV infected mice.
A) Immunoblot analysis of Sp1 levels in total lung tissue of NSG-BLT Mice: Protein was extracted from whole lung tissue of un-infected, mock-infected, or HIV-1 infected humanized mice for immunoblot analysis. B) Sp1 densitometry: Values shown as normalized to β-actin. (un-infected n = 3, mock infected n = 4, HIV-1 infected n = 3). Mean ± SEM of biological replicates (**p ≤ 0.01 by un-paired student’s T-test).
Figure 11.
Figure 11.. SOD2 levels are increased in the lungs of HIV infected humanized NSG-BLT mice while total antioxidant capacity is lower
A) IHC of lung tissue from HIV infected (10 weeks) and uninfected humanized NSG-BLT mice reveals periadventitial increase in S0D2. B) Total antioxidant capacity (TAC) is reduced in the lungs of HIV infected humanized NSG-BLT mice (n = 8) compared to uninfected mice (n = 8). Mean ± SEM of biological replicates (*p ≤ 0.05 by un-paired student’s T-test).
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References

    1. Triplette M, Crothers K, Attia EF, Non-infectious Pulmonary Diseases and HIV, Curr. HIV/AIDS Rep 13 (2016) 140–148. doi:10.1007/s11904-016-0313-0. - DOI - PubMed
    1. Ivanov AV, Valuev-Elliston VT, Ivanova ON, Kochetkov SN, Starodubova ES, Bartosch B, Isaguliants MG, Oxidative Stress during HIV Infection: Mechanisms and Consequences, Oxid. Med. Cell. Longev 2016 (2016) 1–18. doi:10.1155/2016/8910396. - DOI - PMC - PubMed
    1. Flores SC, Marecki JC, Harper KP, Bose SK, Nelson SK, McCord JM, Tat protein of human immunodeficiency virus type 1 represses expression of manganese superoxide dismutase in HeLa cells., Proc. Natl. Acad. Sci. U. S. A 90 (1993) 7632–7636. - PMC - PubMed
    1. Marciniak RA, Calnan BJ, Frankel AD, Sharp PA, HIV-1 Tat Protein Tram-Activates Transcription In Vitro, Cell. 63 (1990) 791–602. http://ac.els-cdn.com.hsl-ezproxy.ucdenver.edu/0092867490901455/1-s2.0-0... (accessed April 25, 2017). - PubMed
    1. Marciniak RA, Sharp PA, HIV-1 Tat protein promotes formation of more-processive elongation complexes, EMBO J. 1013 (1991) 4189–4196. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC453171/pdf/emboj00111-0201.pdf (accessed April 25, 2017). - PMC - PubMed

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