PU.1 binding to ets motifs within the equine infectious anemia virus long terminal repeat (LTR) enhancer: regulation of LTR activity and virus replication in macrophages.
Abstract: Binding of the transcription factor PU.1 to its DNA binding motif regulates the expression of a number of B-cell- and myeloid-specific genes. The long terminal repeat (LTR) of macrophage-tropic strains of equine infectious anemia virus (EIAV) contains three PU.1 binding sites, namely an invariant promoter-proximal site as well as two upstream sites. We have previously shown that these sites are important for EIAV LTR activity in primary macrophages (W. Maury, J. Virol. 68:6270-6279, 1994). Since the sequences present in these three binding motifs are not identical, we sought to determine the role of these three sites in EIAV LTR activity. While DNase I footprinting studies indicated that all three sites within the enhancer were bound by recombinant PU.1, reporter gene assays demonstrated that the middle motif was most important for basal levels of LTR activity in macrophages and that the 5' motif had little impact. The impact of the 3' site became evident in Tat transactivation studies, in which the loss of the site reduced Tat-transactivated expression 40-fold. In contrast, elimination of the 5' site had no effect on Tat-mediated activity. Binding studies were performed to determine whether differences in PU.1 binding affinity for the three sites correlated with the relative impact of each site on LTR transcription. While small differences were observed in the binding affinities of the three sites, with the promoter-proximal site having the strongest binding affinity, these differences could not account for the dramatic differences observed in the transcriptional effects. Instead, the promoter-proximal position of the 3' motif appeared to be critical for its transcriptional impact and suggested that the PU.1 sites may serve different roles depending upon the location of the sites within the enhancer. Infectivity studies demonstrated that an LTR containing an enhancer composed of the three PU.1 sites was not sufficient to drive viral replication in macrophages. These findings indicate that while the promoter-proximal PU.1 site is the most critical site for EIAV LTR activity in the presence of Tat, other elements within the enhancer are needed for EIAV replication in macrophages.
Publication Date: 2004-03-16 PubMed ID: 15016863PubMed Central: PMC371083DOI: 10.1128/jvi.78.7.3407-3418.2004Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- Non-P.H.S.
- Research Support
- U.S. Gov't
- P.H.S.
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
The research examines how the transcription factor PU.1 binds to different motifs in the replication process of equine infectious anemia virus (EIAV) and how it impacts the virus’ ability to activate certain genes. Their findings highlight the complexity and significance of these binding processes in the functioning and control of the virus.
Understanding PU.1 and EIAV
- PU.1 is a transcription factor that helps control gene expression, especially in B-cells and myeloid cells.
- EIAV (equine infectious anemia virus) is a horse disease that can help us understand similar conditions in humans. It uses the long terminal repeat (LTR) as a virus promoter, which contains three PU.1 binding sites.
- Previous research found these sites crucial for EIAV LTR activity in macrophages, types of white blood cells that play key roles in immunity.
The Study’s Methods and Findings
- The researchers evaluated the roles of the three PU.1 binding sites and their contribution to EIAV LTR activity. Their experiments included DNase I footprinting studies and reporter gene assays.
- The middle motif seemed the most important for maintaining basal levels of LTR activity in macrophages, while the 5′ motif impact was minor.
- In the study, they also focused on the effect of Tat, a protein that helps the virus replicate. Removing the 3′ site led to a significant reduction in Tat-transactivated expression, however, the 5′ site’s elimination did not affect this activity.
Observations about PU.1 Binding Affinity
- They attempted to understand whether variations in PU.1 binding affinity for the three sites correlated with each site’s impact on LTR transcription. They noted slight variations in binding affinities, but these could not explain the significant differences in transcriptional impact.
- Notably, the position of the 3′ site, closest to the promoter, was instrumental for its impact, suggesting that the location of PU.1 sites within the enhancer can determine their roles.
Infectivity Studies
- The researchers also conducted infectivity studies and found that an LTR with an enhancer made of the three PU.1 sites was insufficient to drive viral replication in macrophages.
- This highlights that, despite the promoter-proximal PU.1 site being crucial for EIAV LTR activity in the presence of Tat, other elements within the enhancer are necessary for EIAV replication.
Cite This Article
APA
Hines R, Sorensen BR, Shea MA, Maury W.
(2004).
PU.1 binding to ets motifs within the equine infectious anemia virus long terminal repeat (LTR) enhancer: regulation of LTR activity and virus replication in macrophages.
J Virol, 78(7), 3407-3418.
https://doi.org/10.1128/jvi.78.7.3407-3418.2004 Publication
Researcher Affiliations
- Division of Basic Biomedical Science, University of South Dakota, Vermillion, South Dakota 57069, USA.
MeSH Terms
- Base Sequence
- Binding Sites
- Cell Line
- DNA Footprinting
- DNA-Binding Proteins / chemistry
- DNA-Binding Proteins / genetics
- DNA-Binding Proteins / metabolism
- Enhancer Elements, Genetic / genetics
- Gene Expression Regulation, Viral
- HIV Enhancer / genetics
- Humans
- Infectious Anemia Virus, Equine / genetics
- Macrophages / virology
- Models, Molecular
- Molecular Sequence Data
- Mutation / genetics
- Nucleic Acid Conformation
- Promoter Regions, Genetic / genetics
- Protein Binding
- Protein Structure, Tertiary
- Proto-Oncogene Proteins / chemistry
- Proto-Oncogene Proteins / genetics
- Proto-Oncogene Proteins / metabolism
- Proto-Oncogene Proteins c-ets
- Response Elements / genetics
- Terminal Repeat Sequences / genetics
- Trans-Activators / chemistry
- Trans-Activators / genetics
- Trans-Activators / metabolism
- Transcription Factors / metabolism
- Virus Replication
Grant Funding
- CA72063 / NCI NIH HHS
References
This article includes 48 references
- Barboric M, Nissen RM, Kanazawa S, Jabrane-Ferrat N, Peterlin BM. NF-kappaB binds P-TEFb to stimulate transcriptional elongation by RNA polymerase II.. Mol Cell 2001 Aug;8(2):327-37.
- Berkhout B, Jeang KT. Functional roles for the TATA promoter and enhancers in basal and Tat-induced expression of the human immunodeficiency virus type 1 long terminal repeat.. J Virol 1992 Jan;66(1):139-49.
- Carvalho M, Derse D. Mutational analysis of the equine infectious anemia virus Tat-responsive element.. J Virol 1991 Jul;65(7):3468-74.
- Carvalho M, Derse D. The PU.1/Spi-1 proto-oncogene is a transcriptional regulator of a lentivirus promoter.. J Virol 1993 Jul;67(7):3885-90.
- Carvalho M, Kirkland M, Derse D. Protein interactions with DNA elements in variant equine infectious anemia virus enhancers and their impact on transcriptional activity.. J Virol 1993 Nov;67(11):6586-95.
- Cheevers WP, McGuire TC. Equine infectious anemia virus: immunopathogenesis and persistence.. Rev Infect Dis 1985 Jan-Feb;7(1):83-8.
- Chun RF, Jeang KT. Requirements for RNA polymerase II carboxyl-terminal domain for activated transcription of human retroviruses human T-cell lymphotropic virus I and HIV-1.. J Biol Chem 1996 Nov 1;271(44):27888-94.
- Chun RF, Semmes OJ, Neuveut C, Jeang KT. Modulation of Sp1 phosphorylation by human immunodeficiency virus type 1 Tat.. J Virol 1998 Apr;72(4):2615-29.
- Clabough DL, Gebhard D, Flaherty MT, Whetter LE, Perry ST, Coggins L, Fuller FJ. Immune-mediated thrombocytopenia in horses infected with equine infectious anemia virus.. J Virol 1991 Nov;65(11):6242-51.
- Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.. Nucleic Acids Res 1983 Mar 11;11(5):1475-89.
- Dorn PL, Derse D. cis- and trans-acting regulation of gene expression of equine infectious anemia virus.. J Virol 1988 Sep;62(9):3522-6.
- English D, Andersen BR. Single-step separation of red blood cells. Granulocytes and mononuclear leukocytes on discontinuous density gradients of Ficoll-Hypaque.. J Immunol Methods 1974 Aug;5(3):249-52.
- Feng X, Teitelbaum SL, Quiroz ME, Cheng SL, Lai CF, Avioli LV, Ross FP. Sp1/Sp3 and PU.1 differentially regulate beta(5) integrin gene expression in macrophages and osteoblasts.. J Biol Chem 2000 Mar 24;275(12):8331-40.
- Fisher RC, Olson MC, Pongubala JM, Perkel JM, Atchison ML, Scott EW, Simon MC. Normal myeloid development requires both the glutamine-rich transactivation domain and the PEST region of transcription factor PU.1 but not the potent acidic transactivation domain.. Mol Cell Biol 1998 Jul;18(7):4347-57.
- Fisher RC, Scott EW. Role of PU.1 in hematopoiesis.. Stem Cells 1998;16(1):25-37.
- Gorman C, Padmanabhan R, Howard BH. High efficiency DNA-mediated transformation of primate cells.. Science 1983 Aug 5;221(4610):551-3.
- Graham FL, Smiley J, Russell WC, Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5.. J Gen Virol 1977 Jul;36(1):59-74.
- Gross P, Arrowsmith CH, Macgregor RB Jr. Hydroxyl radical footprinting of DNA complexes of the ets domain of PU.1 and its comparison to the crystal structure.. Biochemistry 1998 Apr 14;37(15):5129-35.
- Gross P, Yee AA, Arrowsmith CH, Macgregor RB Jr. Quantitative hydroxyl radical footprinting reveals cooperative interactions between DNA-binding subdomains of PU.1 and IRF4.. Biochemistry 1998 Jul 7;37(27):9802-11.
- Hagemeier C, Bannister AJ, Cook A, Kouzarides T. The activation domain of transcription factor PU.1 binds the retinoblastoma (RB) protein and the transcription factor TFIID in vitro: RB shows sequence similarity to TFIID and TFIIB.. Proc Natl Acad Sci U S A 1993 Feb 15;90(4):1580-4.
- Hines R, Maury W. DH82 cells: a macrophage cell line for the replication and study of equine infectious anemia virus.. J Virol Methods 2001 Jun;95(1-2):47-56.
- Huang LM, Jeang KT. Increased spacing between Sp1 and TATAA renders human immunodeficiency virus type 1 replication defective: implication for Tat function.. J Virol 1993 Dec;67(12):6937-44.
- Johnson ML, Frasier SG. Non-linear least squares analysis. Methods Enzymol 117:301-342.
- Kodandapani R, Pio F, Ni CZ, Piccialli G, Klemsz M, McKercher S, Maki RA, Ely KR. A new pattern for helix-turn-helix recognition revealed by the PU.1 ETS-domain-DNA complex.. Nature 1996 Apr 4;380(6573):456-60.
- Leonard J, Parrott C, Buckler-White AJ, Turner W, Ross EK, Martin MA, Rabson AB. The NF-kappa B binding sites in the human immunodeficiency virus type 1 long terminal repeat are not required for virus infectivity.. J Virol 1989 Nov;63(11):4919-24.
- Li SL, Schlegel W, Valente AJ, Clark RA. Critical flanking sequences of PU.1 binding sites in myeloid-specific promoters.. J Biol Chem 1999 Nov 5;274(45):32453-60.
- Maury W. Monocyte maturation controls expression of equine infectious anemia virus.. J Virol 1994 Oct;68(10):6270-9.
- Maury W, Bradley S, Wright B, Hines R. Cell specificity of the transcription-factor repertoire used by a lentivirus: motifs important for expression of equine infectious anemia virus in nonmonocytic cells.. Virology 2000 Feb 15;267(2):267-78.
- Maury W, Perryman S, Oaks JL, Seid BK, Crawford T, McGuire T, Carpenter S. Localized sequence heterogeneity in the long terminal repeats of in vivo isolates of equine infectious anemia virus.. J Virol 1997 Jul;71(7):4929-37.
- Maury WJ, Carpenter S, Graves K, Chesebro B. Cellular and viral specificity of equine infectious anemia virus Tat transactivation.. Virology 1994 May 1;200(2):632-42.
- Nagulapalli S, Pongubala JM, Atchison ML. Multiple proteins physically interact with PU.1. Transcriptional synergy with NF-IL6 beta (C/EBP delta, CRP3).. J Immunol 1995 Nov 1;155(9):4330-8.
- Oaks JL, McGuire TC, Ulibarri C, Crawford TB. Equine infectious anemia virus is found in tissue macrophages during subclinical infection.. J Virol 1998 Sep;72(9):7263-9.
- Parrott C, Seidner T, Duh E, Leonard J, Theodore TS, Buckler-White A, Martin MA, Rabson AB. Variable role of the long terminal repeat Sp1-binding sites in human immunodeficiency virus replication in T lymphocytes.. J Virol 1991 Mar;65(3):1414-9.
- Payne SL, Rausch J, Rushlow K, Montelaro RC, Issel C, Flaherty M, Perry S, Sellon D, Fuller F. Characterization of infectious molecular clones of equine infectious anaemia virus.. J Gen Virol 1994 Feb;75 ( Pt 2):425-9.
- Perkel JM, Atchison ML. A two-step mechanism for recruitment of Pip by PU.1.. J Immunol 1998 Jan 1;160(1):241-52.
- Pongubala JM, Atchison ML. PU.1 can participate in an active enhancer complex without its transcriptional activation domain.. Proc Natl Acad Sci U S A 1997 Jan 7;94(1):127-32.
- Pongubala JM, Van Beveren C, Nagulapalli S, Klemsz MJ, McKercher SR, Maki RA, Atchison ML. Effect of PU.1 phosphorylation on interaction with NF-EM5 and transcriptional activation.. Science 1993 Mar 12;259(5101):1622-5.
- Reed-Inderbitzin E, Maury W. Cellular specificity of HIV-1 replication can be controlled by LTR sequences.. Virology 2003 Sep 30;314(2):680-95.
- Ross EK, Buckler-White AJ, Rabson AB, Englund G, Martin MA. Contribution of NF-kappa B and Sp1 binding motifs to the replicative capacity of human immunodeficiency virus type 1: distinct patterns of viral growth are determined by T-cell types.. J Virol 1991 Aug;65(8):4350-8.
- Ross IL, Yue X, Ostrowski MC, Hume DA. Interaction between PU.1 and another Ets family transcription factor promotes macrophage-specific Basal transcription initiation.. J Biol Chem 1998 Mar 20;273(12):6662-9.
- Sellon DC. Equine infectious anemia.. Vet Clin North Am Equine Pract 1993 Aug;9(2):321-36.
- Shea MA, Sorensen BR, Pedigo S, Verhoeven AS. Proteolytic footprinting titrations for estimating ligand-binding constants and detecting pathways of conformational switching of calmodulin.. Methods Enzymol 2000;323:254-301.
- Sorensen BR, Shea MA. Interactions between domains of apo calmodulin alter calcium binding and stability.. Biochemistry 1998 Mar 24;37(12):4244-53.
- Strauss-Soukup JK, Maher LJ 3rd. Role of asymmetric phosphate neutralization in DNA bending by PU.1.. J Biol Chem 1997 Dec 12;272(50):31570-5.
- Tenen DG, Hromas R, Licht JD, Zhang DE. Transcription factors, normal myeloid development, and leukemia.. Blood 1997 Jul 15;90(2):489-519.
- van Dijk TB, Baltus B, Caldenhoven E, Handa H, Raaijmakers JA, Lammers JW, Koenderman L, de Groot RP. Cloning and characterization of the human interleukin-3 (IL-3)/IL-5/ granulocyte-macrophage colony-stimulating factor receptor betac gene: regulation by Ets family members.. Blood 1998 Nov 15;92(10):3636-46.
- Yedavalli VS, Benkirane M, Jeang KT. Tat and trans-activation-responsive (TAR) RNA-independent induction of HIV-1 long terminal repeat by human and murine cyclin T1 requires Sp1.. J Biol Chem 2003 Feb 21;278(8):6404-10.
- Ying H, Chang JF, Parnes JR. PU.1/Spi-1 is essential for the B cell-specific activity of the mouse CD72 promoter.. J Immunol 1998 Mar 1;160(5):2287-96.
Citations
This article has been cited 6 times.- Wang XF, Liu Q, Wang YH, Wang S, Chen J, Lin YZ, Ma J, Zhou JH, Wang X. Characterization of Equine Infectious Anemia Virus Long Terminal Repeat Quasispecies In Vitro and In Vivo.. J Virol 2018 Apr 15;92(8).
- Wei L, Fan X, Lu X, Zhao L, Xiang W, Zhang X, Xue F, Shao Y, Shen R, Wang X. Genetic variation in the long terminal repeat associated with the transition of Chinese equine infectious anemia virus from virulence to avirulence.. Virus Genes 2009 Apr;38(2):285-8.
- van Opijnen T, Boerlijst MC, Berkhout B. Effects of random mutations in the human immunodeficiency virus type 1 transcriptional promoter on viral fitness in different host cell environments.. J Virol 2006 Jul;80(13):6678-85.
- Brindley MA, Maury W. Endocytosis and a low-pH step are required for productive entry of equine infectious anemia virus.. J Virol 2005 Dec;79(23):14482-8.
- Goffin V, Demonté D, Vanhulle C, de Walque S, de Launoit Y, Burny A, Collette Y, Van Lint C. Transcription factor binding sites in the pol gene intragenic regulatory region of HIV-1 are important for virus infectivity.. Nucleic Acids Res 2005;33(13):4285-310.
- Maury W, Thompson RJ, Jones Q, Bradley S, Denke T, Baccam P, Smazik M, Oaks JL. Evolution of the equine infectious anemia virus long terminal repeat during the alteration of cell tropism.. J Virol 2005 May;79(9):5653-64.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
