Skip to main content
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems
  • Log in
  • My alerts
  • My Cart

Main menu

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Minireviews
    • JVI Classic Spotlights
    • Archive
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JVI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
  • ASM
    • Antimicrobial Agents and Chemotherapy
    • Applied and Environmental Microbiology
    • Clinical Microbiology Reviews
    • Clinical and Vaccine Immunology
    • EcoSal Plus
    • Infection and Immunity
    • Journal of Bacteriology
    • Journal of Clinical Microbiology
    • Journal of Microbiology & Biology Education
    • Journal of Virology
    • mBio
    • Microbiology and Molecular Biology Reviews
    • Microbiology Resource Announcements
    • Microbiology Spectrum
    • Molecular and Cellular Biology
    • mSphere
    • mSystems

User menu

  • Log in
  • My alerts
  • My Cart

Search

  • Advanced search
Journal of Virology
publisher-logosite-logo

Advanced Search

  • Home
  • Articles
    • Current Issue
    • Accepted Manuscripts
    • COVID-19 Special Collection
    • Minireviews
    • JVI Classic Spotlights
    • Archive
  • For Authors
    • Submit a Manuscript
    • Scope
    • Editorial Policy
    • Submission, Review, & Publication Processes
    • Organization and Format
    • Errata, Author Corrections, Retractions
    • Illustrations and Tables
    • Nomenclature
    • Abbreviations and Conventions
    • Publication Fees
    • Ethics Resources and Policies
  • About the Journal
    • About JVI
    • Editor in Chief
    • Editorial Board
    • For Reviewers
    • For the Media
    • For Librarians
    • For Advertisers
    • Alerts
    • RSS
    • FAQ
  • Subscribe
    • Members
    • Institutions
ANIMAL VIRUSES

In Vivo Protein Binding and Functional Analysis ofcis-Acting Elements in the U3 Region of the Bovine Leukemia Virus Long Terminal Repeat

Jianqiao Xiao, Gertrude C. Buehring
Jianqiao Xiao
School of Public Health, University of California, Berkeley, California 94720
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Gertrude C. Buehring
School of Public Health, University of California, Berkeley, California 94720
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/JVI.72.7.5994-6003.1998
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

  • Tables
  • Fig. 1.
    • Open in new tab
    • Download powerpoint
    Fig. 1.

    cis-acting elements in the U3 region of BLV. (A) Schematic map of the BLV LTR showing the locations of thecis-acting elements in the U3 region and the binding sites for the primers used for in vivo footprinting. (B) Nucleotide sequence comparison of response elements in the BLV U3 region: the TREs, consensus CRE and CRE-like sequences including the AP4 sites, and the consensus GRE and GRE-like sequence. Bases different from the consensus sequences are shown in boldface type.

  • Fig. 2.
    • Open in new tab
    • Download powerpoint
    Fig. 2.

    In vivo DMS footprinting of the BLV U3 region in the presence of Tax. BLV-infected Bat2Cl6 cells were used as the source of DNA. Lanes: 1 and 2, noncoding strand with primer set A; 3 and 4, coding strand with primer sets B and C. In vitro (purified DNA) DMS-treated samples are in lanes 1 and 3. In vivo (living cells) DMS-treated samples are in lanes 2 and 4. The locations of the bands relative to the RNA start site are labeled at the left of each gel (negative numbers for sequences upstream and positive numbers for the downstream of the start site). Increased sensitivity to DMS (band in lane 2 darker than in lane 1, and band in lane 4 darker than in lane 3) is indicated by arrows pointing away from the gel, and decreased sensitivity to DMS (lighter or no bands in lanes 2 and 4) is indicated by arrows pointing toward the gel. cis-acting elements reported in the literature are indicated by open boxes on the right of the gel, the consensus sequences of the AP4 sites are indicated by solid boxes, the core sequences of three CRE-like elements are indicated by the hatched boxes, and the GREs are indicated by dotted boxes.

  • Fig. 3.
    • Open in new tab
    • Download powerpoint
    Fig. 3.

    Gel shift assay to detect in vitro protein binding to wild-type and some mutant cis-acting elements in the BLV LTR. Synthetic double-stranded oligonucleotides were end labeled with [γ-32P]ATP and used as probes. Nuclear protein extracts were prepared from Bat2Cl6 (BLV-producing cell line) and Tb1Lu (BLV-free parental cell line of Bat2Cl6). Labeled probes were incubated with nuclear extracts in the presence (+) or absence (−) of unlabeled probe (100 times the concentration of the labeled probe) and separated on a 5% nondenaturing polyacrylamide gel. Protein-bound and free probes are indicated by the arrowheads. Tb1Lu cell extracts were used for lanes 1, 4, 7, and 10 and Bat2Cl6 cell extracts were used for the remaining lanes.

  • Fig. 4.
    • Open in new tab
    • Download powerpoint
    Fig. 4.

    Basal promoter function of the wild-type BLV U3 and 25 LTR mutants in the absence (A) and presence (B) of p34Tax (solid bars) with values in the absence of p34Tax (open bars) shown for comparison. LTRs were inserted into a CAT reporter construct and transfected into BLV-negative Tb1Lu cells with or without cotransfection with a tax-containing plasmid. Mutants are arranged according to their location on the U3 map (Fig. 1). Promoter activity was measured as nanograms of CAT protein per 10 μg of total protein, and values were standardized by scaling the activity of the wild-type LTR to 1.0. Each value is the mean of five (A) or two (B) trials in separate experiments. Asterisks indicate that the CAT activity of the mutant is significantly different from the wild-type LTR (P ≤ 0.05 [Dunnett t test]). Abbreviations: a, addition; d, deletion; s, substitution; WT, wild type. Acronyms in capital letters refer to sites illustrated in Fig.1.

  • Fig. 5.
    • Open in new tab
    • Download powerpoint
    Fig. 5.

    Promoter function of the wild-type BLV LTR and 11 LTR mutants in six different cell lines: BL3 (bovine B lymphocytes) (A), Raji (human B lymphocytes) (B), FLK (sheep fibroblasts) (C), GR (mouse mammary epithelial cells) (D), MCF-7 cells (human mammary epithelial cells) (E), and Tb1Lu (bat lung epithelium) (F). For BL3 and FLK cells, only the plasmid with the wild-type or mutant LTR sequences was transfected, since these cell lines contain endogenous p34Tax. For the other four lines, wild-type or mutant LTR was cotransfected into cells with the pSGtax plasmid. Bars represent the mean values (nanograms of CAT protein per 10 μg of total protein) from two to four independent experiments. Values were standardized by scaling the activity of the wild-type LTR to 1.0. Asterisks represent statistically significant differences (P ≤ 0.05 [Dunnett t test]) relative to the wild-type LTR.

  • Fig. 6.
    • Open in new tab
    • Download powerpoint
    Fig. 6.

    Functional regions of the BLV U3. The genome structure of the whole BLV provirus is shown at the top. A schematic map of the U3 region is shown in the middle of the figure. The locations of three DRs are indicated by brackets. Possible protein binding sites are marked with boxes of different shapes (indicating different types of proteins). The locations of sequence changes in 15 mutants are indicated by asterisks (14 of the sites had altered in vivo footprints; no footprints were detected at bp −167). Mapping of the U3 region was based on the promoter activity of the mutants. Mutants with increased promoter activity were used to define an NRE, and the ones with decreased activity were used to define a PRE. The core promoter region was defined as reported in the literature (6, 9, 10). Regulatory regions were defined according to the reactions represented in Fig. 4B. Elements within the dually functional region function as NREs in some cell lines and as PREs in other cell lines.

Tables

  • Figures
  • Table 1.

    Sequences of primers and linkers used in this study

    NameLocationSequence
    Gene-specific primers used for in vivo footprinting
     Primer set A (noncoding strand)
      A1(bp −211 to −191)5′-TGTATGAAAGATCATGCCGA-3′
      A2(bp −200 to −180)5′-ATCATGCCGACCTAGGAGCCG-3′
      A3(bp −181 to −156)5′-CCACCGCCCCGTAAACCAGACAGA-3′
     Primer set B (coding strand)
      B1(bp 8 to −16)5′-AGCTCAATCGCCGTGGTCTTCGCAA-3′
      B2(bp −6 to −30)5′-CCGCTAACTCGACAGGGCCGGCATT-3′
      B3(bp −30 to −57)5′-TTATTAATTTATCAGCAGGTGAGGTCAG-3′
     Primer set C (coding strand)
      C1(bp 97 to 74)5′-TACCTGACCGCTGCCGGATAGCCG-3′
      C2(bp 84 to 60)5′-CCGGATAGCCGACCAGAAGGTCTCG-3′
      C3(bp 60 to 34)5′-GGGAGCAAGAGAGCTCAGGACCGA-3′
     Linkers
      15′-GCGGTGACCCGGGAGATCTGAATTC-3′
      25′-GAATTCAGATC-3′
    Primers used for mutagenesis
     Vector primers
      15′-ACGGUUAUCCACAGAAUCA-3′
      25′-ACUGGAACAACACUCAACC-3′
     Universal primers
      15′-AUUCUGUGGAUAACCGUA-3′
      25′-AGUGUUGUUCCAGUTTGG-3′
    Primers for CAT plasmid constructs
      1LTR 5′ primer5′-TGTATGAAAGATCATGCCGAC-3′
      2LTR 3′ primer5′-ATTGTTTGCCGGTCTCTCCT-3′
  • Table 2.

    Sequence changes of 25 BLV LTR mutants made by site-directed mutagenesis

    NameaSite of mutationbOriginal sequenceMutated sequence
    d-CRE1−157 to −153AGACGDeleted
    d-CRE2−131 to −129GACDeleted
    d-CRE3−57 to −54TGACDeleted
    s-CRE3−56GT
    d-CRE 1/2−157 to −153AGACGDeleted
    −131 to −129GACDeleted
    d-CRE 1/3−157 to −153AGACGDeleted
    −57 to −54TGACDeleted
    dCRE 1/s-GRE−157 to −153AGACGDeleted
    −71 to −60ACA---TG-CTTAT---AC--TA
    dCRE2/s-GRE−131 to −129GACDeleted
    −71 to −60ACA---TG--CTTAT---AC--TA
    d-CRE2/3−131 to −129GACDeleted
    −57 to −54TGACDeleted
    d-CRE3/a-GRE−57 to −54TGACDeleted
    −71 to −72Wild typeTATCC added
    d-CRE1/2/3−157 to −153AGACGDeleted
    −131 to −129GACDeleted
    −57 to −54TGACDeleted
    d-CRE1/2/s-GRE−157 to −153AGACGDeleted
    −131 to −129GACDeleted
    −71 to −60ACA---TG--CTTAT---AC--TA
    d-CRE2/3/a-GRE−131 to −129GACDeleted
    −57 to −54TGACDeleted
    −71 to −72Wild typeTATCC added
    d-CRE1/2/3/a-GRE−157 to −153AGACGDeleted
    −131 to −129GACDeleted
    −57 to −54TGACDeleted
    −71 to −72Wild typeTATCC added
    s-GRE−71 to −60ACA---TG--CTTAT---AC--TA
    s-167−167AG
    s-AP4-1−151GT
    s-142,3−142 to −143AGCT
    s-140−140AG
    s-AP4-2−124GT
    s-116−116CA
    s-107,8−107 to −108CCAT
    s-99−99CT
    s-83,4−83 to −84TCCT
    s,a-TATA−41 to −40TAAT
    −37 to −38Wild typeT added
    • ↵a Abbreviations: a, addition; d, deletion; s, substitution; AP4, activating protein 4.

    • ↵b Positions of sequence changes are indicated by the numbers which represent the distance (in base pairs) relative to the RNA start site (+1).

PreviousNext
Back to top
Download PDF
Citation Tools
In Vivo Protein Binding and Functional Analysis ofcis-Acting Elements in the U3 Region of the Bovine Leukemia Virus Long Terminal Repeat
Jianqiao Xiao, Gertrude C. Buehring
Journal of Virology Jul 1998, 72 (7) 5994-6003; DOI: 10.1128/JVI.72.7.5994-6003.1998

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
Print

Alerts
Sign In to Email Alerts with your Email Address
Email

Thank you for sharing this Journal of Virology article.

NOTE: We request your email address only to inform the recipient that it was you who recommended this article, and that it is not junk mail. We do not retain these email addresses.

Enter multiple addresses on separate lines or separate them with commas.
In Vivo Protein Binding and Functional Analysis ofcis-Acting Elements in the U3 Region of the Bovine Leukemia Virus Long Terminal Repeat
(Your Name) has forwarded a page to you from Journal of Virology
(Your Name) thought you would be interested in this article in Journal of Virology.
CAPTCHA
This question is for testing whether or not you are a human visitor and to prevent automated spam submissions.
Share
In Vivo Protein Binding and Functional Analysis ofcis-Acting Elements in the U3 Region of the Bovine Leukemia Virus Long Terminal Repeat
Jianqiao Xiao, Gertrude C. Buehring
Journal of Virology Jul 1998, 72 (7) 5994-6003; DOI: 10.1128/JVI.72.7.5994-6003.1998
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

KEYWORDS

Leukemia Virus, Bovine
Promoter Regions, Genetic
Repetitive Sequences, Nucleic Acid

Related Articles

Cited By...

About

  • About JVI
  • Editor in Chief
  • Editorial Board
  • Policies
  • For Reviewers
  • For the Media
  • For Librarians
  • For Advertisers
  • Alerts
  • RSS
  • FAQ
  • Permissions
  • Journal Announcements

Authors

  • ASM Author Center
  • Submit a Manuscript
  • Article Types
  • Ethics
  • Contact Us

Follow #Jvirology

@ASMicrobiology

       

 

JVI in collaboration with

American Society for Virology

ASM Journals

ASM journals are the most prominent publications in the field, delivering up-to-date and authoritative coverage of both basic and clinical microbiology.

About ASM | Contact Us | Press Room

 

ASM is a member of

Scientific Society Publisher Alliance

 

American Society for Microbiology
1752 N St. NW
Washington, DC 20036
Phone: (202) 737-3600

Copyright © 2021 American Society for Microbiology | Privacy Policy | Website feedback

Print ISSN: 0022-538X; Online ISSN: 1098-5514