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
REPLICATION

Function of Rta Is Essential for Lytic Replication of Murine Gammaherpesvirus 68

Ting-Ting Wu, Leming Tong, Tammy Rickabaugh, Samuel Speck, Ren Sun
Ting-Ting Wu
Department of Molecular and Medical Pharmacology, UCLA AIDS Institute, Jonsson Comprehensive Cancer Center, and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Leming Tong
Department of Molecular and Medical Pharmacology, UCLA AIDS Institute, Jonsson Comprehensive Cancer Center, and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Tammy Rickabaugh
Department of Molecular and Medical Pharmacology, UCLA AIDS Institute, Jonsson Comprehensive Cancer Center, and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Samuel Speck
Department of Pathology and Immunology and Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ren Sun
Department of Molecular and Medical Pharmacology, UCLA AIDS Institute, Jonsson Comprehensive Cancer Center, and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90095, and
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/JVI.75.19.9262-9273.2001
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

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

    Construction and expression of wild-type and mutant Rta proteins. (A) The structures of the wild-type and mutant Rta proteins are shown, with the open boxes representing the DNA binding domains, the hatched boxes representing the dimerization domains (DZ), and the shaded boxes representing the activation domains. The size of each protein is indicated at the right. (B) Wild-type and mutant Rta proteins are expressed in 293T cells. The coding sequences of Rta, Rd1, and Rd2 were individualy cloned into pCMVFLAG. The cells were transfected with the empty vector, pCMVFLAG (lane 1), pCMVFLAG/Rta/KSHV (lane 2), pCMVFLAG/Rta (lane 3), pCMVFLAG/Rd1 (lane 4), or pCMVFLAG /Rd2 (lane 5). Ten percent of the total cell lysates was loaded onto a 10% denaturing polyacrylamide gel. Western blot analysis was carried out using the monoclonal antibody against the FLAG epitope. The masses of individual proteins in the molecular weight standard are indicated at the left.

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

    Rd1 and Rd2 function as dominant-negative mutants of Rta in 293T cells. (A) Rd1 and Rd2 do not activate the ORF57 promoter. 293T cells were cotransfected with 50 ng of the reporter construct, p57Luc, and 5 ng of pCMVFLAG/Rta, or 5 to 500 ng of pCMVFLAG/Rd1, or 5 to 500 ng of pCMVFLAG/Rd2. The total amounts of plasmid DNA were brought up to 600 ng with pCMVFLAG. The control transfection was carried out with 550 ng of pCMVFLAG and 50 ng of p57Luc. Each transfection included 1 ng of pRLCMV containing theRenilla luciferase gene driven by the constitutively active CMV promoter for nomalization of variations among transfections. At 48 h posttransfection, total cell lysates were harvested for analysis of luciferase activity. Normalized luciferase activity was calculated by dividing the level of firefly luciferase activity by the level of Renilla luciferase activity in each transfection. The fold induction was then calculated by dividing the level of normalized luciferase activity by that of the control transfection. Standard deviations derived from four experiments are shown in parentheses. (B) Rd1 and Rd2 inhibit wild-type Rtatrans-activation of the ORF57 promoter. 293T cells were transfected with 50 ng of p57Luc and 5 ng of pCMVFLAG/Rta alone or with 5 to 500 ng of pCMVFLAG/Rd1 or 5 to 500 ng of pCMVFLAG/ Rd2. Total cell lysates were harvested at 48 h posttransfection, and the luciferase activity in each transfection was measured. The fold induction was calculated as described above. Wild-type Rta activity was expressed as the percentage of the fold induction relative to cotransfection of p57Luc and pCMVFLAG/Rta. Standard deviations derived from four experiments are expressed as error bars.

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

    Rd1 and Rd2 function as dominant-negative mutants of Rta in BHK-21 cells. The transfections described in the legend to Fig. 2were repeated in BHK-21 cells. (A) Rd1 and Rd2 do not activate the ORF57 promoter. (B) Rd1 and Rd2 inhibit wild-type Rtatrans-activation of the ORF57 promoter.

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

    Rd1 and Rd2 inhibit MHV-68 lytic protein expression from transfected virion DNA in 293T cells. pCMVFLAG (0.2 μg; lanes 2 to 4), pCMVFLAG/Rta (0.2 μg; lanes 5 to 7), pCMVFLAG/Rd1 (0.2 μg; lanes 8 to 10), or pCMVFLAG/Rd2 (0.2 μg; lanes 11 to 13) was cotransfected into 293T cells with 0.2 μg of virion DNA derived from the recombinant green fluorescent protein-expressing virus tw25. Total cell lysates were harvested at 2, 4, and 6 days posttransfection (as indicated above the lanes), and 10% of each lysate, including a negative control of untransfected cells (lanes 1), was used for Western blot analysis. (A) Expression of MHV-68 lytic proteins is suppressed by Rd1 and Rd2. The membrane was probed with the polyclonal rabbit serum against the MHV-68-infected cell lysates (anti-MHV-68). (B) Expression of MHV-68 M9 protein is suppressed by Rd1 and Rd2. The membrane was probed with anti-M9 polyclonal rabbit serum. (C) Wild-type and mutant Rta proteins are expressed in 293T cells. The membrane was probed with the monoclonal antibody against the FLAG epitope (anti-FLAG). (D) The levels of cellular β-actin were examined (anti-actin). The membrane was probed with monoclonal antibody against cellular β-actin.

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

    Rd1 and Rd2 inhibit the production of infectious viruses after transfection of virion DNA in 293T cells. The supernatants from the transfections described in the legend to Fig. 4 were harvested, and the viral titers were determined using plaque assays. The assays were repeated three times for each transfection. Standard deviations are expressed as error bars.

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

    Rd1 and Rd2 inhibit MHV-68 lytic protein expression from transfected virion DNA in BHK-21 cells. The transfections described in the legend to Fig. 4 were repeated in BHK-21 cells. Total cell lysates were harvested at 2, 4, and 6 days posttransfection (as indicated above the lanes), and 10% of each lysate, including a negative control of untransfected cells (lanes 1), was used for Western blot analysis. (A) Expression of MHV-68 lytic proteins is suppressed by Rd1 and Rd2. The membrane was probed with the polyclonal serum against MHV-68-infected cell lysates (anti-MHV-68). (B) Expression of MHV-68 M9 protein is suppressed by Rd1 and Rd2. The membrane was probed with a rabbit serum against the MHV-68 M9 protein (anti-M9). (C) Wild-type and mutant Rta proteins are expressed in BHK-21 cells. The membrane was probed with the monoclonal antibody against the FLAG epitope (anti-FLAG). (D) The levels of cellular β-actin were examined. The membrane was probed with monoclonal antibody against cellular β-actin (anti-actin).

  • Fig. 7.
    • Open in new tab
    • Download powerpoint
    Fig. 7.

    Rd1 and Rd2 inhibit the production of infectious viruses after transfection of virion DNA in BHK-21 cells. The supernatants from the transfections described in the legend to Fig. 6 were harvested, and the viral titers were determined using plaque assays. The assays were repeated three times for each transfection. Standard deviations are expressed as error bars.

  • Fig. 8.
    • Open in new tab
    • Download powerpoint
    Fig. 8.

    Transcription of tk andrta genes is suppressed in stable cell lines expressing Rd2. Two cell lines, 45-5 and V-30, stably transfected with pCMVFLAG/Rd2 and the parental 293T cells (P) were infected with wild-type MHV-68 (3 PFU/cell). Total RNA from uninfected (U) or infected cells was harvested at 4 or 15 h postinfection, and 30% of each RNA sample was used for Northern blot analysis. (A) Transcription of rta is reduced in 45-5 and V-30 cells. The probe was derived from the rta gene (nt 68651 to 69378). (B) Transcription of tk is reduced in 45-5 and V-30 cells. The same membrane was stripped and rehybridized with a probe derived from the tk gene (nt 32879 to 34813). (C) The RNA loadings were examined by rehybridizing with a probe derived from the cellular GAPDH (glyceraldehyde-3-phosphate dehydrogenase) gene.

  • Fig. 9.
    • Open in new tab
    • Download powerpoint
    Fig. 9.

    Viral protein expression is inhibited in stable cell lines expressing Rd2. 45-5, V-30, and the parental 293T (P) cells were infected with wild-type MHV-68 at the different MOIs (PFU/cell) indicated above the panel. Total cell lysates were harvested at day 2 postinfection, and 10% of each lysate was used for Western blot analyses. (A) Expression of viral lytic proteins is reduced in 45-5 and V-30 cells. The membrane was probed with polyclonal serum against MHV-68-infected cell lysates. (B) Expression of M9 (a viral late protein) is reduced in 45-5 and V-30 cells. The membrane was probed with polyclonal antibody against the recombinant MHV-68 M9 protein. (C) The protein loadings were examined by reprobing with monoclonal antibody against cellular β-actin.

  • Fig. 10.
    • Open in new tab
    • Download powerpoint
    Fig. 10.

    The yield of infectious viruses is reduced in cell lines expressing Rd2. The supernatants were harvested from the infections described in the legend to Fig. 9, and viral titers were determined using plaque assays. The assays were performed three times for each infection, and standard deviations are expressed as error bars.

PreviousNext
Back to top
Download PDF
Citation Tools
Function of Rta Is Essential for Lytic Replication of Murine Gammaherpesvirus 68
Ting-Ting Wu, Leming Tong, Tammy Rickabaugh, Samuel Speck, Ren Sun
Journal of Virology Oct 2001, 75 (19) 9262-9273; DOI: 10.1128/JVI.75.19.9262-9273.2001

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.
Function of Rta Is Essential for Lytic Replication of Murine Gammaherpesvirus 68
(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
Function of Rta Is Essential for Lytic Replication of Murine Gammaherpesvirus 68
Ting-Ting Wu, Leming Tong, Tammy Rickabaugh, Samuel Speck, Ren Sun
Journal of Virology Oct 2001, 75 (19) 9262-9273; DOI: 10.1128/JVI.75.19.9262-9273.2001
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

Gammaherpesvirinae
Immediate-Early Proteins
Trans-Activators

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