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
Virus-Cell Interactions

fus-1, a pH Shift Mutant of Semliki Forest Virus, Acts by Altering Spike Subunit Interactions via a Mutation in the E2 Subunit

Sallie Glomb-Reinmund, Margaret Kielian
Sallie Glomb-Reinmund
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Margaret Kielian
Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/JVI.72.5.4281-4287.1998
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

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

    pH dependence of E1 epitope exposure for wt and mutant viruses. 35S-labeled wt/ic, S1J, fus-1, and fus-1/ic virus preparations were treated at the indicated pH for 10 min at 37°C, adjusted to neutral pH, solubilized in lysis buffer containing 1% Triton X-100, and immunoprecipitated with MAb E1a-1, an acid conformation-specific antibody. E1 precipitated by the MAb was quantitated by SDS-PAGE and phosphorimaging and then compared to the total E1 precipitated by a rabbit polyclonal antibody. Data represent the mean of three separate experiments for each virus. The standard deviations ranged from 1 to 30%.

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

    pH dependence of wt and mutant E1 homotrimer formation.35S-labeled wt/ic, S1J, fus-1, andfus-1/ic virus preparations were treated at the indicated pH for 10 min at 37°C in the presence of liposomes, adjusted to neutral pH, and solubilized in SDS sample buffer for 3 min at 30°C. Samples were analyzed by SDS-PAGE and quantitated by phosphorimaging. Results are shown as percent E1 in the homotrimer band compared to total E1 and represent the mean for three experiments, with standard deviations between 1 and 33%.

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

    Sensitivity of infection by wt, mutant, and revertant viruses to inhibition by NH4Cl. BHK cells were pretreated with the indicated concentrations of NH4Cl for 15 min at 37°C and then infected with wt/ic, S1J, fus-1,fus-1/ic, R43 revertant, or R46 revertant at 1 PFU/cell for 1.5 h in the continued presence of NH4Cl. Following infection, the cells were treated with 2 μg of ACD per ml and 15 mM NH4Cl for 30 min and then labeled with [3H]uridine for 3.5 h in the continued presence of ACD and 15 mM NH4Cl. Infection was quantitated as the percent [3H]uridine incorporation compared to controls infected in the absence of NH4Cl. Background incorporation by uninfected cells was subtracted from all points. Data represent the mean of three separate experiments for each virus, with standard deviations between 1 and 20%.

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

    Sucrose gradient sedimentation profiles of wt andfus-1 viral spike proteins. 35S-labeled wt (A and C) or fus-1 (B and C) virus was mixed with 1 mM liposomes, treated at the indicated pH for 10 min at 37°C, solubilized in 1.0 to 0.2% NP-40, and adjusted to pH 7.0. The samples were analyzed by centrifugation on 5 to 20% (wt/wt) sucrose gradients in buffer containing 0.1% NP-40. Gradients were centrifuged 22 h at 4°C in an SW41 rotor at 39,000 rpm and fractionated, and radioactivity was quantitated by scintillation counting. Fraction 1 represents the bottom of the gradient. The positions of the monomer (m), dimer (d), and E1 homotrimer (h) peaks are indicated. Recoveries ranged from 53 to 29%.

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

    Sucrose gradient sedimentation profiles offus-1 and fus-1/ic viral spike proteins.35S-labeled fus-1 or fus-1/ic virus was mixed with 1 mM liposomes and treated at the indicated pH for 10 min at 37°C. Samples were solubilized, neutralized, and analyzed by sucrose gradient sedimentation as for Fig. 4. Fraction 1 represents the bottom of the gradient. The positions of the monomer (m), dimer (d), and E1 homotrimer (h) peaks are indicated. Recoveries ranged from 32 to 63%.

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

    pH dependence of E1* conformational changes. (A) Reactivity with MAb E1a-1. 35S-labeled wt andfus-1 ectodomains were treated at the indicated pH for 10 min at 37°C in the presence of 1 mM liposomes, adjusted to neutral pH, solubilized in lysis buffer containing 1% Triton X-100, and immunoprecipitated with MAb E1a-1. E1* precipitation was quantitated by SDS-PAGE and phosphorimaging and compared to total E1* precipitated by a rabbit polyclonal antibody. Data represent the mean of three separate experiments. (B) E1* homotrimer formation.35S-labeled wt SFV and fus-1 ectodomains were treated at the indicated pH for 10 min at 37°C in the presence of liposomes, adjusted to neutral pH, and solubilized in SDS sample buffer for 3 min at 30°C. E1 homotrimer formation was quantitated by SDS-PAGE and phosphorimaging. Data represent means of three separate experiments.

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

    Amino acid sequence differences among wt, mutant, and revertant virus structural proteins. The E2 isoleucine 12 change responsible for the fus-1 phenotype is marked (∗). No amino acid sequence differences were observed between wt/ic and wt SFV or between fus-1 and fus-1/ic. The positions of the SFV structural protein coding regions are marked.

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

    Sequence comparison of the E2 T12 region in alphaviruses. Amino acid numbering is given for SFV E2, starting with residue 1 at the amino terminus. The tetrabasic cleavage site which generates E3 and E2 is boxed, invariant residues in E2 are shown in bold, and sequence gaps are shown as dashes. The sequence of this region of fus-1 is identical to that of wt SFV except for the T12→I change. RRV, Ross River virus; CHICK (Chickungunya virus) (NCBI accession no. 576465); EEV, eastern equine encephalitis virus; OMN, O’Nyong-nyong virus; VEE, Venezuelan equine encephalitis virus; WEE, western equine encephalitis virus; SV, Sindbis virus; AURA, Aura virus; OCK, Ockelbo virus, as listed in references16 and 30.

PreviousNext
Back to top
Download PDF
Citation Tools
fus-1, a pH Shift Mutant of Semliki Forest Virus, Acts by Altering Spike Subunit Interactions via a Mutation in the E2 Subunit
Sallie Glomb-Reinmund, Margaret Kielian
Journal of Virology May 1998, 72 (5) 4281-4287; DOI: 10.1128/JVI.72.5.4281-4287.1998

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.
fus-1, a pH Shift Mutant of Semliki Forest Virus, Acts by Altering Spike Subunit Interactions via a Mutation in the E2 Subunit
(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
fus-1, a pH Shift Mutant of Semliki Forest Virus, Acts by Altering Spike Subunit Interactions via a Mutation in the E2 Subunit
Sallie Glomb-Reinmund, Margaret Kielian
Journal of Virology May 1998, 72 (5) 4281-4287; DOI: 10.1128/JVI.72.5.4281-4287.1998
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

mutation
Semliki Forest virus
Viral Envelope Proteins

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