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
Structure and Assembly

Resistance of Native, Oligomeric Envelope on Simian Immunodeficiency Virus to Digestion by Glycosidases

Robert E. Means, Ronald C. Desrosiers
Robert E. Means
Department of Microbiology and Molecular Genetics, New England Regional Primate Research Center, Harvard Medical School, Southborough, Massachusetts 01772-9102
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ronald C. Desrosiers
Department of Microbiology and Molecular Genetics, New England Regional Primate Research Center, Harvard Medical School, Southborough, Massachusetts 01772-9102
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
DOI: 10.1128/JVI.74.23.11181-11190.2000
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

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

    Schematic of experimental design. Stocks of virus containing both free and virion-bound gp120 were incubated with various glycosidases. After digestion, a portion of the virus was used to test for infectivity and neutralization sensitivity using CEM×174 SIV-SEAP cells. Another portion of the treated virus was subjected to high-speed centrifugation. Supernatant was removed from the resulting pellet, and the gp120 within it was immunoprecipitated with sera from SIVmac239-infected rhesus monkeys. The pellet and immunoprecipitated materials were then analyzed by SDS-PAGE and Western blotting. Ab, antibody.

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

    Mobilities of virion-associated, glycosidase-treated gp120 of SIVmac239 (A) and SIVmac316 (B). Virus stock containing 60 ng of p27 was incubated at 37°C with one of the various glycosidases or buffer alone for 3 h. Each of the samples was then spun for 90 min at high speed in a refrigerated microcentrifuge. Supernatant was drawn off from each sample and set aside for immunoprecipitation experiments. Each of the pellets was boiled for 5 min in loading buffer containing 10% SDS and 1% β-mercaptoethanol. Equal amounts of each sample were then subjected to SDS-PAGE and blotted onto Immobilon-P membranes. The blots were sequentially incubated with a mixture of anti-gp120 monoclonal antibodies (KK43, KK52, and KK54) and a horseradish peroxidase-conjugated anti-mouse IgG antibody. Localization of the antibodies was visualized with a SuperSignal Pico West kit (Pierce) according to the manufacturer's recommendations. Lanes: 1, mock treatment; 2, NgF-treatment; 3, N-glycosidase A treatment; 4, α-mannosidase treatment; 5, eF treatment; 6, endoglycosidase H treatment; 7, endo-β-galactosidase-treatment; 8, neuraminidase-treatment. Numbers on the left indicate the relative positions of molecular mass markers and are shown in kilodaltons. An arrow shows the location of gp120.

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

    Mobilities of nonpelletable, glycosidase-treated gp120 of SIVmac239 (A) and SIVmac316 (B). Supernatants from viral pellets (described in the legend to Fig. 2) were subjected to immunoprecipitation with serum from an SIVmac239-infected rhesus macaque. The samples were then electrophoresed in a 5% polyacrylamide–SDS gel, transferred to a membrane, and reacted with a mixture of anti-gp120 monoclonal antibodies KK43, KK52, and KK54. Lanes: 1, mock treatment; 2, NgF treatment; 3, N-glycosidase A treatment; 4, α-mannosidase treatment; 5, eF treatment; 6, endoglycosidase H treatment; 7, endo-β-galactosidase treatment; 8, neuraminidase treatment. Numbers on the left indicate the relative positions of molecular mass markers (in kilodaltons).

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

    Mobility of SIVmac239 gp120 after SDS denaturation and then glycosidase treatment. Equal amounts of SIVmac239 were subjected to high-speed centrifugation for 90 min at 4°C. The supernatant was removed, and the pellets were resuspended in 10% SDS and 1% β-mercaptoethanol. Samples were boiled for 5 min, and then excess NP-40 was added to complex the SDS. Samples were digested with various glycosidases and then electrophoresed in a 5% polyacrylamide–SDS gel, transferred to a membrane, and reacted with a mixture of anti-gp120 monoclonal antibodies KK43, KK52, and KK54. Antibody localization was visualized by Western blotting as described in Materials and Methods. Lanes: 1, mock treatment; 2, NgF treatment; 3, N-glycosidase A treatment; 4, α-mannosidase treatment; 5, eF treatment; 6, endoglycosidase H treatment; 7, endo-β-galactosidase treatment; 8, neuraminidase treatment. Numbers on the left indicate the relative positions of molecular mass markers and are shown in kilodaltons.

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

    Infectivity of glycosidase-treated virus. After digestion with the indicated glycosidase, SIVmac239 (A) or SIVmac316 (B) was used to infect CEM×174 SIV-SEAP cells. Twofold dilutions of virus were added to equal amounts of CEM×174 SIV-SEAP cells and then transferred to a 37°C CO2 incubator. At approximately 60 h postinfection, the cell-free supernatant was harvested and the amount of SEAP expression was measured as described in Material and Methods. The amount of SEAP expression for each dilution was used to generate a curve from which the amount of SEAP activity per nanogram of p27 was calculated. Panel A shows the average SEAP activity per nanogram of p27 SIVmac239 after treatment with the indicated glycosidase, and panel B shows infectivity of SIVmac316 after treatment with the same glycosidases. The standard error for each experiment is indicated.

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

    Neuraminidase treatment increases the infectivity of SIVmac239-EGFP. Equal amounts of SIVmac239-EGFP were either mock treated, treated with neuraminidase, or treated with NgF. The treated virus was used to infect CEM×174 cells (A) or 221 cells (B). At various time points postinfection, the amounts of EGFP-positive cells were quantitated by flow cytometry analysis.

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

    Effects of neuraminidase on cells. CEM×174 SIV-SEAP cells were treated for 6 h with 40 mU of neuraminidase, which is 10 times the largest amount of neuraminidase present during the measurement of neuraminidase-treated virus in the infectivity assay. These cells were then washed with neuraminidase-free medium and infected with either mock- or neuraminidase-treated SIVmac239. SEAP activity in the medium was assayed at approximately 60 h postinfection as described in Materials and Methods.

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

    Differential effects of sialic acid residues linked α2-3, α2-6, or α2-8 on viral infectivity. SIVmac239 was treated with the indicated neuraminidases as described in Materials and Methods. The treated virus was then used to infect CEM×174 SIV-SEAP cells, and the amount of SEAP activity was quantitated at approximately 60 h postinfection. The standard deviation for each experiment is indicated.

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

    Neutralization of viruses by sera from SIVmac239-infected rhesus macaques. SIVmac239 (A) and SIVmac316 (B), treated with the indicated glycosidases as described in Materials and Methods, were used as virus inocula in SEAP neutralization assays as described in Materials and Methods. From the neutralization curves the amount of sera, pooled from SIVmac239-infected rhesus macaques, required to neutralize 50% of the virus-induced SEAP activity as compared with nonneutralized virus was determined. These results are representative of those from three separate experiments performed with each set of viruses, and the standard deviations are indicated.

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

    Neutralization of viruses by sCD4. Neutralization of SIVmac239 (A) or SIVmac316 (B) was tested as described in the legend to Fig. 9, using sCD4 instead of macaque sera to neutralize viral infectivity. The concentration of sCD4 required to reduce SIVmac239- or SIVmac316-induced SEAP activity by 50% is shown. These results are representative of those from three separate experiments performed with each set of viruses, and the standard deviations are indicated.

  • Fig. 11.
    • Open in new tab
    • Download powerpoint
    Fig. 11.

    Mobility of gp120 from SIVmac239 grown in the presence of glycosylation inhibitors. CEM×174 cells were infected with equal amounts of SIVmac239. At 6 days postinfection, medium alone or medium containing dGJ (1 mM), swainsonine (50 μM), or DANA (1.72 mM) was added to the cultures. The medium was completely replaced with fresh inhibitor-containing medium every 24 h for 3 days. At 10 days postinfection, the cells were washed and resuspended in medium without inhibitor. Cell-free virus stocks were collected 24 h later, and the amount of p27 antigen was quantitated by enzyme-linked immunosorbent assay. Equal amounts of each virus were spun for 90 min at high speed in a microcentrifuge. The supernatant was removed, and gp120 was electrophoresed and visualized as described in the legend to Fig. 2. Lanes: 1 and 5, mock treatment; 2, dGJ treatment; 3, swainsonine treatment; 4, DANA treatment.

  • Fig. 12.
    • Open in new tab
    • Download powerpoint
    Fig. 12.

    Infectivity and neutralization sensitivity of SIVmac239 grown in the presence of glycosylation inhibitors. (A) Stocks of SIVmac239 grown in the presence of the indicated inhibitor were used to generate infectivity curves as described in the legend to Fig. 5. The average amounts of SEAP activity induced per nanogram of p27 of each stock from CEM×174 SIV-SEAP cells are shown, along with the standard error from three separate experiments. (B) The same stocks of virus were used as inocula for SEAP neutralization assays, and the 50% reciprocal neutralization titer of pooled SIVmac239-infected rhesus macaque sera against each stock is shown. Similar results were obtained in three separate experiments, and the standard deviations are indicated.

Tables

  • Figures
  • Table 1.

    Summary of expected and observed shifts in molecular masses of virion-associated, free, and SDS-denatured gp120 for each of the glycosidases tested

    GlycosidaseSpecificityaMobility (kDa) of:Predicted mobility (kDa) of gp120 after complete digestion
    Virion-bound gp120dFree gp120eSDS-denatured gp120f
    NgFR1-GlcNAc-GlcNAc⇓Asnb 110–115806060h
    N-glycosidase AR1-GlcNAc-GlcNAc⇓Asnc 12012012060i
    α-Mannosidaseα1-2,3,6-linked mannose115–120115–120115NPj
    eFR2-GlcNAc⇓GlcNAc-Asn12067–7260g 67k
    Endoglycosidase HR3-GlcNAc⇓GlcNAc-Asn110 75–11097–85NP
    Endo-β-galactosidaseR4-(GlcNAc-Galβ1)n⇓4R5 120110100NP
    NeuraminidaseTerminal α2-3,6,8,9-Neu5Ac120120120105–91l
    • ↵a The arrow indicates the site of cleavage. Asn, asparagine; Gal, galactose; Neu5Ac,N-acetylneuraminic acid; R1, high-mannose, hybrid-, or complex-form oligosaccharides; R2, high-mannose, hybrid-, or biantennary-complex-form oligosaccharides; R3, high-mannose or hybrid-form oligosaccharides; R4, Gal or Neu5Ac-Gal; R5, GlcNAc or Gal.

    • ↵b Blocked by core fucosylation.

    • ↵c Blocked by Neu5Ac.

    • ↵d Mobility of pelletable, glycosidase-treated gp120 as described in the text.

    • ↵e Mobility of immunoprecipitated, nonpelletable, glycosidase-treated gp120 as described in the text.

    • ↵f Mobility of SDS-denatured and then glycosidase-treated gp120 as described in the text.

    • ↵g The mobility of denatured, eF-treated SIVmac239 is greater than predicted because of NgF contamination, as described in the text.

    • ↵h Based on the assumption that all 24 potential N-linked carbohydrates, each contributing about 2.5 kDa, are completely removed.

    • ↵i Based on the assumption that all 24 potential N-linked carbohydrates, each contributing about 2.5 kDa, are completely removed.

    • ↵j NP, not possible to predict.

    • ↵k Based on the assumption that all 24 potential N-linked carbohydrates are removed by cleavage in the chitobiose core, removing about 2.2 kDa each.

    • ↵l Based on the assumption that all 24 potential N-linked carbohydrates have two to four sialic residues, each contributing 309 Da, that are completely removed.

PreviousNext
Back to top
Download PDF
Citation Tools
Resistance of Native, Oligomeric Envelope on Simian Immunodeficiency Virus to Digestion by Glycosidases
Robert E. Means, Ronald C. Desrosiers
Journal of Virology Dec 2000, 74 (23) 11181-11190; DOI: 10.1128/JVI.74.23.11181-11190.2000

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.
Resistance of Native, Oligomeric Envelope on Simian Immunodeficiency Virus to Digestion by Glycosidases
(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
Resistance of Native, Oligomeric Envelope on Simian Immunodeficiency Virus to Digestion by Glycosidases
Robert E. Means, Ronald C. Desrosiers
Journal of Virology Dec 2000, 74 (23) 11181-11190; DOI: 10.1128/JVI.74.23.11181-11190.2000
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

Glycoside Hydrolases
simian immunodeficiency 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