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

Second-Site Compensatory Mutations of HIV-1 Capsid Mutations

Colleen M. Noviello, Claudia S. López, Ben Kukull, Henry McNett, Amelia Still, Jacob Eccles, Rachel Sloan, Eric Barklis
Colleen M. Noviello
1Department of Microbiology, Oregon Health & Science University, Mail Code L220, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239-3098
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Claudia S. López
1Department of Microbiology, Oregon Health & Science University, Mail Code L220, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239-3098
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ben Kukull
1Department of Microbiology, Oregon Health & Science University, Mail Code L220, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239-3098
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Henry McNett
1Department of Microbiology, Oregon Health & Science University, Mail Code L220, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239-3098
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Amelia Still
2Department of Biochemistry, University of Wisconsin, Madison, Wisconsin
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jacob Eccles
1Department of Microbiology, Oregon Health & Science University, Mail Code L220, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239-3098
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Rachel Sloan
1Department of Microbiology, Oregon Health & Science University, Mail Code L220, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239-3098
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Eric Barklis
1Department of Microbiology, Oregon Health & Science University, Mail Code L220, 3181 SW Sam Jackson Park Road, Portland, Oregon 97239-3098
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: barklis@ohsu.edu
DOI: 10.1128/JVI.00099-11
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

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

    Effects of mutations on virus particle assembly and release. (A) Shown is a pair of CA proteins (Protein Data Bank [PDB] accession no. 3GV2) in HIV-1 CA hexamers viewed roughly from the center of the hexamer outward. NTDs (blue) and CTDs (green) are marked, as are the β-hairpin (β) and cyclophilin A binding (CypA) loops and helices 1 to 11 on one of the capsid proteins. H62 residues are indicated in red. (B) HEK 293T cells were transfected with the indicated HIVLuc constructs. At 72 h posttransfection, virus samples were collected, and Gag proteins were detected after gel electrophoresis by immunoblotting using a primary anti-CA antibody. Full-length PrGag and CA bands are as indicated. (C) For quantification of viral particle release at 72 h posttransfection, virus and cell samples were collected and subjected to electrophoresis and immunoblotting using an anti-CA antibody. Cell and virus Gag levels were quantitated densitometrically; raw virus release levels (Virus Gag/Cell PrGag) were calculated and are shown, normalized to WT HIV release levels. Results derive from two (H62K and H62W), three (H62C and H62F), four (H62Y), five (H62A), or eight (WT) separate experiments. Standard deviations are as shown.

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

    Analysis of virus infectivity. Luciferase gene-transducing HIV viruses were produced by cotransfection of HEK 293T cells with a VSV-G expression vector plus WT and H62 mutant HIVLuc constructs (A) or the indicated HIV variants that derived from selection of H62A or H62F second-site revertants (B). At 3 days posttransfection, virus-containing medium supernatants were collected, filtered through 0.45-μm sterile filters, and used to infect HiJ cells. At 3 days postinfection, HiJ cells were collected and processed for luciferase assays. Results are depicted as normalized luciferase activity signals in infected cells, relative to signals from cells infected with WT HIVLuc virus in parallel: they are not corrected for variations observed in virus particle release. Results are derived from 46 (WT), 16 (H62A and H62F), 2 (H62C and H62F/G208A), 3 (H62K), 4 (H62W and H62Y), 5 (G208A), 8 (G208R and H62F/SP1P10L), and nine (H62A/G208R) separate experiments. Standard deviations are as shown.

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

    Effects of H62 CA mutations on virus-like-particle morphologies. The indicated HIVLuc viruses from transfected cells were sedimented through 20% sucrose cushions, resuspended, adhered to carbon-coated EM grids, and visualized by EM. Conical and tubular cores in panels A, G, and H are indicated by three white arrowheads each. The size bar for all panels is provided at the bottom of panel G, and observed core morphologies are tabulated in Table 1.

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

    In vitro assembly of HIV capsid proteins. WT or variant CA proteins were expressed in bacteria, purified, and employed in in vitro assembly reactions. Following assembly incubations, samples were processed and imaged by EM at low magnification (A, B, D, E, G, H, J, and K; size bar in panel J) or high magnification (C, F, I, and L; size bar in panel L). For each capsid variant, assembly efficiency was assessed by the appearance of capsid tubes, and tube assembly was quantified by tube coverage in EM images as described in Materials and Methods. Average tube coverage percentages, normalized to the WT level, were calculated from 12 pictures each and are as follows: WT, 100%; H62A, 0%; H62A/G208R, 11%; G208R, 68%.

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

    Replication kinetics of HIV mutants. Stocks of WT and CA variant viruses on an NL4-3 HIV background were generated by transfection of constructs into HEK 293T cells and were normalized with respect to Gag levels. CEM-SS (A) or MT4 (B to D) cells were infected in parallel with normalized virus stocks of WT or variant viruses and passaged at 3- to 4-day intervals. Virus spread was monitored by measuring Gag levels in aliquots of infected cells by immunoblotting and densitometry. Replication is plotted as relative Gag level in infected cells versus number of days postinfection.

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

    Proximity of mutant and revertant residues on models of HIV CA. The locations of CTD helices 10 and 11 and residues H62 (red) and G208 (yellow) on pairs of CA proteins in HIV CA hexamers (PDB accession no. 3GV2) are depicted. Panel A is viewed roughly parallel to the plane of capsid coats, with the predicted outer capsid surface up and the inner surface down. Panel B is viewed roughly perpendicular to the capsid surface from the outside. Light and dark blue N-terminal domains and light and dark green C-terminal domains are as labeled.

Tables

  • Figures
  • Table 1.

    Morphologies of HIV particlesa

    Capsid variant% conical or cylindricalNo. viewed
    WT5564
    H62A1952
    H62C1973
    H62K1741
    H62F1724
    H62W2134
    H62Y4715
    H62A/G208R4730
    • ↵a Morphologies of virus-like particles. Virus-like particles from cells transfected with the indicated HIVLuc viruses were sedimented through 20% sucrose cushions, resuspended, and processed for EM. The percentages of particles with discernibly roughly conical or cylindrical cores were determined from the indicated numbers of separate virus particle images.

PreviousNext
Back to top
Download PDF
Citation Tools
Second-Site Compensatory Mutations of HIV-1 Capsid Mutations
Colleen M. Noviello, Claudia S. López, Ben Kukull, Henry McNett, Amelia Still, Jacob Eccles, Rachel Sloan, Eric Barklis
Journal of Virology Apr 2011, 85 (10) 4730-4738; DOI: 10.1128/JVI.00099-11

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.
Second-Site Compensatory Mutations of HIV-1 Capsid Mutations
(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
Second-Site Compensatory Mutations of HIV-1 Capsid Mutations
Colleen M. Noviello, Claudia S. López, Ben Kukull, Henry McNett, Amelia Still, Jacob Eccles, Rachel Sloan, Eric Barklis
Journal of Virology Apr 2011, 85 (10) 4730-4738; DOI: 10.1128/JVI.00099-11
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
  • Top
  • Article
    • ABSTRACT
    • INTRODUCTION
    • MATERIALS AND METHODS
    • RESULTS
    • DISCUSSION
    • ACKNOWLEDGMENTS
    • FOOTNOTES
    • REFERENCES
  • Figures & Data
  • Info & Metrics
  • PDF

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