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

Importance of both the Coding and the Segment-Specific Noncoding Regions of the Influenza A Virus NS Segment for Its Efficient Incorporation into Virions

Ken Fujii, Yutaka Fujii, Takeshi Noda, Yukiko Muramoto, Tokiko Watanabe, Ayato Takada, Hideo Goto, Taisuke Horimoto, Yoshihiro Kawaoka
Ken Fujii
1Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo
2Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Saitama
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Yutaka Fujii
3Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Takeshi Noda
4Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Yukiko Muramoto
4Laboratory of Microbiology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Tokiko Watanabe
1Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo
2Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Saitama
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Ayato Takada
1Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo
2Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Saitama
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Hideo Goto
1Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo
2Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Saitama
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Taisuke Horimoto
1Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo
2Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Saitama
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Yoshihiro Kawaoka
1Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo
2Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Saitama
5Department of Pathological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • For correspondence: kawaoka@ims.u-tokyo.ac.jp
DOI: 10.1128/JVI.79.6.3766-3774.2005
  • Article
  • Figures & Data
  • Info & Metrics
  • PDF
Loading

Article Figures & Data

Figures

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

    Schematic representation of the system used to determine the efficiency of vRNA incorporation into virions. 293T cells were transfected with plasmids for production of influenza virus VLPs. Forty-eight hours later, aliquots of the supernatant were used to infect MDCK cells. To determine efficiency of incorporation of mutant NS vRNA segments, GFP- and NP-expressing cells were counted at 24 h postinfection.

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

    Efficiency of incorporation of recombinant NS segments. Incorporation efficiencies of recombinant NS segments with different lengths of NS coding regions were determined as described in the legend to Fig. 1. The noncoding and coding regions of the NS vRNA are represented by grey and white bars, respectively, while the green bars represent the coding region of the GFP gene. The dotted line represents deleted sequences of the NS coding region. The results shown are representative data from three independent experiments.

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

    Efficiencies of incorporation of NS segments with nucleotide substitutions in the NS coding regions. The nucleotide sequences at the 3′ (positions 27 to 56) or 5′ (positions 832 to 864) end of the NS coding region are shown. The silent mutations introduced are shown by asterisks. The coding regions of the NS vRNA and GFP are represented by white and green bars, respectively. The results shown are representative data for three independent experiments. (A) Efficiencies of incorporation of the NS-GFP segment with nucleotide substitutions in the NS coding regions. Incorporation efficiencies were determined as described in the legend to Fig. 1. (B) Efficiencies of incorporation of the NS segment with nucleotide substitutions in the NS coding regions. To determine the incorporation efficiency of mutant NS vRNA segments, NS2- and NP-expressing cells were counted at 24 h postinfection.

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

    Growth properties of mutant viruses containing silent mutations in the coding regions of the NS segment. MDCK cells were infected with virus at an multiplicity of infection of 0.001. At the indicated times after infection, the virus titer in the supernatant was determined. The values are means and standard deviations from triplicate experiments. •, m1; ○, m2; ▪, m3; □, wild type.

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

    Importance of NS segment-specific noncoding regions for incorporation of recombinant NS segments. (A) Schematic diagram of the NS vRNA. Segment-specific complementary sequences are underlined. The poly(U) sequence for the addition of the poly(A) tail to the mRNA is shown in italics. The grey boxes represent U12 and U13. (B) Efficiencies of incorporation of recombinant NS segments with deletions in the NS segment-specific noncoding regions. U12 and U13 are represented by grey boxes. The coding regions of the NS vRNA and GFP gene are represented by white and green bars, respectively. The dotted lines represent sequences deleted from the NS segment. The results shown are representative data from three independent experiments.

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

    Comparison of test NS vRNA amounts and GFP expression levels in plasmid-transfected cells. (A) Quantitative analysis of NS(0)GFP(0), NSΔNTR, and NS(150)GFP(150) vRNAs produced from PolI plasmids in 293T cells by real-time quantitative reverse transcription-PCR. 293T cells (106 cells) were transfected with 18 plasmids. At 48 h posttransfection, total RNA was extracted from 293T cells and quantified by real-time PCR with GFP and NP sequence-specific primers and probes as described in Materials and Methods. The primers and probes for the NS-GFP segments detect NS-GFP but not the authentic NS segment. Experiments were performed in the presence (+) or absence (−) of reverse transcriptase (RT) to demonstrate that the results are not affected by the plasmids used for the experiments. NS2KO represents NS2 knockout virus, which does not produce NS2(NEP) protein and does not undergo multiple cycles of replication (27). It therefore serves as a negative control for detection of NS-GFP and a positive control for the NP segment. The results shown are representative data from three independent experiments. Error bars indicate standard deviations. (B) Comparison of GFP expression levels in PolI plasmid-transfected 293T cells by fluorescence microscopy. At 48 h after transfection, the cells were observed by fluorescence microscopy.

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

    Sequence alignment of the NS incorporation signals among influenza A viruses. The red, orange, and purple lines represent U12, incorporation signal, and U13, respectively. The conserved sequences among all strains are shown in black. The NS sequences were obtained from the influenza A virus sequence database at Los Alamos National Laboratory. (A) Comparison of the incorporation signals among allele A NS segments. Human viruses: WSN, A/WSN/33; PR834, A/Puerto Rico/8/34; HK481, A/Hong Kong/481/97; FM47, A/Fort Monmouth/1/47; AL77, A/Alaska/6/77; SWCO77, A/swine/Colorado/1/77; BM18, A/Brevig Mission/1/18. Eurasian avian viruses: SWNE85, A/swine/Netherlands/12/85; SWCH78, A/swine/China/8/78. North American avian viruses: RTNJ85, A/ruddy turnstone/New Jersey/47/85; WHME84, A/whale/Marine/328/84; GDE471-86, A/gull/Delaware/471/86. Equine viruses: EQKY76, A/equine/Kentucky/76; EQCO76, A/equine/Cordaba/4/76. Classic swine viruses: SWIA30, A/swine/Iowa/15/30; SWIA88, A/swine/Italy/671/87. EQPR56-like equine viruses: EQPR56, A/equine/Prague/1/56; EQDET64, A/equine/Detroit/3/64. Gull viruses: GDE87, A/gull/Delaware/2838/87; GMN81, A/gull/Minnesota/1352/81. (B) Comparison of the incorporation signals among allele B NS segments. DKAL60, A/duck/Alberta/60/76; MAL827-78, A/mallard/Alberta/827/78; MAL88-76, A/mallard/Alberta/88/76; PAL121-79, A/pintail/Alberta/121/79; PAL358-79, A/pintail/Alberta/358/79. The sequence data for BM18, MAL827-78, MAL88-76, PAL121-79, and PAL358-79 are not complete in the influenza virus database.

PreviousNext
Back to top
Download PDF
Citation Tools
Importance of both the Coding and the Segment-Specific Noncoding Regions of the Influenza A Virus NS Segment for Its Efficient Incorporation into Virions
Ken Fujii, Yutaka Fujii, Takeshi Noda, Yukiko Muramoto, Tokiko Watanabe, Ayato Takada, Hideo Goto, Taisuke Horimoto, Yoshihiro Kawaoka
Journal of Virology Feb 2005, 79 (6) 3766-3774; DOI: 10.1128/JVI.79.6.3766-3774.2005

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.
Importance of both the Coding and the Segment-Specific Noncoding Regions of the Influenza A Virus NS Segment for Its Efficient Incorporation into Virions
(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
Importance of both the Coding and the Segment-Specific Noncoding Regions of the Influenza A Virus NS Segment for Its Efficient Incorporation into Virions
Ken Fujii, Yutaka Fujii, Takeshi Noda, Yukiko Muramoto, Tokiko Watanabe, Ayato Takada, Hideo Goto, Taisuke Horimoto, Yoshihiro Kawaoka
Journal of Virology Feb 2005, 79 (6) 3766-3774; DOI: 10.1128/JVI.79.6.3766-3774.2005
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

influenza A virus
RNA, Viral
viral nonstructural proteins
virus assembly

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