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In vitro assembly of alphavirus cores by using nucleocapsid protein expressed in Escherichia coli

Academic Article
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Overview

authors

  • Tellinghuisen, Timothy
  • Hamburger, A. E.
  • Fisher, B. R.
  • Ostendorp, R.
  • Kuhn, R. J.

publication date

  • July 1999

journal

  • Journal of Virology  Journal

abstract

  • The production of the alphavirus virion is a multistep event requiring the assembly of the nucleocapsid core in the cytoplasm and the maturation of the glycoproteins in the endoplasmic reticulum and the Golgi apparatus. These components associate during the budding process to produce the mature virion. The nucleocapsid proteins of Sindbis virus and Ross River virus have been produced in a T7-based Escherichia coli expression system and purified. In the presence of single-stranded but not double-stranded nucleic acid, the proteins oligomerize in vitro into core-like particles which resemble the native viral nucleocapsid cores. Despite their similarities, Sindbis virus and Ross River virus capsid proteins do not form mixed core-like particles. Truncated forms of the Sindbis capsid protein were used to establish amino acid requirements for assembly. A capsid protein starting at residue 19 [CP(19-264)] was fully competent for in vitro assembly, whereas proteins with further N-terminal truncations could not support assembly. However, a capsid protein starting at residue 32 or 81 was able to incorporate into particles in the presence of CP(19-264) or could inhibit assembly if its molar ratio relative to CP(19-264) was greater than 1:1. This system provides a basis for the molecular dissection of alphavirus core assembly.

subject areas

  • Base Sequence
  • Capsid
  • DNA, Viral
  • Electrophoresis, Agar Gel
  • Escherichia coli
  • Molecular Sequence Data
  • Nucleocapsid Proteins
  • RNA, Viral
  • Recombinant Proteins
  • Ross River virus
  • Serine Endopeptidases
  • Sindbis Virus
  • Viral Core Proteins
  • Virus Assembly
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Identity

PubMed Central ID

  • PMC112586

International Standard Serial Number (ISSN)

  • 0022-538X

PubMed ID

  • 10364277
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Additional Document Info

start page

  • 5309

end page

  • 5319

volume

  • 73

issue

  • 7

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