In Vitro Assembly of Alphavirus Cores by Using Nucleocapsid Protein Expressed in Escherichia coli
| Autor: | Timothy L. Tellinghuisen, Richard J. Kuhn, Agnes E. Hamburger, Bonnie R. Fisher, Ralf Ostendorp |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Sindbis virus
viruses Molecular Sequence Data Immunology Alphavirus Microbiology symbols.namesake Capsid Virology Escherichia coli Ross River virus Electrophoresis Agar Gel chemistry.chemical_classification Base Sequence biology Structure and Assembly Viral Core Proteins Virus Assembly Endoplasmic reticulum Serine Endopeptidases Viral nucleocapsid Nucleocapsid Proteins Golgi apparatus biology.organism_classification Recombinant Proteins Biochemistry chemistry Cytoplasm Insect Science DNA Viral symbols RNA Viral Sindbis Virus Glycoprotein |
| Zdroj: | Journal of Virology. 73:5309-5319 |
| ISSN: | 1098-5514 0022-538X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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