Genetic analysis of the yellow fever virus NS1 protein: identification of a temperature-sensitive mutation which blocks RNA accumulation.
The flavivirus NS1 protein is a highly conserved nonstructural glycoprotein that is capable of eliciting protective immunity. NS1 homodimers are secreted from virus-infected mammalian cells, but the protein is also present at the plasma membrane and in the lumen of intracellular vesicles. Based on these properties, it has been speculated that NS1 may function in virus maturation or release. To gain further insight into NS1 function, we used clustered charged-amino-acid-to-alanine mutagenesis to create 28 clustered substitutions in the NS1 protein of yellow fever virus. To screen for conditional mutations, full-length RNAs containing each mutation were assayed for plaque formation at 32 and 39 degrees C after RNA transfection. We found that 9 mutations were lethal, 18 allowed plaque formation at both temperatures, and 1, ts25, was strongly heat sensitive and was unable to form plaques at 39 degrees C. Lethal mutations clustered in the amino-terminal half of NS1, whereas those leading to impaired replication relative to the parent were distributed throughout the protein. High-multiplicity infections at 39 degrees C demonstrated that ts25 was defective for RNA accumulation, leading to depressed viral protein synthesis and delayed virus production. Although ts25 secreted less NS1 than did the parent, temperature shift experiments failed to demonstrate any temperature-dependent differences in polyprotein processing, NS1 stability and secretion, or release of infectious virus. The ts lesion of ts25 was shown to be due to a single alanine substitution for Arg-299, a residue which is conserved among flaviviruses. These results argue that NS1 plays an essential but as yet undefined role in flavivirus RNA amplification.
Muylaert
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Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093, USA.
Galler
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Rice
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CM
D000409:Alanine; D001120:Arginine; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism; D012367:RNA, Viral / Q000096:biosynthesis*; D013696:Temperature; D014407:Tumor Cells, Cultured; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014764:Viral Proteins / Q000032:analysis; D015005:Yellow fever virus / Q000235:genetics*
C100315:NS1 protein, Flavivirus; D011506:Proteins; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D001120:Arginine; D000409:Alanine