FUNCTIONAL ANALYSIS OF SIGNAL SEQUENCES OF INFLUENZA VIRUS HEMAGGLUTININ

流感病毒血凝素信号序列的功能分析

• 批准号: 4688500
• 负责人: C J LAI
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/4688500
• 关键词: Orthomyxoviridae; gene expression; genetic manipulation; genetic recombination; hemagglutination test; immunofluorescence technique; kidney cell; microorganism hemagglutinin; mutant; nucleic acid sequence; point mutation; simian virus 40

The amino acid requirements of a functional influenza virus signal peptide were investigated using the influenza hemagglutinin (HA) cDNA-SV40 expression system in African green monkey kidney (AGMK) cells. Local site-specific mutagenesis was carried out to generate a series of recombinants of HA-SV40 containing point mutations in the region of the influenza virus hemagglutinin (HA) gene that codes for the signal peptide sequences. These mutant HA-SV40 recombinants were used to transfect AGMK cells in order to achieve expression of mutant hemagglutinins. Functional characterization of such HA products by cell surface immunofluorescence assay, hemadsorption and analysis of glycosylation showed that a majority of the mutations had no effect on functional properties of HA. However, one isolate (mutant 28) that sustained several mutations including an amino acid substitution at the signal cleavage site was defective with regard to cell surface expression. Amino acid sequence analysis of the NH2-terminus of mutant HA showed that the intracellularly accumulated HA failed to undergo signal cleavage. Also, the defective mutant HA contained only endoglycosidase H sensitive carbohydrate components that are added in the endoplasmic reticulum. These findings suggest that HA containing an uncleaved hydrophobic signal sequence translocates across the microsomal membrane but fails to proceed to the Golgi apparatus where endoglycosidase H resistant carbohydrates are incorporated. Point mutagenesis using a defined oligonucleotide primer has been attempted with the intention of isolating a specific cleavage mutant that will allow us to confirm that the signal cleavage defect present in mutant 28 is indeed responsible for its defect in HA translocation and cell surface expression.

功能性流感病毒信号肽的氨基酸需求