臺灣博碩士論文加值系統

A型流行性感冒病毒隸屬正黏液病毒科，具有八條單股負極性分節的核醣核酸基因體，在病毒的生命週期中可轉錄出十一個蛋白質。其中第八片段即NS（nonstructural）gene可以轉譯出兩個非結構性蛋白，NS1A與NEP。其中NS1A是最主要之非結構性蛋白，具有高度抗原性，與病毒的複製和抑制宿主干擾素反應有關。前人研究發現，以A型流感病毒H3N2亞型(A/Udron/72)感染細胞，其NS1A蛋白會分佈於核仁中。在本篇的研究，主要探討NS1A分佈情形在其他亞型的A型流感是否具有一致性。我們將A型流感病毒H1N1亞型(A/PR/8/33)的NS1A與S-tag接合(S-NS1A)，以transient expression的模式在細胞中轉錄出融合蛋白，再利用免疫螢光染色觀察病毒蛋白質表現及在細胞中的分佈情形。實驗結果發現，在只有轉染(transfection) 的細胞中，S-NS1A會分佈於粒線體和核仁中，但在轉染後再以病毒感染的細胞中，大部分的S-NS1A只會分佈於核仁中。因此推測NS1A確實會分佈於核仁中，而此分佈可能需要其他病毒蛋白質或核酸參與。

Influenza A virus, a member of the family Orthomyxoviridae, is characterized by segmented RNA genomes of negative polarity. The viral genome consists of eight single-stranded RNA segments which encode 11 proteins. The viral protein NS1A is a major virulence factor during virus infection. Previous studies show that NS1A derived from A/Udron/72 H3N2 strain localized to nucleolus during virus infection. Our aim is to know whether the nucleolar localization of NS1A is a general or a strain specific feature. In this study an N-terminal S-tag-fused NS1A protein (S-NS1A) from A/PR/8/34 H1N1 strain was transiently expressed in MDCK and A549 cells. Immunofluorescence assay (IFA) was performed to observe localization of S-NS1A. It was interesting to note that NS1A localizes to the mitochondria and nucleolus in transfected cells but relocalizes to the nucleolus when transfected cells were infected with virus. This indicates that NS1A has the inherent ability to localize to the nucleolus but its localization could be influenced by other factors directly or indirectly.

摘要…………………………….…………………………………………….…… i
Abstract………………………………………………………………… ii
Background
Influenza virus………………………………………………………………… 1
The life cycle of influenza virus……………………………………………… 2
The NS1 Protein of Influenza A Virus (NS1A Protein)
I. RNA segment 8 of Influenza virus: unspliced and spliced mRNA
encode the NS1 and NEP protein…………………………………….…… 3
II. Functional domains of NS1A ……………………………………….… 4
III. Two nuclear location signals and a latent nuclear export signal in NS1A 5
Reverse genetics………………………………………….…………………… 6
Introduction……………………………………………………………….……… 8
Materials and methods
Cell and Virus…………………………………………………………………. 9
Plasmids………………………………………………………………………. 9
Antibodies and reagents……………………………………………………. 10
Transfection and virus infection……………………………………………. 11
Immunofluorescence assay…………………………………………………… 11
In vitro translation…………………………………………………………….. 12
Reverse genetic system……………………………………………………….. 12
Plaque assay…………………………………………………………………… 13
Result
NS1A and MitoTracker co-localize in the mitochondria in transfected cells… 14
S-NS1A and nucleolin co-localize in the nucleolus in transfected and infected
cells…………………………………………………………………………… 15
What factors affect the mitochondrial localization of NS1A protein…….… 16
A reverse genetics system for influenza virus A/PR/8/34…………………… 16
Discussion……………………………………………………………………..... 19
Figures………………………………………………………………...................... 22
References………………………………………………………………………… 40

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