COMPOSITION, ASSEMBLY AND REPLICATION OF RNA VIRUSES

我们实验室将研究甲型流感病毒感染的机制(S) RNA依赖的RNA聚合酶被修改为从转录转换 (病毒特异的mRNAs的合成，这些mRNAs被封顶、甲基化和 多腺苷)到复制(全长(+)和(-)义的合成 RNAs)。我们将利用最近的一项重大突破，它应该 使我们能够重建包含以下内容的体外RNA合成系统 与NP组装的流感RNA(全长和截短的T7转录本) 作为流感转录酶亚基模板的蛋白质 从病毒粒子RNPs或从高水平的Sf9细胞纯化 杆状病毒重组体的表达。我们将研究生物化学 分离纯化的聚合酶亚基(PB1、PB2、PA)的功能 指认CAP内切酶、酪蛋白激酶、ATPase、GTP结合、起始和 伸长和启动子结合的功能。我们亦会研究 全酶与(+)和(-)义3‘端的相互作用 使用合成寡核苷酸和重组的流感模板 RNP复合体，以及酶的引发和延伸。通过使用 含流感病毒3‘端非翻译区的嵌合RNA构建 一个基因组片段和来自另一个基因组片段的下游编码序列 在体外检查流感转录和翻译对照。 这个重组的流感RNA合成系统也将使我们能够 探讨流感聚合酶的错误频率，以及 甲型和乙型流感的特殊性，禁止它们重新分类。 使用含有流感特异性3‘和5’的重组RNPs TERMINI和插入的报告基因(荧光素酶)作为单链RNA(-)正义 在RNP复合体中构建我们将感染流感病毒(Wt)的 细胞，看看这个含有报告基因的结构是否可以转录， 复制并打包成病毒粒子。 流感病毒感染细胞的核提取物，用以下任一种方法处理 用于抑制复制或转录的放线菌亚胺或α-氨基丁酸 和抗多聚酶亚基和流感的抗血清。 非结构蛋白将被用来试图确定 性质的酶修饰(S)参与了从 转录到复制与细胞的可能参与 影响这一转变的因素。

Our laboratory will study the mechanism(s) by which the influenza A virus RNA-dependent RNA polymerase is modified to switch from transcription (synthesis of virus-specific mRNAs which are capped, methylated and polyadenylated) to replication (synthesis of full length (+) and (-) sense RNAs). We will take advantage of a recent major breakthrough which should enable us to reconstitute an in vitro RNA synthesis system containing influenza RNA (full length and truncated T7 transcripts) assembled with NP protein which serves as template for influenza transcriptase subunits purified from either virion RNPs or from Sf9 cells with high levels of expression from baculovirus recombinants. We will study the biochemical functions of the isolated purified polymerase subunits (PB1, PB2, PA), to assign cap endonuclease, casein kinase, ATPase, GTP-binding, initiation and elongation, and promoter binding functions. We will also study the interaction of the holoenzyme with the 3' termini of (+) and (-) sense influenza templates using synthetic oligoribonucleotides and reconstituted RNP complexes, and initiation and elongation by the enzyme. By using chimeric influenza RNA constructs containing the 3' untranslated region of one genome segment and the downstream coding sequence from another we will examine influenza transcriptional and translational controls in vitro. This reconstituted system for influenza RNA synthesis will also enable us to explore the error frequency of the influenza polymerase, and the specificities of influenza A and B which prohibit their reassortment. Using reconstituted RNPs which contain the influenza-specific 3' and 5' termini and an inserted reporter gene (luciferase) as ssRNA (-) sense constructs in RNP complexes we will transfect influenza virus (wt) infected cells to see if this reporter gene-containing construct can be transcribed, replicated and packaged into virions. Nuclear extracts from influenza virus infected-cells, treated with either cycloheximide or alpha-amanatin to inhibit replication or transcription respectively, and anti-sera against polymerase subunits and influenza nonstructural proteins will be utilized in an attempt to determine the nature of the enzyme modification(s) involved in the switch from transcription to replication and the possible involvement of cellular factors in this switch.