COMPOSITION, ASSEMBLY AND REPLICATION OF RNA VIRUSES

我们的实验室将研究甲型流感病毒 RNA依赖性RNA聚合酶被修饰， （病毒特异性mRNA的合成，其被加帽、甲基化和 多腺苷酸化）复制（合成全长（+）和（-）正义 RNA）。 我们将利用最近的一项重大突破， 使我们能够重建体外RNA合成系统， 与NP组装的流感RNA（全长和截短的T7转录物） 作为流感病毒转录酶亚单位模板的蛋白质 从病毒体RNP或从具有高水平的RNA的Sf 9细胞纯化。 从杆状病毒重组体表达。 我们将研究 分离纯化的聚合酶亚基（PB 1，PB 2，PA）的功能， 分配帽核酸内切酶，酪蛋白激酶，ATP酶，GTP结合，起始和 延伸和启动子结合功能。 我们亦会研究 全酶与（+）和（-）正义3'末端的相互作用 使用合成的寡核糖核苷酸和重构的流感模板 RNP复合物，以及酶的引发和延伸。 通过使用 嵌合流感病毒RNA构建体，其含有 一个基因组片段和另一个基因组片段的下游编码序列， 在体外检测流感病毒的转录和翻译控制。 这种重组的流感RNA合成系统也将使我们能够 探索流感聚合酶的错误频率， A型和B型流感病毒的特异性，这阻止了它们的重配。 使用含有流感特异性3'和5'核苷酸的重构RNP， 末端和插入的报告基因（荧光素酶）作为ssRNA（-）正义 构建在RNP复合物中，我们将检测流感病毒（wt）感染 细胞以观察这种含有报告基因的构建体是否可以被转录， 复制并包装成病毒体。 来自流感病毒感染细胞的核提取物，用 放线菌酮或α-鹅膏苷来抑制复制或转录 以及针对聚合酶亚单位和流感病毒的抗血清 将利用非结构蛋白来试图确定 参与从蛋白质转变的酶修饰的性质 从转录到复制， 这一转变中的因素。

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.