Nucleotide Pools and the Replication of Sindbis Virus

描述(申请人提供)：近400种节肢动物传播的病毒 (虫媒病毒)已被发现，其中许多会导致严重的人类疾病。 蚊媒虫媒病毒Sindbis Virus(SV)及其原型病毒 托格韦里科的艾帕病毒属是这项提案的主题。 细胞内的rNTP是SV RNA的重要构建块 聚合酶合成病毒RNA。然而，人们对此知之甚少 操纵这些底物的水平可以影响复制 SV，或其他细胞质RNA病毒的情况。那里也没有 是否有任何研究表明感染甲型病毒会如何影响 蚊子或脊椎动物宿主细胞中的核苷酸池。我们已经展示了 吡唑呋喃(PZF)，一种阻止嘧啶合成的化合物 核苷酸可抑制病毒在蚊子细胞中的复制。随后，我们 分离出了一株SV VZf‘突变体，与亲本病毒不同，它是 能够在PZF处理过的蚊子细胞中生长；然后我们展示了，正如我们预期的那样， 导致对PZF产生耐药性的突变映射到该序列 编码NSP4(病毒RNA聚合酶)。我们将比较核糖核酸聚合酶 由SVPZF和亲本病毒编码，以测试前者是否 对UTP和CTP具有较低的Km(较高亲和力)。我们还发现，SVPZF具有 第二种表型。它在BHK细胞中受到限制，但限制是 被腺苷缓解了。这一限制与 亚基因组(SG)RNA的合成。单个突变就足以导致 限制表型，但这个单一突变改变了 NSP4和SG启动子的序列。我们建议用实验来解释 SVPZF对BHK细胞的抑制作用及其相关作用 NSP4和SG启动子在产生这种表型过程中的变化。其中一些 这些实验将涉及使用双重SG病毒和SV复制子。我们 还将测试NSP4中的氨基酸是否发生变化，我们认为NSP4 病毒RDRP与UTP和CTP的亲和力，影响病毒的保真度 病毒RDRP。最后，我们将寻找SV的突变体，根据 它们在蚊子细胞中生长的能力只会耗尽CTP，或者只会耗尽UTP， 并与SVPZF的性能进行了比较。拟议的研究将 首次告知我们主机中rNTP池的大小 细胞可以影响病毒RNA聚合酶的活性，以及病毒RNA是如何 聚合酶可以改变，这样它就可以在面对 S。这些研究也将带来更好的 了解调节SG RNA合成的因素。我们要做的是 将学到自甲型病毒表达系统以来将有实际应用 从SG中表达外源基因的情况很好 推动者。我们的发现也将应用于开发 抗病毒治疗。

DESCRIPTION (provided by applicant): Close to 400 arthropod-borne viruses (arboviruses) have been identified, many of which cause serious human disease. Sindbis virus (SV), a mosquito-transmitted arbovirus, and the prototype virus of the family Togaviridae, genus aiphavirus, is the subject of this proposal. The intracellular rNTP's are the vital building blocks from which the SV RNA polymerase synthesizes the viral RNA. Yet, little is known concerning how the manipulation of the levels of theses substrates can affect the replication of SV, or for that matter of other cytoplasmic RNA viruses. Neither have there been any studies indicating how infection with an alphavirus affects the nucleotide pools in either a mosquito or vertebrate host cell. We have shown that pyrazofurin (PZF), a compound that prevents the synthesis of pyrimidine nucleotides inhibits the replication of SV in mosquito cells. Subsequently, we isolated a mutant of SV, SV VZF' which in contrast to the parental virus, is able to grow in PZF-treated mosquito cells; we then showed, as we expected, that mutations responsible for the resistance to PZF mapped to the sequence encoding nsP4 (the viral RNA polymerase). We shall compare the RNA polymerases encoded by SVPZF and by the parental virus, in order to test whether the former has a lower Km (higher affinity) for UTP and CTP. We also found that SVPZF has a second phenotype. It is restricted in BHK cells, but the restriction is relieved by adenosine. The restriction is associated with a decrease in the synthesis of subgenomic (SG) RNA. A single mutation is sufficient to cause the restriction phenotype, but this single mutation changes both an amino acid in nsP4 and the sequence of the SG promoter. Experiments are proposed to explain the restriction of SVPZF in BHK cells, and to evaluate the relative roles of the changes in nsP4 and the SG promoter in producing this phenotype. Some of these experiments will involve the use of double SG virus, and SV replicons. We shall also test whether the amino acid changes in nsP4, which we believe modify the affinity of the viral RDRP for UTP and CTP, affect the fidelity of the viral RDRP. Finally, we shall seek mutants of SV, isolated on the basis of their ability to grow in mosquito cells depleted only of CTP, or only of UTP, and compare their properties with those of SVPZF. The proposed studies will inform us for the first time as to how the size of the rNTP pools in the host cell can affect the activity of the viral RNA polymerase, and how a viral RNA polymerase can change so that it can function efficiently in the face of decreased levels of rNTP' s. These studies will also lead to a better understanding of the factors that regulate the synthesis of SG RNA. What we will learn will have practical application since alphavirus expression systems are being well developed in which foreign genes are expressed from the SG promoter. Our findings will also have application for the development of antiviral therapy.