Nucleotide Pools and the Replication of Sindbis Virus

核苷酸池和辛德比斯病毒的复制

• 批准号: 6619848
• 负责人: VICTOR STOLLAR
• 金额: $ 23.18万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-28 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6619848
• 关键词: DNA directed RNA polymerase; Sindbis virus; cytosine nucleotides; enzyme activity; host organism interaction; tissue /cell culture; uridine triphosphate; virus protein; virus replication

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.

描述（由申请人提供）：近400种节肢动物传播病毒 （虫媒病毒）已被鉴定，其中许多引起严重的人类疾病。 辛德毕斯病毒（SV），一种蚊子传播的虫媒病毒，以及原型病毒 披膜病毒科的甲病毒属是本提议的主题。 细胞内的rNTP是SV RNA的重要组成部分， 聚合酶合成病毒RNA。但很少有人知道， 对这些底物水平的操纵可以影响 SV，或其它胞质RNA病毒。那里也没有 是否有任何研究表明甲病毒感染如何影响 蚊子或脊椎动物宿主细胞中的核苷酸库。我们已经表明 阿佐弗林（PZF），一种阻止嘧啶合成的化合物， 核苷酸抑制蚊子细胞中SV的复制。后续我们 分离出SV的突变体，SV VZF'，与亲本病毒相反， 能够在PZF处理过的蚊子细胞中生长;然后，正如我们预期的那样， 对PZF产生抗性的突变对应于 编码nsP 4（病毒RNA聚合酶）。我们将比较RNA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（较高的亲和力）。我们还发现SVPZF具有 第二个表型。它在BHK细胞中受到限制，但限制是 由腺苷缓解。这种限制与减少 亚基因组（SG）RNA的合成。一个突变就足以导致 限制性表型，但这个单一的突变改变了一个氨基酸， nsP 4和SG启动子的序列。提出实验来解释 SVPZF在BHK细胞中的限制，并评估SVPZF的相对作用。 nsP 4和SG启动子在产生这种表型中的变化。一些 这些实验将涉及使用双SG病毒和SV复制子。我们 还将测试nsP 4中的氨基酸是否发生变化，我们认为nsP 4修饰了 病毒RDRP对UTP和CTP的亲和力，影响病毒RDRP的保真度。 病毒RDRP。最后，我们将寻找SV的突变体，这些突变体是根据以下条件分离的： 它们在仅耗尽CTP或仅耗尽UTP的蚊子细胞中生长的能力， 并与SVPZF的性质进行了比较。拟议的研究将 首次通知我们主机中rNTP池的大小 细胞可以影响病毒RNA聚合酶的活性， 聚合酶可以改变，以便它可以在面对 降低rNTP的水平。这些研究也将导致更好的 了解调节SG RNA合成的因素。我们 将学习将有实际应用，因为甲病毒表达系统 正在很好地开发，其中外源基因从SG表达 启动子我们的研究结果也将应用于开发 抗病毒治疗

项目成果

A cell-free system for the synthesis of Sindbis virus subgenomic RNA: importance of the concentration of the initiating NTP.

用于合成辛德比斯病毒亚基因组 RNA 的无细胞系统：起始 NTP 浓度的重要性。

• DOI: 10.1016/j.virol.2005.07.007
• 发表时间: 2005
• 期刊: Virology
• 影响因子: 3.7
• 作者: Li,Mei-Ling;Lin,Yen-Huei;Stollar,Victor
• 通讯作者: Stollar,Victor

A mutant of Sindbis virus which is able to replicate in cells with reduced CTP makes a replicase/transcriptase with a decreased Km for CTP.

辛德比斯病毒的一种突变体能够在 CTP 降低的细胞中复制，从而产生 CTP Km 降低的复制酶/转录酶。

• DOI: 10.1128/jvi.78.18.9645-9651.2004
• 发表时间: 2004
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Li,Mei-Ling;Lin,Yen-Huei;Simmonds,HAnne;Stollar,Victor
• 通讯作者: Stollar,Victor