A mutant of Sindbis virus that is resistant to pyrazofurin encodes an altered RNA polymerase
A mutant of Sindbis virus that is resistant to pyrazofurin encodes an altered RNA polymerase
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DOI：
10.1006/viro.2000.0329
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发表时间：
2000-06-20
期刊：
影响因子：
3.7
通讯作者：
Stollar, V
中科院分区：
文献类型：
--
作者：
Lin, YH;Yadav, P;Stollar, V
吡唑呋林 (PZF) 是一种胞苷类似物和乳清酸单磷酸脱羧酶抑制剂，已被证明可以降低处理细胞中 UTP 和 CTP 的水平。当用 PZF 处理感染辛德毕斯病毒 (SV) 的白纹伊蚊细胞时，病毒产量减少 100 至 1000 倍。通过我们在 Ae 中的标准 SVSTD 的连续传递。在白纹伊蚊细胞中存在浓度不断增加的 PZF 时，衍生出一种突变体 SVPZF，该突变体不受 PZF 抑制。 SVPZF 还对腺苷、鸟苷和膦酰乙酰基-N-天冬氨酸具有抗性，所有这些都已被证明可以降低 UTP 和 CTP 的水平。对含有 SVPZF 和亲本基因组序列的嵌合病毒的分析表明，nt 5262 和 7999 之间的序列赋予 PZF 抗性表型。该区域的测序发现了四个突变（nt 5750、6627、7543 和 7593），预计这些突变会导致氨基酸变化：nsP3 中的 opal550L 以及 nsP4 中的 M287L、K592I 和 P609T。含有一种或多种这些突变的病毒的表征表明，nsP4 编码区的所有三种突变都是产生对 PZF 的完全抗性所必需的。使用基于 HIV 逆转录酶结构的 nsP4 分子模型，我们将氨基酸变化 M287L 定位在病毒 RNA 聚合酶的手指结构域的尖端，将 K592I 和 P609T 定位在病毒 RNA 聚合酶的拇指结构域的基部。我们认为 nsP4 中的这三个氨基酸变化改变了 NTP 结合袋的几何形状，从而增加了酶对 CTP 和 UTP 的亲和力，(C) 2000 学术出版社。
Pyrazofurin (PZF), a cytidine analog and an inhibitor of orotate monophosphate decarboxylase, has been shown to decrease the levels of UTP and CTP in treated cells. When Sindbis Virus (SV)-infected Aedes albopictus cells were treated with PZF, the yield of Virus was reduced 100- to 1000-fold. By serial passage of our standard SVSTD in Ae. albopictus cells in the presence of increasing concentrations of PZF, a mutant, SVPZF was derived, which was not inhibited by PZF. SVPZF is also resistant to adenosine, guanosine, and phosphono-acetyl-N-aspartate, all of which have been shown to decrease levels of UTP and CTP. Analysis of chimeric Viruses containing sequences from the SVPZF and parental genomes showed that the sequence between nt 5262 and 7999 conferred the PZF-resistant phenotype. Sequencing of this region identified four mutations (nt 5750, 6627 7543, and 7593), which are predicted to lead to amino acid changes: opal550L in nsP3 and M287L, K592I, and P609T in nsP4. Characterization of viruses containing one or more of these mutations demonstrated that all three mutations in the nsP4 coding region are required to produce full resistance to PZF. Using a molecular model of nsP4 based on the structure of HIV reverse transcriptase, we located amino acid change M287L at the tip of the fingers domain and K592I and P609T at the base of the thumb domain of the viral RNA polymerase. We suggest that these three amino acid changes in nsP4 alter the geometry of the NTP binding pocket so as to increase the affinity of the enzyme for CTP and UTP, (C) 2000 Academic Press.