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.
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.
复制标题
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
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发表时间：
2004
影响因子：
5.4
通讯作者：
Stollar,Victor
中科院分区：
文献类型：
--
作者：
Li,Mei-Ling;Lin,Yen-Huei;Simmonds,HAnne;Stollar,Victor
我们先前报道了辛德毕斯病毒突变体SVPZF的分离和特征，该突变体可以在用干扰嘧啶生物合成的化合物阿佐弗林（PZF）处理的蚊子细胞中生长（Y. H. Lin，P. Yadav，R. Ravatn和V. Stollar，Virology 272：61-71，2000; Y. H. Lin，H. A. Simmonds和V. Stollar，Virology 292：78-86，2002）。病毒RNA聚合酶nsP 4中的三个氨基酸变化是产生这种表型所必需的。我们现在描述一个突变的辛德毕斯病毒，SVCPC，这是耐环戊烯胞嘧啶（CPC），一种化合物，只干扰CTP的合成。因此，与SVPZF相反，选择SVPZF是因为其在具有低水平UTP和CTP的蚊子细胞中生长的能力，选择SVCPC是因为其在仅CTP水平降低的细胞中生长的能力。SVPZF与CPC交叉耐药，而SVCPC对PZF不耐药。只有一个氨基酸的变化，nsP 4，Leu 585苯丙氨酸，需要的CPC抗性表型。SVCPC感染的蚊子细胞中产生的病毒复制酶/转录酶对CTP的Km值低于SVSTD感染的蚊子细胞中产生的酶对UTP的Km值。SVPZF和SVCPCrepresent的第一个例子的病毒突变体选择的能力，在细胞中生长的低水平的核糖核苷三磷酸（rNTPs）。对这些突变体的进一步研究和nsP 4结构的确定应该证明RNA依赖性RNA聚合酶的改变如何使其在rNTPs水平异常低的细胞中发挥作用。
We reported earlier the isolation and characterization of a Sindbis virus mutant, SVPZF, that can grow in mosquito cells treated with pyrazofurin (PZF), a compound that interferes with pyrimidine biosynthesis (Y. H. Lin, P. Yadav, R. Ravatn, and V. Stollar, Virology 272:61-71, 2000; Y. H. Lin, H. A. Simmonds, and V. Stollar, Virology 292:78-86, 2002). Three amino acid changes in nsP4, the viral RNA polymerase, were required to produce this phenotype. We now describe a mutant of Sindbis virus, SVCPC, that is resistant to cyclopentenylcytosine (CPC), a compound that interferes only with the synthesis of CTP. Thus, in contrast to SVPZF, which was selected for its ability to grow in mosquito cells with low levels of UTP and CTP, SVCPCwas selected for its ability to grow in cells in which only the level of CTP was reduced. Although SVPZFwas cross-resistant to CPC, SVCPCwas not resistant to PZF. Only one amino acid change in nsP4, Leu 585 to Phe, was required for the CPC resistance phenotype. The viral replicase/transcriptase generated in SVCPC-infected mosquito cells had a lowerKmfor CTP (but not for UTP) than did the enzyme made in SVSTD-infected mosquito cells. SVPZFand SVCPCrepresent the first examples of viral mutants selected for the ability to grow in cells with low levels of ribonucleoside triphosphates (rNTPs). Further study of these mutants and determination of the structure of nsP4 should demonstrate how alterations in an RNA-dependent RNA polymerase permit it to function in cells with abnormally low levels of rNTPs.