Development of an in vivo assay to identify structural determinants in murine leukemia virus reverse transcriptase important for fidelity
Development of an in vivo assay to identify structural determinants in murine leukemia virus reverse transcriptase important for fidelity
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DOI：
10.1128/jvi.74.1.312-319.2000
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发表时间：
2000-01-01
影响因子：
5.4
通讯作者：
Pathak, VK
中科院分区：
文献类型：
--
作者：
Halvas, EK;Svarovskaia, ES;Pathak, VK
逆转录病毒逆转录酶（RT）的易错DNA合成是逆转录病毒群体变异的主要原因。逆转录病毒RT的结构特征对体内DNA合成的准确性很重要，但尚不清楚。为了鉴定小鼠白血病病毒（MLV）RT的结构元件对体内保真度的重要性，我们开发了一种基于D17的细胞系（ANGIE P），该细胞系被设计为表达嗜中性MLV包膜。- 你好P还含有基于MLV的逆转录病毒载体（GA-1），其编码野生型细菌β-半乳糖苷酶基因（lacZ）和新霉素磷酸转移酶基因。用野生型或突变的MLV gag-pol表达构建体转染ANGIE P细胞产生GA-1病毒，其在感染D17细胞后仅能够经历一个病毒复制周期。通过用5-溴-4-氯-3-吲哚基-β-D-吡喃半乳糖苷（X-Gal）染色来表征感染的D17细胞克隆，并定量lacZ中失活突变的频率。YVDD基序中的三个突变（V223 M、V223 S和V223 A）和RNase H结构域中的两个突变（S526 A和R657 S）显示lacZ失活频率比野生型MLV RT高1.2至2.3倍（P < 0.005）。两个突变（V223 I和Y 598 V）不影响lacZ失活的频率。这些结果建立了一种灵敏的体内测定方法，用于鉴定对DNA合成准确性重要的结构决定因素，并表明几种结构决定因素可能对MLV RT的体内保真度产生影响。
Error-prone DNA synthesis by retroviral reverse transcriptases (RTs) is a major contributor to variation in retroviral populations. Structural features of retroviral RTs that are important fur accuracy of DNA Synthesis in vivo are not known. To identify structural elements of murine leukemia virus (MLV) RT important for fidelity in vivo, we developed a D17-based encapsidating cell line (ANGIE P) which is designed to express the amphotropic MLV envelope. ANGIE: P also contains an MLV-based retroviral vector (GA-1) which encodes a wild-type bacterial beta-galactosidase gene (lacZ) and a neomycin phosphotransferase gene. Transfection of ANGIE P cells with wild-type or mutated MLV gag-pol expression constructs generated GA-1 virus that was able to undergo only one cycle of viral replication upon infection of D17 cells. The infected D17 cell clones were characterized by staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), and the frequencies of inactivating mutations in lacZ were quantified. Three mutations in the YVDD motif (V223M, V223S, and V223A) and two mutations in the RNase H domain (S526A and R657S) exhibited frequencies of lacZ inactivation 1.2- to 2.3-fold higher than that for the wild-type MLV RT (P < 0.005). Two mutations (V223I and Y598V) did not affect the frequency of lacZ inactivation. These results establish a sensitive in vivo assay for identification of structural determinants important for accuracy of DNA synthesis and indicate that several structural determinants may have an effect on the in vivo fidelity of MLV RT.