Functional analysis of N-terminal residues of ty1 integrase.
Functional analysis of N-terminal residues of ty1 integrase.
复制标题
ty1整合酶N端残基的功能分析。
DOI：
10.1128/jvi.00159-09
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发表时间：
2009
影响因子：
5.4
通讯作者：
Garfinkel,DavidJ
中科院分区：
文献类型：
--
作者：
Moore,SharonP;Garfinkel,DavidJ
酵母Ty 1逆转录转座子由结构和酶蛋白组成，其功能与逆转录病毒相似。尽管整体序列差异，但某些基序是高度保守的。我们通过突变IN-H22和IN-C55之间的固定组氨酸和半胱氨酸残基以及间插（X32）序列中的13个残基，检查了Ty 1整合酶（IN）锌结合结构域。锌配位组氨酸或半胱氨酸残基的突变使转座减少了4,000倍以上，并导致IN和逆转录酶（RT）不稳定以及蛋白水解加工效率低下。在X32区域中的疏水残基I28、L32、I37和V45的丙氨酸取代使转座减少85至688倍。这些残基中的三个，L32，I37和V45，在逆转录病毒中是高度保守的，尽管它们对整合或病毒感染性的影响尚未被表征。与HHCC突变体相比，所有X32突变体的IN和RT均稳定，蛋白质加工和cDNA生产不受影响。然而，谷胱甘肽S-转移酶下拉和基因内互补分析选定的转座缺陷型X32突变体显示减少IN-IN相互作用。此外，-L32 A和-V45 A突变内的病毒样颗粒在体外没有表现出实质水平的协同整合产物。我们的研究结果表明，组氨酸/半胱氨酸残基是重要的转座前整合的步骤，而疏水残基的功能在IN多聚化。
The Ty1 retrotransposon ofSaccharomyces cerevisiaeis comprised of structural and enzymatic proteins that are functionally similar to those of retroviruses. Despite overall sequence divergence, certain motifs are highly conserved. We have examined the Ty1 integrase (IN) zinc binding domain by mutating the definitive histidine and cysteine residues and thirteen residues in the intervening (X32) sequence between IN-H22 and IN-C55. Mutation of the zinc-coordinating histidine or cysteine residues reduced transposition by more than 4,000-fold and led to IN and reverse transcriptase (RT) instability as well as inefficient proteolytic processing. Alanine substitution of the hydrophobic residues I28, L32, I37 and V45 in the X32region reduced transposition 85- to 688-fold. Three of these residues, L32, I37, and V45, are highly conserved among retroviruses, although their effects on integration or viral infectivity have not been characterized. In contrast to the HHCC mutants, all the X32mutants exhibited stable IN and RT, and protein processing and cDNA production were unaffected. However, glutathioneS-transferase pulldowns and intragenic complementation analysis of selected transposition-defective X32mutants revealed decreased IN-IN interactions. Furthermore, virus-like particles within-L32Aandin-V45Amutations did not exhibit substantial levels of concerted integration products in vitro. Our results suggest that the histidine/cysteine residues are important for steps in transposition prior to integration, while the hydrophobic residues function in IN multimerization.