A MUTANT MURINE LEUKEMIA-VIRUS WITH A SINGLE MISSENSE CODON IN POL IS DEFECTIVE IN A FUNCTION AFFECTING INTEGRATION
A MUTANT MURINE LEUKEMIA-VIRUS WITH A SINGLE MISSENSE CODON IN POL IS DEFECTIVE IN A FUNCTION AFFECTING INTEGRATION
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DOI：
10.1073/pnas.81.20.6461
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发表时间：
1984-01-01
期刊：
影响因子：
--
通讯作者：
VARMUS, HE
中科院分区：
文献类型：
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作者：
DONEHOWER, LA;VARMUS, HE
使用克隆的莫洛尼鼠白血病病毒（MuLV）DNA的定点诱变来确定病毒pol基因3“区编码的功能，该功能是病毒DNA有效整合所需的。一种突变体MuLV-SF 1含有一个碱基替换（碱基495处的C变为T），导致逆转录病毒中高度保守的区域中精氨酸变为半胱氨酸。通过与单纯疱疹病毒胸苷激酶基因（HSV tk）共转染引入大鼠细胞的突变体DNA，指导具有逆转录酶活性的病毒颗粒的产生。用这些颗粒感染细胞导致未整合的病毒DNA的全长线性和环状形式的合成;当通过DNA杂交检查时，整合的病毒DNA减少至少10倍，并且突变体颗粒在建立至少300倍的感染方面比野生型病毒效率低。用MuLV-SF 1的蛋白质和携带HSV tk基因的复制缺陷标记MuLV的基因组形成的假型在产生tk+菌落方面比用野生型病毒编码的蛋白质形成的假型的有效性低至少100倍。当MuLV-SF 1假型确实产生tk+细胞时，大多数前病毒异常整合。显然，MuLV-SF 1 pol基因在正常整合重组所需的功能方面存在缺陷，并且与DNA合成分离。
Site-directed mutagenesis of cloned Moloney murine leukemia virus (MuLV) DNA was used to define a function encoded in the 3'' region of the viral pol gene and required for efficient integration of viral DNA. One mutant, MuLV-SF1, contained a single base substitution (C to T at base 495) that resulted in an arginine to cysteine change in a region highly conserved among retroviruses. Mutant DNA, introduced into rat cells by cotransfection with a herpes simplex virus thymidine kinase gene (HSV tk), directed production of virus particles with reverse transcriptase activity. Infection of cells with these particles led to synthesis of full-length linear and circular forms of unintegrated viral full-length linear and circular forms of unintegrated viral DNA; integrated viral DNA was decreased at least by a factor of 10 when examined by DNA hybridization, and the mutant particles were less efficient than wild-type virus at establishing an infection by a factor of at least 300. Pseudotypes formed with the proteins of MuLV-SF1 and the genome of a replication defective marker MuLV, carrying the HSV tk gene, were less effective by at least a factor of 100 in producing tk+ colonies than pseudotypes formed with proteins encoded by wild-type virus. When the MuLV-SF1 pseudotypes did produce tk+ cells, most of the proviruses were integrated aberrantly. Evidently, the MuLV-SF1 pol gene is defective for a function that is required for normal integrative recombination and dissociable from DNA synthesis.