Replication defect of Moloney murine leukemia virus with a mutant reverse transcriptase that can incorporate ribonucleotides and deoxyribonucleotides
Replication defect of Moloney murine leukemia virus with a mutant reverse transcriptase that can incorporate ribonucleotides and deoxyribonucleotides
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DOI：
10.1128/jvi.72.7.5905-5911.1998
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发表时间：
1998-07-01
影响因子：
5.4
通讯作者：
Goff, SP
中科院分区：
文献类型：
--
作者：
Gao, GX;Goff, SP
逆转录酶（RT）在逆转录病毒复制中起关键作用，指导病毒RNA基因组的双链DNA拷贝的合成。我们先前已经描述了莫洛尼鼠白血病病毒的突变体RT，其中F155被缬氨酸取代，我们证明了这种取代允许酶掺入核糖核苷酸形成RNA，同时仍然保留其正常的掺入能力，当引入病毒基因组时，这种突变使病毒无法复制。对突变病毒的鉴定表明，该酶仍具有活性，能合成负链强终止DNA和一些较长的产物，但不能合成全长负链DNA。我们认为，该酶不能在体内完成DNA合成的原因是它能将核糖核苷酸掺入到产物中，这些核苷酸对DNA合成起抑制作用，我们还测试了其他7个氨基酸残基在病毒复制中取代F155的能力;其中，只有酪氨酸可以支持病毒复制。在尝试选择第二位点抑制突变时，对F155 V突变体进行随机诱变，并将其用作分离回复突变病毒的亲本。发现两个独立的回复突变体已将位置155处的缬氨酸残基变回野生型苯丙氨酸。这些结果表明，在这个位置上的芳香环是重要的病毒复制。
Reverse transcriptase (RT) plays a critical role in retrovirus replication, directing the synthesis of a double-stranded DNA copy of the viral RNA genome, We have previously described a mutant RT of the Moloney murine leukemia virus in which F155 was replaced by valine, and we demonstrated that this substitution allowed the enzyme to incorporate ribonucleotides to form RNA while still retaining its normal ability to incorporate deoxyribonucleotides to form DNA, When introduced into the viral genome, this mutation rendered the virus incapable of replication. Characterization of the mutant virus revealed that the enzyme was still active and able to synthesize minus-strand strong stop DNA and some longer products but failed to make full-length minus-strand DNA, We propose that the failure of the enzyme to complete DNA synthesis in vivo resulted from its ability to incorporate ribonucleotides into the products, which served as inhibitors for DNA synthesis, We also tested seven other amino acid residues for their abilities to substitute for F155 in virus replication; of these, only tyrosine could support virus replication. In an attempt to select for second-site suppressor mutations, the F155V mutant was subjected to random mutagenesis and was used as a parent for the isolation of revertant viruses. Two independent revertants were found to have changed the valine residue at position 155 back to the wild type phenylalanine. These results suggest that an aromatic ring at this position is important for virus replication.