Jørgensen E C, Kjeldgaard N O, Pedersen F S, Jørgensen P
Institute of Molecular Biology and Plant Physiology, Aarhus University, Denmark.
J Virol. 1988 Sep;62(9):3217-23. doi: 10.1128/JVI.62.9.3217-3223.1988.
The endogenous ecotropic provirus Emv-3 present in DBA/2 mice is poorly expressed in the animal, as well as in cell cultures. Transfection of proviral DNA into NIH 3T3 cells localized the expression defect to the 5' region of the viral genome, spanning the untranslated region and the N-terminal part of the gag gene. Comparison of the nucleotide sequence of the Emv-3 provirus with the sequence of the highly infectious Akv murine leukemia virus revealed three nucleotide differences within the gag coding region. One of these differences was found in codon 3 of the gag polyprotein, where a Gln codon is seen in Akv and a Pro codon is differences was found in codon 3 of the gag polyprotein, where a Gln codon is seen in Akv and a Pro codon is seen in Emv-3. By site-directed mutagenesis, we showed that the defect of Emv-3 expression indeed is localized to codon 3 of the gag gene. The gag polyprotein of mammalian type C retrovirus contains myristic acid covalently linked to the N-terminal glycine. This myristylation is not seen in the Emv-3-coded gag polyprotein. We showed that the in vitro-mutagenized Emv-3 genome containing a Gln codon at position 3 of the gag gene yields a myristylated gag polyprotein. Thus, it seems most likely that the defect of expression of the Emv-3 provirus is due to the presence of a proline is position 3 of the gag polyprotein, preventing the myristylation.
DBA/2小鼠体内存在的内源性亲嗜性前病毒Emv-3在动物以及细胞培养物中表达水平很低。将前病毒DNA转染到NIH 3T3细胞中，发现表达缺陷定位于病毒基因组的5'区域，该区域跨越非翻译区和gag基因的N端部分。将Emv-3前病毒的核苷酸序列与高感染性的Akv鼠白血病病毒的序列进行比较，发现在gag编码区内有三个核苷酸差异。其中一个差异出现在gag多聚蛋白的第3密码子处，在Akv中该密码子为谷氨酰胺密码子，而在Emv-3中为脯氨酸密码子。通过定点诱变，我们证明Emv-3表达缺陷确实定位于gag基因的第3密码子。哺乳动物C型逆转录病毒的gag多聚蛋白含有与N端甘氨酸共价连接的肉豆蔻酸。在Emv-3编码的gag多聚蛋白中未观察到这种肉豆蔻酰化。我们表明，在gag基因第3位含有谷氨酰胺密码子的体外诱变Emv-3基因组产生了一种肉豆蔻酰化的gag多聚蛋白。因此，Emv-3前病毒表达缺陷最有可能是由于gag多聚蛋白第3位存在脯氨酸，从而阻止了肉豆蔻酰化。
