Reichert M, Winnicka A, Willems L, Kettmann R, Cantor G H
National Veterinary Research Institute, Pulawy, Warsaw, Poland.
J Virol. 2001 Sep;75(17):8082-9. doi: 10.1128/jvi.75.17.8082-8089.2001.
The cytoplasmic tail of bovine leukemia virus (BLV) transmembrane protein gp30 has multiple amino acid motifs that mimic those present in signaling proteins associated with B-cell and T-cell receptors. The proline-rich motif of gp30, PX(2)PX(4-5)P, is analogous to the recognition site of Src homology 3 (SH3) domains of signaling molecules. Using site-directed mutagenesis of an infectious molecular clone of BLV, point mutations were introduced which changed three of the prolines of the motif to alanines. The influence of these mutations on the pathogenicity of BLV was studied in sheep which received either (i) plasmid DNA with provirus containing proline-to-alanine mutations (pppBLV), (ii) plasmid DNA with wild-type provirus (wtBLV), or (iii) transfection reagent alone. Although all of the BLV-injected animals seroconverted at approximately the same time, viral loads at later time points were high in five of five of the wtBLV group and two of five of the pppBLV group but low in three of five of the pppBLV group, as determined by semiquantitative PCR. Viral expression was lower in the pppBLV-transfected sheep, as measured by p24 antigen enzyme-linked immunosorbent assay in cultured cells, and serologic titers were lower. Thirty-one months after transfection, four of four wtBLV-transfected sheep had died of leukemia and lymphoma, and all five of the pppBLV-transfected sheep were clinically healthy and had normal peripheral blood lymphocyte counts. These data indicate that the proline-rich motif of gp30 is not required for viral infectivity but is important for high viral load in vivo, suggesting that SH3-mediated gp30 interactions are critical for viral pathogenesis following infection. Absence of interactions with the proline-rich motif may prevent or delay tumorigenesis in sheep.
牛白血病病毒（BLV）跨膜蛋白gp30的胞质尾具有多个氨基酸基序，这些基序与B细胞和T细胞受体相关的信号蛋白中的基序相似。gp30富含脯氨酸的基序PX(2)PX(4-5)P类似于信号分子Src同源3（SH3）结构域的识别位点。利用BLV感染性分子克隆的定点诱变技术，引入点突变，将该基序中的三个脯氨酸突变为丙氨酸。在绵羊中研究了这些突变对BLV致病性的影响，这些绵羊分别接受了（i）含有脯氨酸到丙氨酸突变的前病毒的质粒DNA（pppBLV）、（ii）含有野生型前病毒的质粒DNA（wtBLV）或（iii）单独的转染试剂。尽管所有注射BLV的动物大约在同一时间血清转化，但通过半定量PCR测定，wtBLV组的五只动物中有五只以及pppBLV组的五只动物中有两只在后期时间点的病毒载量较高，而pppBLV组的五只动物中有三只较低。通过在培养细胞中进行p24抗原酶联免疫吸附测定，发现pppBLV转染的绵羊中病毒表达较低，血清学滴度也较低。转染31个月后，四只wtBLV转染的绵羊中有四只死于白血病和淋巴瘤，而五只pppBLV转染的绵羊在临床上均健康，外周血淋巴细胞计数正常。这些数据表明，gp30富含脯氨酸的基序对于病毒感染性不是必需的，但对于体内高病毒载量很重要，这表明SH3介导的gp30相互作用对于感染后的病毒发病机制至关重要。与富含脯氨酸基序缺乏相互作用可能会预防或延迟绵羊的肿瘤发生。
