Multiple domains of the Jaagsiekte sheep retrovirus envelope protein are required for transformation of rodent fibroblasts
Multiple domains of the Jaagsiekte sheep retrovirus envelope protein are required for transformation of rodent fibroblasts
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DOI：
10.1128/jvi.78.19.10479-10489.2004
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发表时间：
2004-10-01
影响因子：
5.4
通讯作者：
Fan, H
中科院分区：
文献类型：
--
作者：
Hofacre, A;Fan, H
绵羊肺腺瘤病毒（Jaagsiekte sheep retrovirus，JSRV）是一种可引起绵羊肺腺癌的外源性逆转录病毒。我们先前表明，编码JSRV包膜蛋白（Env）的基因似乎是一种癌基因，因为它可以转化小鼠NIH 3 T3细胞。Env跨膜蛋白（TM）的胞质尾区是转化所必需的。然而，以前的实验并不排除Env表面蛋白（SU）参与转化。在这项研究中，我们通过SU结构域创建了一系列巢式缺失突变体，并评估了它们转化啮齿动物成纤维细胞的能力。预测的信号肽下游的所有SU缺失突变体不能转化鼠NIH 3 T3或大鼠208 F细胞。运输到质膜的选定删除的Env蛋白的血凝素标记的版本的共聚焦免疫荧光显微镜证实。其他连续SU缺失突变体缺乏整个SU的50个氨基酸（aa）块也无法转化。此外，在SU的不同位置的两个氨基酸（Leu/Gln）的最小插入突变体也废除了转化。这些数据表明，域SU促进有效的JSRV转换。这可能反映了SU对Env蛋白的适当构型或转化所需的信号传导途径的SU独立激活的必要性。SU和TM突变体之间的互补转化支持后者的假设。用DeltaGP Y 590 F（TM胞质尾中的突变体）与DeltaGP SUDelta 103 -352（缺乏大部分SU）共转染导致有效转化。所得转化体显示出两种突变质粒的存在和表达的证据。
Jaagsiekte sheep retrovirus (JSRV) is an exogenous retrovirus of sheep that induces a contagious lung cancer, ovine pulmonary adenocarcinoma. We previously showed that the gene encoding JSRV envelope protein (Env) appears to function as an oncogene, since it can transform mouse NIH 3T3 cells. The cytoplasmic tail of the Env transmembrane protein (TM) is necessary for the transformation. However, previous experiments did not exclude the involvement of the Env surface protein (SU) in transformation. In this study, we created a series of nested deletion mutants through the SU domain and assessed their ability to transform rodent fibroblasts. All SU deletion mutants downstream of the predicted signal peptide were unable to transform murine NIH 3T3 or rat 208F cells. Transport to the plasma membrane of selected deleted Env proteins was confirmed by confocal immunofluorescence microscopy of hemagglutinin-tagged versions. Additional sequential SU deletion mutants lacking 50-amino-acid (aa) blocks throughout SU also were unable to transform. Furthermore, minimal insertion mutants of two amino acids (Leu/Gln) at various positions in SU also abolished transformation. These data indicate that domains in SU facilitate efficient JSRV transformation. This could reflect a necessity of SU for appropriate configuration of the Env protein or independent activation by SU of a signaling pathway necessary for transformation. Complementation between SU and TM mutants for transformation supported the latter hypothesis. Cotransfection with DeltaGP Y590F (mutant in the TM cytoplasmic tail) with DeltaGP SUDelta103-352 (lacking most of SU) resulted in efficient transformation. The resulting transformants showed evidence for the presence and expression of both mutant plasmids.