Strong J E, Lee P W
Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Centre, Alberta, Canada.
J Virol. 1996 Jan;70(1):612-6. doi: 10.1128/JVI.70.1.612-616.1996.
We have previously demonstrated that two mouse cell lines that are poorly infectible by reovirus become highly susceptible upon transfection with the gene encoding the epidermal growth factor receptor (EGFR) (J. E. Strong, D. Tang, and P. W. K. Lee, Virology 197:405-411, 1993). This enhancement of infection efficiency requires a functional EGFR, since such an enhancement is not observed in cells expressing a mutated (kinase-inactive) EGFR. The additional finding that reovirus is capable of directly binding to the N-terminal ectodomain of the EGFR (D. Tang, J. E. Strong, and P. W. K. Lee, Virology 197:412-414, 1993) has led us to question whether this interaction is required for the activation of a signalling cascade that somehow augments the ensuing infection process. In the present study, we address this question, using cells transfected with the v-erbB oncogene, which encodes a protein structurally related to the EGFR but lacking a large portion of the N-terminal ligand-binding domain. The v-erbB protein also possesses ligand-independent, constitutive tyrosine kinase activity. Control NIH 3T3 cells, which are poorly infectible by reovirus (serotype 3, strain Dearing), and NIH 3T3 cells transfected with the v-erbB oncogene (THC-11) were assayed for their susceptibilities to reovirus infection. Infectivity was determined by immunofluorescent detection of viral proteins, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of radiolabeled cells, and plaque titration. All three assays demonstrated a drastically higher degree of susceptibility to infection in the THC-11 cell line. This enhanced susceptibility was found to be abrogated by treatment of the cells with genistein, an inhibitor of tyrosine protein kinases, but only partially by treatment with daidzein, an inactive analog of genistein. We propose that the mechanism of enhancement of infection efficiency conferred by EGFR and v-erbB is through the opportunistic utilization by the virus of an already activated signal transduction pathway.
我们之前已经证明,两种对呼肠孤病毒感染性较差的小鼠细胞系,在用编码表皮生长因子受体(EGFR)的基因转染后会变得高度易感(J. E. 斯特朗、D. 唐和P. W. K. 李,《病毒学》197:405 - 411,1993年)。感染效率的这种提高需要功能性的EGFR,因为在表达突变型(激酶失活型)EGFR的细胞中未观察到这种提高。另外还发现呼肠孤病毒能够直接结合EGFR的N端胞外结构域(D. 唐、J. E. 斯特朗和P. W. K. 李,《病毒学》197:412 - 414,1993年),这使我们质疑这种相互作用是否是激活某种信号级联反应所必需的,而这种信号级联反应以某种方式增强了随后的感染过程。在本研究中,我们使用转染了v-erbB癌基因的细胞来解决这个问题,该基因编码一种结构上与EGFR相关但缺少大部分N端配体结合结构域的蛋白质。v-erbB蛋白还具有不依赖配体的组成型酪氨酸激酶活性。检测了对呼肠孤病毒(3型,迪林株)感染性较差的对照NIH 3T3细胞以及转染了v-erbB癌基因的NIH 3T3细胞(THC-11)对呼肠孤病毒感染的易感性。通过病毒蛋白的免疫荧光检测、放射性标记细胞的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析以及蚀斑滴定来确定感染性。所有这三种检测方法都表明THC-11细胞系对感染的易感性程度大幅提高。发现用酪氨酸蛋白激酶抑制剂染料木黄酮处理细胞可消除这种增强的易感性,但用染料木黄酮的无活性类似物黄豆苷元处理只能部分消除。我们提出,EGFR和v-erbB赋予感染效率提高的机制是病毒机会性地利用了已激活的信号转导途径。