Burgon Trever B, Jenkins Jomaquai A, Deitz Stephen B, Spagnolo Jeannie F, Kirkegaard Karla
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305-5402, USA.
J Virol. 2009 Oct;83(19):10129-39. doi: 10.1128/JVI.00642-09. Epub 2009 Jul 22.
The rate of protein secretion in host cells is inhibited during infection with several different picornaviruses, with consequences likely to have significant effects on viral growth, spread, and pathogenesis. This Sin(+) (secretion inhibition) phenotype has been documented for poliovirus, foot-and-mouth disease virus, and coxsackievirus B3 and can lead to reduced cell surface expression of major histocompatibility complex class I and tumor necrosis factor receptor as well as reduced extracellular secretion of induced cytokines such as interleukin-6 (IL-6), IL-8, and beta interferon. The inhibition of protein secretion is global, affecting the movement of all tested cargo proteins through the cellular secretion apparatus. To test the physiological significance of the Sin(-) and Sin(+) phenotypes in animal models, Sin(-) mutant viruses are needed that fail to inhibit host protein secretion and also exhibit robust growth properties. To identify such Sin(-) mutant polioviruses, we devised a fluorescence-activated cell sorter-based screen to select virus-infected cells that nevertheless expressed newly synthesized surface proteins. After multiple rounds of selection, candidate Sin(-) mutant viruses were screened for genetic stability, increased secretion of cargo molecules and wild-type translation and growth properties. A newly identified Sin(-) mutant poliovirus that contained coding changes in nonstructural proteins 2A (N32D) and 2C (E253G) was characterized. In this virus, the 2C mutation is responsible for the Sin(-) phenotype and the 2A mutation suppresses a resulting growth defect by increasing the rate of cell death and therefore the rate of viral spread. The 2A-N32D suppressor mutation was not allele specific and, by increasing the rate of cellular apoptosis, affected a completely different pathway than the 2C-E253G Sin(-) mutation. Therefore, the 2A mutation suppresses the 2C-E253G mutant phenotype by a bypass suppression mechanism.
在感染几种不同的小核糖核酸病毒期间，宿主细胞中的蛋白质分泌速率受到抑制，其后果可能会对病毒的生长、传播及发病机制产生重大影响。脊髓灰质炎病毒、口蹄疫病毒和柯萨奇病毒B3都有这种Sin(+)（分泌抑制）表型记录，它可导致主要组织相容性复合体I类分子和肿瘤坏死因子受体的细胞表面表达减少，以及白细胞介素-6（IL-6）、IL-8和β干扰素等诱导细胞因子的细胞外分泌减少。蛋白质分泌的抑制是全局性的，影响所有测试的货物蛋白通过细胞分泌装置的移动。为了在动物模型中测试Sin(-)和Sin(+)表型的生理意义，需要Sin(-)突变病毒，这些病毒不能抑制宿主蛋白分泌，并且还具有强大的生长特性。为了鉴定此类Sin(-)突变脊髓灰质炎病毒，我们设计了一种基于荧光激活细胞分选仪的筛选方法，以选择那些仍能表达新合成表面蛋白的病毒感染细胞。经过多轮筛选，对候选Sin(-)突变病毒进行了遗传稳定性、货物分子分泌增加以及野生型翻译和生长特性的筛选。鉴定出一种新的Sin(-)突变脊髓灰质炎病毒，其非结构蛋白2A（N³²D）和2C（E²⁵³G）存在编码变化。在这种病毒中，2C突变导致Sin(-)表型，而2A突变通过增加细胞死亡速率从而增加病毒传播速率来抑制由此产生的生长缺陷。2A-N³²D抑制突变不是等位基因特异性的，并且通过增加细胞凋亡速率，影响了与2C-E²⁵³G Sin(-)突变完全不同的途径。因此，2A突变通过旁路抑制机制抑制2C-E²⁵³G突变表型。
