Claus Claudia, Tzeng Wen-Pin, Liebert Uwe Gerd, Frey Teryl K
Institute of Virology, University of Leipzig, Leipzig, Germany.
Virology. 2007 Dec 5;369(1):19-34. doi: 10.1016/j.virol.2007.06.047. Epub 2007 Aug 15.
During serial passaging of rubella virus (RUB) in cell culture, the dominant species of defective-interfering RNA (DI) generated contains an in-frame deletion between the capsid protein (C) gene and E1 glycoprotein gene resulting in production of a C-E1 fusion protein that is necessary for the maintenance of the DI [Tzeng, W.P., Frey, T.K. (2006). C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage. Virology 356 198-207.]. A BHK cell line stably expressing the RUB structural proteins was established which was used to package DIs into virus particles following transfection with in vitro transcripts from DI infectious cDNA constructs. Packaging of a DI encoding an in-frame C-GFP-E1 reporter fusion protein corresponding to the C-E1 fusion protein expressed in a native DI was only marginally more efficient than packaging of a DI encoding GFP, indicating that the C-E1 fusion protein did not function by enhancing packaging. However, infection with the DI encoding the C-GFP-E1 fusion protein (in the absence of wt RUB helper virus) resulted in formation of clusters of GFP-positive cells and the percentage of GFP-positive cells in the culture following infection remained relatively constant. In contrast, a DI encoding GFP did not form GFP-positive clusters and the percentage of GFP-positive cells declined by roughly half from 2 to 4 days post-infection. Cluster formation and sustaining the percentage of infected (GFP-positive) cells required the C part of the fusion protein, including the downstream but not the upstream of two arginine clusters (both of which are associated with RNA binding and association with mitochondrial p32 protein) and the E1 part through the transmembrane sequence, but not the C-terminal cytoplasmic tail. Among a collection of mutant DI constructs, cluster formation and sustaining infected cell percentage correlated with maintenance during serial passage with wt RUB. We hypothesize that cluster formation and sustaining infected cell percentage increase the likelihood of co-infection by a DI and wt RUB during serial passage thus enhancing maintenance of the DI. Cluster formation and sustaining infected cell percentage were found to be due to a combination of attenuated cytopathogenicity of DIs that express the C-E1 fusion protein and cell-to-cell movement of the DI. In infected cells, the C-GFP-E1 fusion protein was localized to potentially novel vesicular structures that appear to originate from ER-Golgi transport vacuoles. This species of DI expressing a C-E1 fusion protein that exhibits attenuated cytopathogenicity and the ability to increase the number of infected cells through cell-to-cell movement could be the basis for development of an attractive vaccine vector.
在风疹病毒（RUB）在细胞培养中的连续传代过程中，产生的主要缺陷干扰RNA（DI）种类在衣壳蛋白（C）基因和E1糖蛋白基因之间存在框内缺失，导致产生一种C-E1融合蛋白，该蛋白对于DI的维持是必需的[Tzeng, W.P., Frey, T.K. (2006). C-E1融合蛋白由风疹病毒DI RNA在连续传代过程中合成并维持。病毒学356 198-207。]。建立了一个稳定表达RUB结构蛋白的BHK细胞系，在用DI感染性cDNA构建体的体外转录本转染后，该细胞系用于将DIs包装到病毒颗粒中。包装一个编码与天然DI中表达的C-E1融合蛋白相对应的框内C-GFP-E1报告融合蛋白的DI，其效率仅比包装一个编码GFP的DI略高，这表明C-E1融合蛋白并非通过增强包装来发挥作用。然而，用编码C-GFP-E1融合蛋白的DI感染（在没有野生型RUB辅助病毒的情况下）导致形成GFP阳性细胞簇，并且感染后培养物中GFP阳性细胞的百分比保持相对恒定。相比之下，编码GFP的DI没有形成GFP阳性簇，并且GFP阳性细胞的百分比在感染后2至4天下降了大约一半。簇的形成和维持感染（GFP阳性）细胞的百分比需要融合蛋白的C部分，包括两个精氨酸簇下游但不包括上游（这两个簇都与RNA结合以及与线粒体p32蛋白的结合有关）以及通过跨膜序列的E1部分，但不需要C末端细胞质尾巴。在一系列突变DI构建体中，簇的形成和维持感染细胞百分比与野生型RUB连续传代期间的维持相关。我们假设簇的形成和维持感染细胞百分比增加了在连续传代过程中DI与野生型RUB共同感染的可能性，从而增强了DI的维持。发现簇的形成和维持感染细胞百分比是由于表达C-E1融合蛋白的DIs的细胞病变效应减弱和DI的细胞间移动的组合。在感染细胞中，C-GFP-E1融合蛋白定位于可能源自内质网-高尔基体运输泡的潜在新型囊泡结构。这种表达C-E1融合蛋白的DI种类表现出减弱的细胞病变效应以及通过细胞间移动增加感染细胞数量的能力，可能是开发一种有吸引力的疫苗载体的基础。
