Identification of host factors involved in the replication of an RNA virus

本研究鉴定并分析了烟草花叶病毒（TMV）单细胞高效增殖所需的拟南芥TOM1和TOM2基因。通过定位克隆，我们发现TOM1编码一个由291个氨基酸组成的七通跨膜蛋白。我们还发现，在tom2突变体中，两个独立的基因TOM2A和TOM2B参与了TMV增殖减少表型的表达。TOM2A编码280个氨基酸的4通跨膜蛋白，而TOM2B编码122个氨基酸的蛋白。为了了解这些宿主蛋白是如何支持TMV增殖的，我们在酵母中通过Sos招募系统检测了这些宿主蛋白与TMV编码蛋白之间的相互作用。通过这些分析，我们发现TOM1蛋白可以与tmv编码的130k蛋白的解旋酶结构域相互作用。包括TMV在内的高级真核正链RNA病毒的复制复合体已知与细胞膜有关。然而，tmv编码的复制蛋白在膜上定位的机制尚不清楚。根据我们的研究结果，我们认为TOM1可能存在于TMV复制复合体中，并发挥连接该复合体与膜的功能。到目前为止获得的三个tom1突变等位基因很可能已经完全失去了功能。然而，TMV仍然可以在这些tom1突变体中繁殖到低水平。在本研究中，我们发现这种支持TMV在tom1突变体中增殖的残留活性来自TOM3，这是拟南芥基因组中存在的tom1同源物，因此，tom1 /TOM3的功能是TMV增殖所必需的。

In this research, we identified and analyzed Arabidopsis TOM1 and TOM2 genes which are necessary for the efficient multiplication of tobacco mosaic virus (TMV) within a single cell. By positional cloning, we found TOM1 encodes a putative seven-pass transmembrane protein of 291 amino acids. We also revealed that two independent genes named TOM2A and TOM2B are involved in the expression of the phenotype of reduced multiplication of TMV in the tom2 mutant. TOM2A encodes a putative 4-pass transmembrane protein of 280 amino acids, whereas TOM2B encodes a 122 amino-acid protein. In order to know how these host proteins support TMV multiplication, we examined the interaction between these host proteins and TMV-encoded proteins by the Sos recruitment system in yeast. Through these analyses, we found that TOM1 protein could interact with the helicase domain of TMV-encoded 130 k protein. The replication complexes of higher eukaryotic positive-strand RNA viruses including TMV are known to be associated with intracellular membranes. However, the mechanisms by which TMV-encoded replication proteins are localized on membranes had not been known. From our results, it was suggested that TOM1 may be present in the TMV replication complex and function to connect the complex with membranes. All three tom1 mutant alleles obtained so far are likely to have lost their function completely.Nevertheless, TMV can still multiply to low levels in these tom1 mutants. In this study, we found that this residual activity to support TMV multiplication in tom1 mutants is from TOM3, a TOM1 homologue present in the Arabidopsis genome, and therefore, the function of TOM1/TOM3 is necessary for TMV multiplication.