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

本研究对烟草花叶病毒（tobacco mosaic virus，TMV）在拟南芥中高效增殖所必需的基因TOM 1和TOM 2进行了鉴定和分析。通过定位克隆，我们发现TOM 1编码一种推定的七次跨膜蛋白，由291个氨基酸组成。我们还发现，两个独立的基因TOM 2A和TOM 2B参与表达的表型减少的TMV在tom 2突变体的增殖。TOM 2A编码280个氨基酸的推定的4次跨膜蛋白，而TOM 2B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，我们通过酵母中的Sos募集系统研究了这些宿主蛋白与TMV编码蛋白之间的相互作用。通过这些分析，我们发现TOM 1蛋白可以与TMV编码的130 k蛋白的解旋酶结构域相互作用。包括TMV在内的高等真核正链RNA病毒的复制复合物与细胞内膜相关。然而，TMV编码的复制蛋白定位在膜上的机制尚不清楚。从我们的研究结果，这表明，TOM 1可能存在于TMV复制复合物和功能连接复合物与膜。目前所获得的三个tom 1突变体等位基因很可能已经完全丧失功能，但TMV在这些tom 1突变体中仍能低水平繁殖。在这项研究中，我们发现，这种残留的活性，以支持TMV增殖tom 1突变体是从TOM 3，TOM 1同源存在于拟南芥基因组中，因此，TOM 1/TOM 3的功能是必要的TMV增殖。

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.