Recognition of RNA templates by viral replicase proteins is one of the key steps in the replication process of all RNA viruses. However, the mechanisms underlying this phenomenon, including primary RNA elements that are recognized by the viral replicase proteins, are not well understood. Here, we used aptamer pulldown assays with membrane fractionation and protein-RNA coimmunoprecipitation in a cell-free viral translation/ replication system to investigate how viral replicase proteins recognize the bipartite genomic RNAs of the Red clover necrotic mosaic virus (RCNMV). RCNMV replicase proteins bound specifically to a Y-shaped RNA element (YRE) located in the 3 untranslated region (UTR) of RNA2, which also interacted with the 480-kDa replicase complexes that contain viral and host proteins. The replicase-YRE interaction recruited RNA2 to the membrane fraction. Conversely, RNA1 fragments failed to interact with the replicase proteins supplied in trans. The results of protein-RNA coimmunoprecipitation assays suggest that RNA1 interacts with the replicase proteins coupled with their translation. Thus, the initial template recognition mechanisms employed by the replicase differ between RCNMV bipartite genomic RNAs and RNA elements are primary determinants of the differential replication mechanism.After entry into host cells, the genomic RNA of a positivestrand RNA virus is translated using host translational machinery, to produce the replicase proteins. Then, the replicase proteins synthesize negative-strand RNAs, which function as a template for positive-strand RNA synthesis. In an early replication phase, the viral replicase proteins must recognize the viral genomic RNAs rapidly and specifically in a pool of abundant cellular RNAs (e.g., rRNA, tRNA, and mRNA) to recruit them to replication sites on intracellular membranes before viral RNAs are degraded by antiviral mechanisms. For example, the 1a protein of Brome mosaic virus (BMV) recruits BMV RNA2 and RNA3 to the membrane of the endoplasmic reticulum (ER) in Saccharomyces cerevisiae, depending on cis-acting RNA elements that are present in the 5Ј proximal region of RNA2 and in the intergenic region of RNA3 (6,20,50,52). The replication protein A of Flock House virus (FHV) also recruits FHV RNA1 to the mitochondrial membrane in yeast and Drosophila melanogaster cells, depending on a 5Ј cis element (58, 59). The p33 accessory protein of tombusviruses (Tomato bushy stunt virus [TBSV] and Cucumber necrosis virus [CNV]) binds directly to the internal replication element located in the coding region of the p92 RNA-dependent RNA polymerase (RdRP) in vitro, and CNV p33 recruits defective interfering RNAs to the peroxisomal membrane in yeast (43,44,46,47). However, the detailed mechanisms via which viral RNAs are specifically recognized and recruited to appropriate membranes by replicase proteins are not well understood.