Genomic RNA of positive-strand RNA viruses replicate via complementary (i.e., negative-strand) RNA in membrane-bound replication complexes. Before replication complex formation, virusencoded replication proteins specifically recognize genomic RNA molecules and recruit them to sites of replication. Moreover, in many of these viruses, selection of replication templates by the replication proteins occurs preferentially in cis. This property is advantageous to the viruses in several aspects of viral replication and evolution, but the underlying molecular mechanisms have not been characterized. Here, we used an in vitro translation system to show that a 126-kDa replication protein of tobacco mosaic virus (TMV), a positive-strand RNA virus, binds a 5′-terminal ∼70-nucleotide region of TMV RNA cotranslationally, but not posttranslationally. TMV mutants that carried nucleotide changes in the 5′-terminal region and showed a defect in the binding were unable to synthesize negative-strand RNA, indicating that this binding is essential for template selection. A C-terminally truncated 126-kDa protein, but not the full-length 126-kDa protein, was able to posttranslationally bind TMV RNA in vitro, suggesting that binding of the 126-kDa protein to the 70-nucleotide region occurs during translation and before synthesis of the C-terminal inhibitory domain. We also show that binding of the 126-kDa protein prevents further translation of the bound TMV RNA. These data provide a mechanistic explanation of how the 126-kDa protein selects replication templates in cis and how fatal collision between translating ribosomes and negative-strand RNA-synthesizing polymerases on the genomic RNA is avoided. V irions of positive-strand RNA viruses contain genomic RNA of messenger sense. After infection, genomic RNA is released from the virions into the cytoplasm and translated to produce viral proteins, including viral RNA-dependent RNA polymerases and other replication-related proteins. These proteins are collectively called "replication proteins." In eukaryotic positive-strand RNA viruses, replication proteins recruit genomic RNA to the cytoplasmic face of intracellular membranes to form replication complexes (1, 2). Negative-strand RNAs that are complementary to genomic RNAs are synthesized in the replication complexes, and then, using the negative-strand RNAs as templates, genomic RNA is copied and released into the cytoplasm. The recognition of template RNAs and their recruitment to the replication complexes are key processes in selective amplification of genomic RNA by positive-strand RNA viruses. In several positive-strand RNA viruses, cis-acting elements for replication-template selection have been identified, and, for some of them, it was demonstrated that replication proteins directly bind to these elements (3).Replication of tobacco mosaic virus (TMV), poliovirus, and many other positive-strand RNA viruses is cis-preferential: i.e., replication proteins recognize their own translation templates for replication (4-13). Because viral RN...