Sequence, necessary for initiating RNA synthesis, in the 3'-noncoding region of the classical swine fever virus genome

Classical swine fever virus (CSFV) is the causative agent of swine fever, which represents an economically important disease in hogs. We previously made a prediction about the recognition sites of replication initiation of CSFV by using the information content method, and it was predicted that the 21 nucleotides located at 3' end of the CSFV genome 3'UTR were essential to CSFV replication. In this paper, we experimentally studied these 21 nucleotides by site-directed mutagenesis. It was found that the 3'UTRs with the 21 nucleotides had the function of initiating RNA synthesis, while the 3'UTRs without the 21 nucleotides did not. The 21 nucleotides alone, without the rest of 3'UTR, were able to initiate RNA synthesis, though with a slump. It was demonstrated that the 21 nucleotides were essential to the replication of CSFV genome. The other part of 3'UTR was also required for sufficient RNA synthesis. It is highly likely that the 21 nucleotides were the necessary site for the CSFV genome replication initiation, and that the elements required for sufficient RNA synthesis were in the other part of 3'UTR. It was assumed that the CSFV replicase bound to the site and initiated the replication of the CSFV genome. In the 21 nucleotides, it was found that the mutation of position 216 and destruction of the 3' terminus in the 3'UTR precluded initiation of RNA synthesis, that the mutation of position 212 did not affect the capacity for initiation of RNA synthesis but attenuated the synthesis of RNA. Among the four mutants of 3'UTR at position 219, three produced the 3'UTR without initiation of RNA synthesis, and the other one produced the 3'UTR with initiation of less RNA synthesis. Therefore, it could be concluded that T216 was the most important while T212 was the least important, and that G219 and C228 were also important for RNA synthesis. The normal base component within the 21 nucleotides was essential to sufficient RNA synthesis.