Date Published: December 12, 2013
Publisher: Public Library of Science
Author(s): Ting-Yung Ke, Wei-Yu Liao, Hung-Yi Wu, Volker Thiel.
The establishment of persistent viral infection is often associated with the selection of one or more mutant viruses. For example, it has been found that an intraleader open reading frame (ORF) in genomic and subgenomic mRNA (sgmRNA) molecules is selected during bovine coronavirus (BCoV) persistence which leads to translation attenuation of the downstream ORF. Here, we report the unexpected identification of leaderless genomes, in addition to leader-containing genomes, in a cell culture persistently infected with BCoV. The discovery was made by using a head-to-tail ligation method that examines genomic 5′-terminal sequences at different times postinfection. Functional analyses of the leaderless genomic RNA in a BCoV defective interfering (DI) RNA revealed that (1) the leaderless genome was able to serve as a template for the synthesis of negative-strand genome, although it cannot perform replicative positive-strand genomic RNA synthesis, and (2) the leaderless genome retained its function in translation and transcription, although the efficiency of these processes was impaired. Therefore, this previously unidentified leaderless genome is associated with the attenuation of genome expression. Whether the leaderless genome contributes to the establishment of persistent infection remains to be determined.
The families Coronaviridae (Coronavirus and Torovirus genera) and Arteriviridae, together with Roniviridae, are members of order Nidovirales in which a nested set of subgenomic RNA (sgmRNA) molecules are made that are 3′-coterminal with the genome during transcription –. In arteriviruses and coronaviruses, sgmRNAs are both 3′-coterminal and contain a common 5′ leader sequence derived from the 5′ end of the genome –. However, all sgmRNAs in gill-associated virus (GAV), and sgmRNAs 3, 4, and 5 in equine torovirus (EToV), lack a leader sequence identical to the 5′ end of the genome –. The mechanism of leader acquisition during discontinuous negative-strand ((−)-strand) synthesis from the positive-strand ((+)-strand) genomic RNA template has gained favor to explain how sgmRNAs acquire a leader sequence from the 5′ end of the genome –. The mechanism has been applied to explain high-frequency leader-switching events during the replication of the coronavirus defective interfering (DI) RNA genome –.
Synthesis of a 3′ co-terminal nested set of sgmRNAs is a common feature of members of the Nidovirales , , –. In addition to this feature, the 5′ end of all sgmRNAs in arteri- and coronaviruses possesses a common leader sequence derived from the 5′ terminus of the genomic RNA , –, , , . Although ronivirus GAV sgmRNAs and torovirus EToV sgmRNAs 3, 4, and 5 have been identified as lacking a leader sequence, leaderless genomic RNA has not been documented in all members of Nidovirales –. In this study, we found for the first time a leaderless genomic RNA in coronavirus-infected cells at a late stage of persistent coronavirus infection, and this finding prompted us to analyze the functional role of the leaderless genome in coronavirus infection.