The positive-strand coronavirus genome of ~30 kilobase in length and subgenomic (sg) mRNAs of shorter lengths, are 5’ and 3’-co-terminal by virtue of a common 5’-capped leader and a common 3’-polyadenylated untranslated region. Here, by ligating head-to-tail viral RNAs from bovine coronavirus-infected cells and sequencing across the ligated junctions, it was learned that at the time of peak viral RNA synthesis [6 hours postinfection (hpi)] the 3’ poly(A) tail on genomic and sgmRNAs is ~65 nucleotides (nt) in length. Surprisingly, this length was found to vary throughout infection from ~45 nt immediately after virus entry (at 0 to 4 hpi) to ~65 nt later on (at 6 h to 9 hpi) and from ~65 nt (at 6 h to 9 hpi) to ~30 nt (at 120-144 hpi). With the same method, poly(U) sequences of the same lengths were simultaneously found on the ligated viral negative-strand RNAs. Functional analyses of poly(A) tail length on specific viral RNA species, furthermore, revealed that translation, in vivo, of RNAs with the longer poly(A) tail was enhanced over those with the shorter poly(A). Although the mechanisms by which the tail lengths vary is unknown, experimental results together suggest that the length of the poly(A) and poly(U) tails is regulated. One potential function of regulated poly(A) tail length might be that for the coronavirus genome a longer poly(A) favors translation. The regulation of coronavirus translation by poly(A) tail length resembles that during embryonal development suggesting there may be mechanistic parallels.

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.