A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression

Abstract

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.

Interaction of coronavirus nucleocapsid protein with the 5′‐ and 3′‐ends of the coronavirus genome is involved in genome circularization and negative‐strand RNA synthesis

“…Several studies have suggested that cis-acting RNA elements located at the 5 0 -terminus of the genome are also critical for (À)-strand RNA synthesis [8,10,49,50]. In the current study, we found that in addition to the 3 0 -terminal sequence or structure, the binding affinity of N protein to the 5 0 -terminal sequence also correlates with (À)-strand RNA synthesis.…”

“…RNA was then collected at 8 h posttransfection (hpt) and subjected to RT-qPCR to evaluate the effect of poly(A) tail length on (À)-strand synthesis efficiency. It should be noted that there is no possibility of DI RNA-BCoV genome recombinant synthesized at 8 hpt because no RT-PCR product was observed (data not shown) using previously described methods [8,16,39]. Surprisingly, (À)-strand RNA synthesis was still detected using DI RNA with no poly(A) tail (BM0A, Fig.…”

“…applied as internal controls for normalization of (À)-strand DI RNA synthesis [8,10,16]. The reactions were conducted with an initial preincubation at 95°C for 5 min, followed by 35 amplification cycles as follows: 95°C for 15 s and 60°C for 60 s.…”

Using Biosecurity Measures to Combat Respiratory Disease in Cattle: The Norwegian Control Program for Bovine Respiratory Syncytial Virus and Bovine Coronavirus

“…Infective BRSV has been isolated from lymph nodes 71 days post infection (74). BCoV persistence has been demonstrated in cell culture (75). Long-term PCR positivity in calves has been shown in one experimental study, but transmission potential was not confirmed by virus isolation/sentinel trials nor was sequencing of virus done to exclude new infection (76).…”

Effects of Coronavirus Persistence on the Genome Structure and Subsequent Gene Expression, Pathogenicity and Adaptation Capability

“…However, variations at the 5′ terminal sequence in mouse hepatitis virus-A59 (MHV-A59), a mouse coronavirus, have not been identified, but instead a mutation at nucleotide (nt) 77 in the genome leads to the synthesis of the start codon AUG and subsequent translation enhancement [ 18 ]. The recently identified leaderless genome in BCoV has also been suggested to be associated with an attenuation of translation during BCoV persistence [ 24 ]. These studies focus only on the structural changes at the 5′ end of the genome and subgenome during persistence [ 18 , 23 , 24 ].…”