An Intraleader Open Reading Frame is Selected from a Hypervariable 5’ Terminus during Persistent Infection by the Bovine Coronavirus
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In an effort to unambiguously establish the 5’ terminal nucleotides on bovine coronavirus (BCV) mRNAs, we developed a method that employs the head-to-tail ligation of single-stranded cDNA from extended primers before PCR amplification, cloning and sequencing1,2. Only the mRNAs for the N, M, and S structural proteins were studied in this manner, and mRNAs isolated from cells at various times postinfection through 432 days were studied. We learned that the 5’ terminal five nucleotides were hypervariable among four major terminus types, and that certain types predominated at various times postinfection. The type I terminus (GAUUGUG…) predominated within the first 4 days postinfection and was considered the wild type terminus. At 296 days postinfection and beyond, the type II terminus (GAUUAUG…) predominated. Types II, III and IV all possessed a G → A mutation that gave rise to an AUG codon and established an intraleader ORF for 11 amino acids. Although we have not yet been able to show the existence of the peptide in persistently infected cells, we have demonstrated that the intraleader ORF attenuates translation of downstream ORFs during translation in vitro.
KeywordsPersistent Infection Virus Inoculum Terminal Nucleotide Result Amino Acid Terminus Type