Reselection of a Genomic Upstream Open Reading Frame in Mouse Hepatitis Coronavirus 5′-Untranslated-Region Mutants

Abstract

An AUG-initiated upstream open reading frame (uORF) encoding a potential polypeptide of 3 to 13 amino acids (aa) is found within the 5= untranslated region (UTR) of >75% of coronavirus genomes based on 38 reference strains. Potential CUG-initiated uORFs are also found in many strains. The AUG-initiated uORF is presumably translated following genomic 5=-end cap-dependent ribosomal scanning, but its function is unknown. Here, in a reverse-genetics study with mouse hepatitis coronavirus, the following were observed. (i) When the uORF AUG-initiating codon was replaced with a UAG stop codon along with a U112A mutation to maintain a uORF-harboring stem-loop 4 structure, an unimpaired virus with wild-type (WT) growth kinetics was recovered. However, reversion was found at all mutated sites within five virus passages. (ii) When the uORF was fused with genomic (main) ORF1 by converting three in-frame stop codons to nonstop codons, a uORF-ORF1 fusion protein was made, and virus replicated at WT levels. However, a frameshifting G insertion at virus passage 7 established a slightly 5=-extended original uORF. (iii) When uAUG-eliminating deletions of 20, 30, or 51 nucleotides (nt) were made within stem-loop 4, viable but debilitated virus was recovered. However, a C80U mutation in the first mutant and an A77G mutation in the second appeared by passage 10, which generated alternate uORFs that correlated with restored WT growth kinetics. In vitro, the uORF-disrupting nondeletion mutants showed enhanced translation of the downstream ORF1 compared with the WT. These results together suggest that the uORF represses ORF1 translation yet plays a beneficial but nonessential role in coronavirus replication in cell culture.

Alternative promoters drive human cytomegalovirus reactivation from latency

“…For example, the discrete 5′ untranslated regions (UTRs) of the iP-derived transcripts contain multiple alternative AUG translation start sites which may be posited to suppress translational activity via leaky scanning under basal conditions while promoting translation during cell stress (42,43). Such regulatory elements are common in viruses and appear to regulate translation in a similar manner (44)(45)(46). Furthermore, Arend et al (47) have previously demonstrated that the 5′ UTR of MIE mRNAs plays a role in their translation and is required for efficient HCMV replication during lytic infection.…”

Dependence of Coronavirus RNA Replication on an NH 2 -Terminal Partial Nonstructural Protein 1 in cis

“…Although discovered in BCoV DI RNA, the requirement for the WAPEFPWM domain in cis for DI RNA replication may have implications for genome replication, although this should be viewed with the caveat that cis-acting replication elements for DI RNA are not always the same for the viral genome. An example is the 5=-proximal stem-loop 4 in MHV (29,30). If the WAPEFPWM domain does function in the genome, it would likely not interact with a replicase protein as postulated for the DI RNA (see below).…”

“…(i) With regard to the 5=-terminal cis-acting RNA structures, stem-loops 1, 2, and 3 map (almost entirely) within the most 5= 74 nt (7, 27) and may not be components unique to the function of the 322-nt region. Similarly, stem-loop 4 (28), which is nearly identical to its homolog in MHV that has been recently shown not to be required for virus replication (29,30), also may not be a component unique to the function of the 322-nt region. All of cis-acting stem-loops 5 (31, 32), 6 (33), and 7 (26) and possibly a small stem-loop 8, which has been predicted to be but not tested as a cis-acting replication signal (26), however, may contribute uniquely to the replication function of the 322-nt region.…”

Reinitiation after Translation of Two Upstream Open Reading Frames (ORF) Governs Expression of the ORF35-37 Kaposi's Sarcoma-Associated Herpesvirus Polycistronic mRNA

“…Short uORFs are common regulatory elements in mammalian transcripts that generally function to dampen translation of the major ORF by capturing a population of the scanning ribosomes (8). They are prevalent in several viruses as well, and those that have been characterized appear to function in an analogous manner (9)(10)(11). However, KSHV has adopted this cellular regulatory feature to instead facilitate translation of multiple proteins from a single mRNA.…”