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Abstract
The influenza A virus contains 8 segmented genomic RNAs and was considered to encode 10 viral proteins until investigators identified the 11th viral protein, PB1-F2, which uses an alternative reading frame of the PB1 gene. The recently identified PB1-N40, PA-N155 and PA-N182 influenza A proteins have shown the potential for using a leaking ribosomal scanning mechanism to generate novel open reading frames (ORFs). These novel ORFs provide examples of the manner in which the influenza A virus expands its coding capacity by using overlapping reading frames. In this study, we performed a computational search, based on a ribosome scanning mechanism, on all influenza A coding sequences to identify possible forward-reading ORFs that could be translated into novel viral proteins. We specified that the translated products had a prevalence ≥5% to eliminate sporadic ORFs. A total of 1,982 ORFs were thus identified and presented in terms of their locations, lengths and Kozak sequence strengths. We further provided an abridged list of ORFs by requiring every candidate an upstream start codon (within the upstream third of the primary transcript), a strong Kozak consensus sequence and high prevalence (≥95% and ≥50% for in-frame and alternative-frame ORFs, respectively). The PB1-F2, PB1-N40, PA-N155 and PA-N182 proteins all fulfilled our filtering criteria. Subject to these three stringent settings, we additionally named 16 novel ORFs for all influenza A genomes except for HA and NA, for which 43 HA and 11 NA ORFs from their respective subtypes were also recognized.
Highlights
In molecular biology, an open reading frame (ORF) is a nucleic acid sequence that does not contain a stop codon in a given reading frame, and can potentially be translated into a functional protein product
We based our hypothesis for identifying novel ORFs on the leaky ribosomal scanning mechanism, which uses a downstream AUG to initiate the translation rather than the first AUG encountered in the mRNA
We performed a computational search, based on ribosome scanning, of all full-length coding sequences to investigate all possible ORFs in 3 forwardreading frames
Summary
An open reading frame (ORF) is a nucleic acid sequence that does not contain a stop codon in a given reading frame, and can potentially be translated into a functional protein product. Gibbs et al identified that the PB1-F2 protein localizes to mitochondria, causing cell death, and exemplifies the manner in which a virus can expand the coding capacity of its genome by using overlapping reading frames [11]. The Influenza Virus Resource (IVR) [15] of the National Center for Biotechnology Information (NCBI) estimated an average prevalence of approximately 90% for all published PB1 sequences, assuming that a functional PB1-F2 contains $79 amino acids (aa). This proportion varies among hosts (humans 90%, swine 76%, other mammals 100% and birds 95%) [16, 17]. The PB1-F2 of this virus is only 11 aa in length
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