Computational Analysis and Mapping of Novel Open Reading Frames in Influenza A Viruses

<div><p>The influenza A virus contains 8 segmented genomic RNAs and was considered to encode 10 viral proteins until investigators identified the 11^th 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.</p></div

MDA5 plays a crucial role in EV71 RNA-mediated IRF3 activation.

<p>(A) HeLa cells were transfected with siRNA against MDA5 or scrambled siRNA for 6 h, followed by transfection with EV71 RNA for 24 h. Extracts from the transfected cells were collected, and the expression of MDA5 and RIG-I was analyzed by immunoblotting using an anti-MDA5 or anti-RIG-I antibody. IRF3 activation was assayed by immunoblotting using anti-phospho-IRF3 and anti-total-IRF3 antibodies. (B) Total RNA was isolated from the transfected HeLa cells. Relative amount of the IFN-β mRNA was measured using real-time RT-PCR. (C) HeLa cells were transfected with scrambled siRNA or siRNA against RIG-I for 24 h, followed by transfection with EV71 RNA for 20 h. Cell extracts and total RNA were collected from the transfected HeLa cells. The expression of RIG-I, MDA5, phosphorylated IRF3, total IRF3, and β-actin was detected by immunoblotting. (D) Relative amount of IFN-β mRNA in RIG-I knockdown cells was measured by real-time RT-PCR.</p

Additive Promotion of Viral Internal Ribosome Entry Site-Mediated Translation by Far Upstream Element-Binding Protein 1 and an Enterovirus 71-Induced Cleavage Product

<div><p>The 5' untranslated region (5' UTR) of the enterovirus 71 (EV71) RNA genome contains an internal ribosome entry site (IRES) that is indispensable for viral protein translation. Due to the limited coding capacity of their RNA genomes, EV71 and other picornaviruses typically recruit host factors, known as IRES <i>trans</i>-acting factors (ITAFs), to mediate IRES-dependent translation. Here, we show that EV71 viral proteinase 2A is capable of cleaving far upstream element-binding protein 1 (FBP1), a positive ITAF that directly binds to the EV71 5' UTR linker region to promote viral IRES-driven translation. The cleavage occurs at the Gly-371 residue of FBP1 during the EV71 infection process, and this generates a functional cleavage product, FBP1^1-371. Interestingly, the cleavage product acts to promote viral IRES activity. Footprinting analysis and gel mobility shift assay results showed that FBP1^1-371 similarly binds to the EV71 5' UTR linker region, but at a different site from full-length FBP1; moreover, FBP1 and FBP1^1-371 were found to act additively to promote IRES-mediated translation and virus yield. Our findings expand the current understanding of virus-host interactions with regard to viral recruitment and modulation of ITAFs, and provide new insights into translational control during viral infection.</p></div

HHblits hits against PDB database for alternative-frame ORFs.

<p>HHblits hits were grouped by PDB ID or putative ORF. E-value, sequence identity and probability were provided by averaging ones from HHblits hits. The alignments between putative ORFs and structures were generated by HHblits tool. The number in parenthesis following a sequence alignment is the length of either query or subject. Consensus sequences are shown in HMM format for both the query and the subject, in which capital and lowercase letters are used to represent conserved columns with ≥60 and ≥40 probabilities, respectively, and a tilde “∼” is used to represent non-conserved column. Based on default settings with two iterations, HHblits tool adds significant hit(s) from the previous search/iteration to the HMM query for the next search/iteration. It results to that our query may not be single sequence. Five symbols are used to show the alignment quality, in which “|”, “+”, “.”, “−” and “ = ” each represents a quality from the perfect to the worst.</p>a<p>Two groups of putative ORFs from PB1 and NP genes hit to 1WRG structure.</p><p>HHblits hits against PDB database for alternative-frame ORFs.</p

Length variability of PB1-F2.

<p>Of 19,378 PB1 sequences analyzed, 19,339 contain a start codon at position 95, signaling the beginning of the PB1-F2 ORF. The downstream stop codons vary, resulting in different ORF sizes. The 3 major PB1-F2 lengths are 90, 57 and 11 aa. It is generally assumed that an intact PB1-F2 is ≥79 aa in length.</p

BLASTP hits against the NR database for all putative ORFs.

<p>All hits for the putative ORFs listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115016#pone-0115016-t001" target="_blank">Table 1</a> are grouped into 8 cases according to the queried ORFs and hits returned from the database.</p><p>BLASTP hits against the NR database for all putative ORFs.</p

An abridged ORF list with additional constraints.

<p>The ORFs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115016#pone-0115016-t001" target="_blank">Table 1</a> were screened with additional filters, strong Kozak consensus sequence, upstream AUG location (within the first third of the transcript), and high prevalence (≥95% and ≥50% for in-frame and alternative-frame ORFs, respectively), to obtain 16 novel ORFs. These ORFs were named, according to conventions, as PB1-N40, PA-N155, PA-N182 (in-frame) and PB1-F2 (alternative-frame). The 4 known ORFs are excluded from this table.</p><p>An abridged ORF list with additional constraints.</p

ORF data for influenza A viruses.

<p>An ORF is defined as containing a start codon AUG at a given genomic position, with ≥5% prevalence from all analyzed sequences from the NCBI.</p><p>ORF data for influenza A viruses.</p

Pharmacological interventions targeting PI3K signaling affect EV-A71 replication in SF268 neural cells.

<p>The effect of PI3K (LY294002 and wortmannin), P38 MAPK (SB203580) (A) and GSK3β (AR-A014418 and LiCl) (B) inhibitors as pharmacological interventions on the virus yields of EV-A71-infected cells was assessed. SF268 cells were infected with EV-A71 (moi 0.5), and various concentrations of compounds were added to the infected cells after 1 h viral adsorption. At 48 h post-infection, the culture supernatants and cell lysates were collected for virus titration using plaque assays. Data are displayed as mean ± s.e.m. from at least two independent experiments performed in duplicates.</p

Overexpression of MDA5 or RIG-I reverses the inhibition of IRF3 activation during EV71 infection.

<p>HeLa cells were transfected with an empty plasmid or a plasmid expressing the FLAG-MDA5 protein for 36 h, and were subsequently infected with the MP4 strain of the EV71 virus at 2 MOI. At 9 h post-infection, cell extracts were analyzed by immunoblotting using anti-MDA5, anti-RIG-I, anti-phospho-IRF3, anti-total IRF3, anti-EV71 3C, and anti-β-actin antibodies (A). (B) Relative amount of IFN-β mRNA produced in the cells was measured using real-time RT-PCR. (C) HeLa cells were transfected with an empty plasmid or a plasmid expressing the FLAG-RIG-I protein for 36 h, and were subsequently infected with the EV71 at 2 MOI. At 9 h post-infection, cell extracts were analyzed by immunoblotting using anti-FLAG, anti-MDA5, anti-RIG-I, anti-phospho-IRF3, anti-total IRF3, anti-EV71 3C, and anti-β-actin antibodies. The asterisk indicates the putative cleavage product of the MDA5 protein. The relative amount of IFN-β mRNA was quantitated by real-time RT-PCR (D). (E) HeLa cells were transfected with RIG-I expressing plasmid for 24 h, and then transfected with MDA5 siRNA for 18 h. The co-transfected cells were infected with EV71 at MOI 2. At 9 h post-infection, cell extracts were detected the RIG-I, MDA5, phosphorylated IRF3, total IRF3, and β-actin by immunoblotting. Total RNA was also collected for detecting IFN-β mRNA by real-time RT-PCR (F).</p