Search

In this study, we characterized an emerging porcine reproductive and respiratory syndrome virus (PRRSV) isolate UIL21-0712, which is a lineage 1C variant with ORF5 restriction fragment length polymorphism (RFLP) cutting pattern of 1-4-4. The UIL21-0712 genome sequence has 85.3% nucleotide identity with the prototypic PRRSV-2 strain VR2332. The nsp2 region is the most variable, and the -2/-1 programmed ribosome frameshifting (PRF) signal therein is distinct from historical PRRSV strains. Analysis of PRRSV sequences in GenBank revealed that the majority of the emerging PRRSV variants contain substitutions that disrupt the -1 PRF stop codon to generate a nsp2N protein with a C-terminal extension. Two of the -1 PRF stop codon variant patterns were identified to be predominantly circulating in the field. They demonstrated higher growth kinetics than the other variants, suggesting that the most dominant -1 PRF stop codon variant patterns may provide enhanced growth fitness for the virus.

Funder: Helmholtz Association; doi: https://doi.org/10.13039/501100009318, Programmed -1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to a pseudoknot-like conformation of the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we demonstrate that 2A stabilises this RNA element, likely explaining the increase in PRF efficiency in the presence of 2A. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, show how 2A-mediated stabilisation of an RNA pseudoknot promotes PRF, and reveal how 2A accumulation may shut down translation during virus infection.

B.1.1.7 lineage SARS-CoV-2 is more transmissible, leads to greater clinical severity, and results in modest reductions in antibody neutralization. Subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome. Applying our tool (periscope) to ARTIC Network Oxford Nanopore Technologies genomic sequencing data from 4400 SARS-CoV-2 positive clinical samples, we show that normalised sgRNA is significantly increased in B.1.1.7 (alpha) infections (n = 879). This increase is seen over the previous dominant lineage in the UK, B.1.177 (n = 943), which is independent of genomic reads, E cycle threshold and days since symptom onset at sampling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median sgRNA abundance. We demonstrate that ORF9b protein levels are increased 6-fold in B.1.1.7 compared to a B lineage virus in vitro. We hypothesise that increased ORF9b in B.1.1.7 is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT > CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3' of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles to evaluate emerging potential variants of concern.

The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) causes significant economic losses to the swine industry worldwide. Here we apply ribosome profiling (RiboSeq) and parallel RNA sequencing (RNASeq) to characterise the transcriptome and translatome of both species of PRRSV and to analyse the host response to infection. We calculated programmed ribosomal frameshift (PRF) efficiency at both sites on the viral genome. This revealed the nsp2 PRF site as the second known example where temporally regulated frameshifting occurs, with increasing -2 PRF efficiency likely facilitated by accumulation of the PRF-stimulatory viral protein, nsp1β. Surprisingly, we find that PRF efficiency at the canonical ORF1ab frameshift site also increases over time, in contradiction of the common assumption that RNA structure-directed frameshift sites operate at a fixed efficiency. This has potential implications for the numerous other viruses with canonical PRF sites. Furthermore, we discovered several highly translated additional viral ORFs, the translation of which may be facilitated by multiple novel viral transcripts. For example, we found a highly expressed 125-codon ORF overlapping nsp12, which is likely translated from novel subgenomic RNA transcripts that overlap the 3' end of ORF1b. Similar transcripts were discovered for both PRRSV-1 and PRRSV-2, suggesting a potential conserved mechanism for temporally regulating expression of the 3'-proximal region of ORF1b. We also identified a highly translated, short upstream ORF in the 5' UTR, the presence of which is highly conserved amongst PRRSV-2 isolates. These findings reveal hidden complexity in the gene expression programmes of these important nidoviruses.Viruses have tiny genomes. Rather than carry all the genetic information they need, they rely on the cells they infect. This makes the few genes they do have all the more important. Many viruses store their genes not in DNA, but in a related molecule called RNA. When the virus infects cells, it uses the cells’ ribosomes – the machines in the cells that make proteins – to build its own proteins. One of the central ideas in biology is that one molecule of RNA carries the instructions for just one type of protein. But many viruses break this rule. The ribosomes in cells read RNA instructions in blocks of three: three RNA letters correspond to one protein building block. But certain sequences in the RNA of viruses act as hidden signals that affect how ribosomes read these molecules. These signals make the ribosomes skip backward by one or two letters on the viral RNA, restarting part way through a three-letter block. Scientists call this a ‘frameshift’, and it is a bit like changing the positions of the spaces in a sentence. The virus causes these frameshifts using proteins or by folding its RNA into a knot-like structure. The frameshifts result in the production of different viral proteins over time. The porcine reproductive and respiratory syndrome virus (PRRSV) uses frameshifts to cause devastating disease in pigs. Besides the sequences in its RNA that allow the ribosomes to skip backwards, the viral enzyme that copies the RNA can also skip forward. This results in shortened copies of its genes, which also changes the proteins they produce. To find out exactly how PRRSV uses these frameshifting techniques, Cook et al. examined infected cells in the laboratory. They monitored the RNA made by the virus and looked closely at the way the cells read it using a technique called ribosome profiling. This revealed that frameshifting increases over the course of an infection. This is partly because the viral protein that causes frameshifts builds up as infection progresses, but it also happened with frameshifts caused by RNA knots. The reason for this is less clear. Cook et al. also discovered several new RNAs made later in infection, which could also change the proteins the virus makes. RNA viruses cause disease in humans as well as pigs. Examples include coronaviruses and HIV. Many of these also have frameshift sites in their genomes. A better understanding of how frameshifts change during infection may aid drug development. Future work could help researchers to understand which proteins viruses make at which stage of infection. This could lead to new treatments for viruses like PRRSV.

Funder: Kay Kendall Leukaemia Fund (KKLF); doi: https://doi.org/10.13039/501100000402, Funder: EC | EC Seventh Framework Programm | FP7 People: Marie-Curie Actions (FP7-PEOPLE - Specific Programme "People" Implementing the Seventh Framework Programme of the European Community for Research, Technological Development and Demonstration Activities (2007 to 2013)); doi: https://doi.org/10.13039/100011264; Grant(s): Mitobiopath-705560, Many cellular processes, including ribosome biogenesis, are regulated through post-transcriptional RNA modifications. Here, a genome-wide analysis of the human mitochondrial transcriptome shows that 2'-O-methylation is limited to residues of the mitoribosomal large subunit (mtLSU) 16S mt-rRNA, introduced by MRM1, MRM2 and MRM3, with the modifications installed by the latter two proteins being interdependent. MRM2 controls mitochondrial respiration by regulating mitoribosome biogenesis. In its absence, mtLSU particles (visualized by cryo-EM at the resolution of 2.6 ��) present disordered RNA domains, partial occupancy of bL36m and bound MALSU1:L0R8F8:mtACP anti-association module, allowing five mtLSU biogenesis intermediates with different intersubunit interface configurations to be placed along the assembly pathway. However, mitoribosome biogenesis does not depend on the methyltransferase activity of MRM2. Disruption of the MRM2 Drosophila melanogaster orthologue leads to mitochondria-related developmental arrest. This work identifies a key checkpoint during mtLSU assembly, essential to maintain mitochondrial homeostasis.

The 2A protein of Theiler’s murine encephalomyelitis virus (TMEV) acts as a switch to stimulate programmed −1 ribosomal frameshifting (PRF) during infection. Here we present the X-ray crystal structure of TMEV 2A and define how it recognises the stimulatory RNA element. We demonstrate a critical role for bases upstream of the originally predicted stem-loop, providing evidence for a pseudoknot-like conformation and suggesting that the recognition of this pseudoknot by beta-shell proteins is a conserved feature in cardioviruses. Through examination of PRF in TMEV-infected cells by ribosome profiling, we identify a series of ribosomal pauses around the site of PRF induced by the 2A-pseudoknot complex. Careful normalisation of ribosomal profiling data with a 2A knockout virus facilitated the identification, through disome analysis, of ribosome stacking at the TMEV frameshifting signal. These experiments provide unparalleled detail of the molecular mechanisms underpinningTheilovirusprotein-stimulated frameshifting.

Many viruses utilize programmed –1 ribosomal frameshifting (–1 PRF) to express additional proteins or to produce frameshift and non-frameshift protein products at a fixed stoichiometric ratio. PRF is also utilized in the expression of a small number of cellular genes. Frameshifting is typically stimulated by signals contained within the mRNA: a ‘slippery’ sequence and a 3′-adjacent RNA structure. Recently, we showed that −1 PRF in encephalomyocarditis virus (EMCV) is trans-activated by the viral 2A protein, leading to a temporal change in PRF efficiency from 0% to 70% during virus infection. Here we analyzed PRF in the related Theiler's murine encephalomyelitis virus (TMEV). We show that 2A is also required for PRF in TMEV and can stimulate PRF to levels as high as 58% in rabbit reticulocyte cell-free translations and 81% during virus infection. We also show that TMEV 2A trans-activates PRF on the EMCV signal but not vice versa. We present an extensive mutational analysis of the frameshift stimulators (mRNA signals and 2A protein) analysing activity in in vitro translation, electrophoretic mobility shift and in vitro ribosome pausing assays. We also investigate the PRF mRNA signal with RNA structure probing. Our results substantially extend previous characterization of protein-stimulated PRF.

A metagenomics approach was used to detect novel and recognized RNA viruses in mosquitoes from the Yucatan Peninsula of Mexico. A total of 1359 mosquitoes of 7 species and 5 genera (Aedes, Anopheles, Culex, Mansonia and Psorophora) were sorted into 37 pools, homogenized and inoculated onto monolayers of Aedes albopictus (C6/36) cells. A second blind passage was performed and then total RNA was extracted and analysed by RNA-seq. Two novel viruses, designated Uxmal virus and Mayapan virus, were identified. Uxmal virus was isolated from three pools of Aedes (Ochlerotatus) taeniorhynchus and phylogenetic data indicate that it should be classified within the recently proposed taxon Negevirus. Mayapan virus was recovered from two pools of Psorophora ferox and is most closely related to unclassified Nodaviridae-like viruses. Two recognized viruses were also detected: Culex flavivirus (family Flaviviridae) and Houston virus (family Mesoniviridae), with one and two isolates being recovered, respectively. The in vitro host ranges of all four viruses were determined by assessing their replicative abilities in cell lines of avian, human, monkey, hamster, murine, lepidopteran and mosquito (Aedes, Anopheles and Culex) origin, revealing that all viruses possess vertebrate replication-incompetent phenotypes. In conclusion, we report the isolation of both novel and recognized RNA viruses from mosquitoes collected in Mexico, and add to the growing plethora of viruses discovered recently through the use of metagenomics.

The herpes simplex virus (HSV)-1 protein pUL21 is essential for efficient virus replication and dissemination. While pUL21 has been shown to promote multiple steps of virus assembly and spread, the molecular basis of its function remained unclear. Here we identify that pUL21 is a virus-encoded adaptor of protein phosphatase 1 (PP1). pUL21 directs the dephosphorylation of cellular and virus proteins, including components of the viral nuclear egress complex, and we define a conserved non-canonical linear motif in pUL21 that is essential for PP1 recruitment. In vitro evolution experiments reveal that pUL21 antagonises the activity of the virus-encoded kinase pUS3, with growth and spread of pUL21 PP1-binding mutant viruses being restored in adapted strains where pUS3 activity is disrupted. This study shows that virus-directed phosphatase activity is essential for efficient herpesvirus assembly and spread, highlighting the fine balance between kinase and phosphatase activity required for optimal virus replication., Author summary Herpes simplex virus (HSV)-1 is a highly prevalent human virus that causes life-long infections. While the most common symptom of HSV-1 infection is orofacial lesions (‘cold sores’), HSV-1 infection can also cause fatal encephalitis and it is a leading cause of infectious blindness. The HSV-1 genome encodes many proteins that dramatically remodel the environment of infected cells to promote virus replication and spread, including enzymes that add phosphate groups (kinases) to cellular and viral proteins in order to fine-tune their function. Here we identify that pUL21 is an HSV-1 protein that binds directly to protein phosphatase 1 (PP1), a highly abundant cellular enzyme that removes phosphate groups from proteins. We demonstrate that pUL21 stimulates the specific dephosphorylation of both cellular and viral proteins, including a component of the viral nuclear egress complex that is essential for efficient assembly of new HSV-1 particles. Furthermore, our in vitro evolution experiments demonstrate that pUL21 antagonises the activity of the HSV-1 kinase pUS3. Our work highlights the precise control that herpesviruses exert upon the protein environment within infected cells, and specifically the careful balance of kinase and phosphatase activity that HSV-1 requires for optimal replication and spread.

RNA structural elements occur in numerous single-stranded positive-sense RNA viruses. The stem-loop 2 motif (s2m) is one such element with an unusually high degree of sequence conservation, being found in the 3' untranslated region (UTR) in the genomes of many astroviruses, some picornaviruses and noroviruses, and a variety of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. The evolutionary conservation and its occurrence in all viral subgenomic transcripts imply a key role for s2m in the viral infection cycle. Our findings indicate that the element, while stably folded, can nonetheless be invaded and remodeled spontaneously by antisense oligonucleotides (ASOs) that initiate pairing in exposed loops and trigger efficient sequence-specific RNA cleavage in reporter assays. ASOs also act to inhibit replication in an astrovirus replicon model system in a sequence-specific, dose-dependent manner and inhibit SARS-CoV-2 replication in cell culture. Our results thus permit us to suggest that the s2m element is readily targeted by ASOs, which show promise as antiviral agents. IMPORTANCE The highly conserved stem-loop 2 motif (s2m) is found in the genomes of many RNA viruses, including SARS-CoV-2. Our findings indicate that the s2m element can be targeted by antisense oligonucleotides. The antiviral potential of this element represents a promising start for further research into targeting conserved elements in RNA viruses.

BackgroundThroughout biology, multiple sequence alignments (MSAs) form the basis of much investigation into biological features and relationships. These alignments are at the heart of many bioinformatics analyses. However, sequences in MSAs are often incomplete or very divergent, which can lead to poor alignment and large gaps. This slows down computation and can impact conclusions without being biologically relevant. Cleaning the alignment by removing common issues such as gaps, divergent sequences, large insertions and deletions and poorly aligned sequence ends can substantially improve analyses. Manual editing of MSAs is very widespread but is time-consuming and difficult to reproduce.ResultsWe present a comprehensive, user-friendly MSA trimming tool with multiple visualisation options. Our highly customisable command line tool aims to give intervention power to the user by offering various options, and outputs graphical representations of the alignment before and after processing to give the user a clear overview of what has been removed. The main functionalities of the tool include removing regions of low coverage due to insertions, removing gaps, cropping poorly aligned sequence ends and removing sequences that are too divergent or too short. The thresholds for each function can be specified by the user and parameters can be adjusted to each individual MSA. CIAlign is designed with an emphasis on solving specific and common alignment problems and on providing transparency to the user.ConclusionCIAlign effectively removes problematic regions and sequences from MSAs and provides novel visualisation options. This tool can be used to fine-tune alignments for further analysis and processing. The tool is aimed at anyone who wishes to automatically clean up parts of an MSA and those requiring a new, accessible way of visualising large MSAs.

At least six small alternative-frame open reading frames (ORFs) overlapping well-characterized SARS-CoV-2 genes have been hypothesized to encode accessory proteins. Researchers have used different names for the same ORF or the same name for different ORFs, resulting in erroneous homological and functional inferences. We propose standard names for these ORFs and their shorter isoforms, developed in consultation with the Coronaviridae Study Group of the ICTV. We recommend calling the 39 codon Spike-overlapping ORF ORF2b; the 41, 57, and 22 codon ORF3a-overlapping ORFs ORF3c, ORF3d, and ORF3b; the 33 codon ORF3d isoform ORF3d-2; and the 97 and 73 codon Nucleocapsid-overlapping ORFs ORF9b and ORF9c. Finally, we document conflicting usage of the name ORF3b in 32 studies, and consequent erroneous inferences, stressing the importance of reserving identical names for homologs. We recommend that authors referring to these ORFs provide lengths and coordinates to minimize ambiguity due to prior usage of alternative names., Graphical abstract Image 1

Identification of the full complement of genes in SARS-CoV-2 is a crucial step towards gaining a fuller understanding of its molecular biology. However, short and/or overlapping genes can be difficult to detect using conventional computational approaches, whereas high throughput experimental approaches – such as ribosome profiling – cannot distinguish translation of functional peptides from regulatory translation or translational noise. By studying regions showing enhanced conservation at synonymous sites in alignments of SARS-CoV and related viruses (subgenus Sarbecovirus), and correlating the results with the conserved presence of an open reading frame and a plausible translation mechanism, a putative new gene – ORF3c – was identified. ORF3c overlaps ORF3a in an alternative reading frame. A recently published ribosome profiling study confirmed that ORF3c is indeed translated during infection. ORF3c is conserved across the subgenus Sarbecovirus, and encodes a 40–41 amino acid predicted transmembrane protein.

RNA viruses are a major human health threat. The life cycles of many highly pathogenic RNA viruses like influenza A virus (IAV) and Lassa virus depends on host mRNA, as viral polymerases cleave 5’-m7G-capped host transcripts to primeviral mRNA synthesis (‘cap-snatching’). We hypothesized that start codons within cap-snatched host transcripts could generate chimeric human-viral mRNAs with coding potential. We report the existence of this mechanism of gene origination, that we named ‘start-snatching’. Depending on the reading frame, start-snatching allows the translation of host and viral “untranslated regions” (UTRs) to create Nterminallyextended viral proteins or entirely novel polypeptides by geneticoverprinting. We show that both types of chimeric proteins are made in IAVinfectedcells, generate T cell responses and contribute to virulence. Our resultsindicate that during infection with IAV, and likely a multitude of other human-,animal- and plant-viruses, a host-dependent mechanism allows the genesis ofhybrid genes.

Positive-sense single-stranded RNA viruses form the largest and most diverse group of eukaryote-infecting viruses. Their genomes comprise one or more segments of coding-sense RNA that function directly as messenger RNAs upon release into the cytoplasm of infected cells. Positive-sense RNA viruses are generally accepted to encode proteins solely on the positive strand. However, we previously identified a surprisingly long (∼1,000-codon) open reading frame (ORF) on the negative strand of some members of the family Narnaviridae which, together with RNA bacteriophages of the family Leviviridae, form a sister group to all other positive-sense RNA viruses. Here, we completed the genomes of three mosquito-associated narnaviruses, all of which have the long reverse-frame ORF. We systematically identified narnaviral sequences in public data sets from a wide range of sources, including arthropod, fungal, and plant transcriptomic data sets. Long reverse-frame ORFs are widespread in one clade of narnaviruses, where they frequently occupy >95 per cent of the genome. The reverse-frame ORFs correspond to a specific avoidance of CUA, UUA, and UCA codons (i.e. stop codon reverse complements) in the forward-frame RNA-dependent RNA polymerase ORF. However, absence of these codons cannot be explained by other factors such as inability to decode these codons or GC3 bias. Together with other analyses, we provide the strongest evidence yet of coding capacity on the negative strand of a positive-sense RNA virus. As these ORFs comprise some of the longest known overlapping genes, their study may be of broad relevance to understanding overlapping gene evolution and de novo origin of genes.

A novel Tymoviridae-like virus, designated Ek Balam virus, was isolated from male Culex quinquefasciatus mosquitoes collected in Yucatan, Mexico. The genome was fully sequenced and shown to have no more than 69% nt sequence identity to its closest known relative. Mosquito cells were permissive to Ek Balam virus replication, but mammalian and avian cells were refractory, suggesting that vertebrates are not involved in the maintenance of the virus in nature.

Sequence accessions attributable to novel plant amalgaviruses have been found in the Transcriptome Shotgun Assembly database. Sixteen accessions, derived from 12 different plant species, appear to encompass the complete protein-coding regions of the proposed amalgaviruses, which would substantially expand the size of genus Amalgavirus from 4 current species. Other findings include evidence for UUU_CGN as a +1 ribosomal frameshifting motif prevalent among plant amalgaviruses; for a variant version of this motif found thus far in only two amalgaviruses from solanaceous plants; for a region of α-helical coiled coil propensity conserved in a central region of the ORF1 translation product of plant amalgaviruses; and for conserved sequences in a C-terminal region of the ORF2 translation product (RNA-dependent RNA polymerase) of plant amalgaviruses, seemingly beyond the region of conserved polymerase motifs. These results additionally illustrate the value of mining the TSA database and others for novel viral sequences for comparative analyses.

Translational control through programmed ribosomal frameshifting (PRF) is exploited widely by viruses and increasingly documented in cellular genes. Frameshifting is induced by mRNA secondary structures that compromise ribosome fidelity during decoding of a heptanucleotide ‘slippery’ sequence. The nsp2 PRF signal of porcine reproductive and respiratory syndrome virus is distinctive in directing both −2 and −1 PRF and in its requirement for a trans-acting protein factor, the viral replicase subunit nsp1β. Here we show that the the trans-activation of frameshifting is carried out by a protein complex composed of nsp1β and a cellular poly(C) binding protein (PCBP). From the results of in vitro translation and electrophoretic mobility shift assays, we demonstrate that a PCBP/nsp1β complex binds to a C-rich sequence downstream of the slippery sequence and here mimics the activity of a structured mRNA stimulator of PRF. This is the first description of a role for a trans-acting cellular protein in PRF. The discovery broadens the repertoire of activities associated with poly(C) binding proteins and prototypes a new class of virus–host interactions.

Background: Programmed ribosomal frameshifting (PRF) is a gene expression mechanism which enables the translation of two N-terminally coincident, C-terminally distinct protein products from a single mRNA. Many viruses utilize PRF to control or regulate gene expression, but very few phylogenetically conserved examples are known in vertebrate genes. Additional sex combs-like (ASXL) genes 1 and 2 encode important epigenetic and transcriptional regulatory proteins that control the expression of homeotic genes during key developmental stages. Here we describe an ~150-codon overlapping ORF (termed TF) in ASXL1 and ASXL2 that, with few exceptions, is conserved throughout vertebrates. Results: Conservation of the TF ORF, strong suppression of synonymous site variation in the overlap region, and the completely conserved presence of an EH[N/S]Y motif (a known binding site for Host Cell Factor-1, HCF-1, an epigenetic regulatory factor), all indicate that TF is a protein-coding sequence. A highly conserved UCC_UUU_CGU sequence (identical to the known site of +1 ribosomal frameshifting for influenza virus PA-X expression) occurs at the 5′ end of the region of enhanced synonymous site conservation in ASXL1. Similarly, a highly conserved RG_GUC_UCU sequence (identical to a known site of −2 ribosomal frameshifting for arterivirus nsp2TF expression) occurs at the 5′ end of the region of enhanced synonymous site conservation in ASXL2. Conclusions: Due to a lack of appropriate splice forms, or initiation sites, the most plausible mechanism for translation of the ASXL1 and 2 TF regions is ribosomal frameshifting, resulting in a transframe fusion of the N-terminal half of ASXL1 or 2 to the TF product, termed ASXL-TF. Truncation or frameshift mutants of ASXL are linked to myeloid malignancies and genetic diseases, such as Bohring-Opitz syndrome, likely at least in part as a result of gain-of-function or dominant-negative effects. Our hypothesis now indicates that these disease-associated mutant forms represent overexpressed defective versions of ASXL-TF.

Recently, we identified a unique -2/-1 ribosomal frameshift mechanism in PRRSV, which yields two truncated forms of nonstructural protein (nsp) 2 variants, nsp2TF and nsp2N. Here, in vitro expression of individual PRRSV nsp2TF and nsp2N demonstrated their ability to suppress cellular innate immune responses in transfected cells. Two recombinant viruses were further analyzed, in which either nsp2TF was C-terminally truncated (vKO1) or expression of both nsp2TF and nsp2N was knocked out (vKO2). Host cellular mRNA profiling showed that a panel of cellular immune genes, in particular those involved in innate immunity, was upregulated in cells infected with vKO1 and vKO2. Compared to the wild-type virus, vKO1 and vKO2 expedited the IFN-α response and increased NK cell cytotoxicity, and subsequently enhanced T cell immune responses in infected pigs. Our data strongly implicate nsp2TF/nsp2N in arteriviral immune evasion and demonstrate that nsp2TF/nsp2N-deficient PRRSV is less capable of counteracting host innate immune responses., Highlights • In vitro expression of PRRSV nsp2TF/nsp2N affects cellular innate immune response. • Nsp2TF/nsp2N suppressed immune genes expression in PRRSV-infected cells. • Nsp2TF/nsp2N-deficient mutants showed attenuated growth in vivo. • Nsp2TF/nsp2N-deficient mutants induced augmented immune responses in infected pigs.

Because so few viruses in the family Barnaviridae have been reported, we searched for more of them in public sequence databases. Here, we report the complete coding sequence of Colobanthus quitensis associated barnavirus 1, mined from a transcriptome of the Antarctic pearlwort Colobanthus quitensis. The 4.2-kb plus-strand sequence of this virus encompasses four main open reading frames (ORFs), as expected for barnaviruses, including ORFs for a protease-containing polyprotein, an RNA-dependent RNA polymerase whose translation appears to rely on − 1 ribosomal frameshifting, and a capsid protein that is likely to be translated from a subgenomic RNA. The possible derivation of this virus from a fungus associated with C. quitensis is discussed. Instituto de Patología Vegetal Fil: Nibert, Max L. Harvard Medical School. Department of Microbiology and Immunobiology; Estados Unidos. Harvard University. Ph.D. Program in Virology; Estados Unidos Fil: Manny, Austin R. Harvard University. Ph.D. Program in Virology; Estados Unidos Fil: Debat, Humberto Julio. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Patología Vegetal; Argentina Fil: Firth, Andrew E. University of Cambridge. Division of Virology. Department of Pathology; Gran Bretaña Fil: Bertini, Laura. Universita degli Studi della Tuscia. Department of Ecological and Biological Sciences; Italia Fil: Caruso, Carla. Universita degli Studi della Tuscia. Department of Ecological and Biological Sciences; Italia

Theiler's murine encephalomyelitis virus (TMEV) is a member of the genus Cardiovirus in the Picornaviridae , a family of positive-sense single-stranded RNA viruses. Previously, we demonstrated that in the related cardiovirus, Encephalomyocarditis virus , a programmed −1 ribosomal frameshift (−1 PRF) occurs at a conserved G_GUU_UUU sequence within the 2B-encoding region of the polyprotein open reading frame (ORF). Here we show that −1 PRF occurs at a similar site during translation of the TMEV genome. In addition, we demonstrate that a predicted 3′ RNA stem-loop structure at a noncanonical spacing downstream of the shift site is required for efficient frameshifting in TMEV and that frameshifting also requires virus infection. Mutating the G_GUU_UUU shift site to inhibit frameshifting results in an attenuated virus with reduced growth kinetics and a small-plaque phenotype. Frameshifting in the virus context was found to be extremely efficient at 74 to 82%, which, to our knowledge, is the highest frameshifting efficiency recorded to date for any virus. We propose that highly efficient −1 PRF in TMEV provides a mechanism to escape the confines of equimolar expression normally inherent in the single-polyprotein expression strategy of picornaviruses. IMPORTANCE Many viruses utilize programmed −1 ribosomal frameshifting (−1 PRF) to produce different protein products at a defined ratio, or to translate overlapping ORFs to increase coding capacity. With few exceptions, −1 PRF occurs on specific “slippery” heptanucleotide sequences and is stimulated by RNA structure beginning 5 to 9 nucleotides (nt) downstream of the slippery site. Here we describe an unusual case of −1 PRF in Theiler's murine encephalomyelitis virus (TMEV) that is extraordinarily efficient (74 to 82% of ribosomes shift into the alternative reading frame) and, in stark contrast to other examples of −1 PRF, is dependent upon a stem-loop structure beginning 14 nt downstream of the slippery site. Furthermore, in TMEV-based reporter constructs in transfected cells, efficient frameshifting is critically dependent upon virus infection. We suggest that TMEV evolved frameshifting as a novel mechanism for removing ribosomes from the message (a “ribosome sink”) to downregulate synthesis of the 3′-encoded replication proteins.

Programmed −1 ribosomal frameshifting is a mechanism of gene expression whereby specific signals within messenger RNAs direct a proportion of ribosomes to shift −1 nt and continue translating in the new reading frame. Such frameshifting normally depends on an RNA structure stimulator 3$^\prime$-adjacent to a ‘slippery’ heptanucleotide shift site sequence. Recently we identified an unusual frameshifting mechanism in encephalomyocarditis virus, where the stimulator involves a $\textit{trans}$-acting virus protein. Thus, in contrast to other examples of −1 frameshifting, the efficiency of frameshifting in encephalomyocarditis virus is best studied in the context of virus infection. Here we use metabolic labelling to analyse the frameshifting efficiency of wild-type and mutant viruses. Confirming previous results, frameshifting depends on a G_GUU_UUU shift site sequence and a 3$^\prime$-adjacent stem-loop structure, but is not appreciably affected by the ‘StopGo’ sequence present ~30 nt upstream. At late timepoints, frameshifting was estimated to be 46–76 % efficient.

Human mitochondria contain a genome (mtDNA) that encodes essential subunits of the oxidative phosphorylation system. Expression of mtDNA entails multi-step maturation of precursor RNA. In other systems, the RNA life cycle involves surveillance mechanisms, however, the details of RNA quality control have not been extensively characterised in human mitochondria. Using a mitochondrial ribosome profiling and mitochondrial poly(A)-tail RNA sequencing (MPAT-Seq) assay, we identify the poly(A)-specific exoribonuclease PDE12 as a major factor for the quality control of mitochondrial non-coding RNAs. The lack of PDE12 results in a spurious polyadenylation of the 3’ ends of the mitochondrial (mt-) rRNA and mt-tRNA. While the aberrant adenylation of 16S mt-rRNA did not affect the integrity of the mitoribosome, spurious poly(A) additions to mt-tRNA led to reduced levels of aminoacylated pool of certain mt-tRNAs and mitoribosome stalling at the corresponding codons. Therefore, our data uncover a new, deadenylation-dependent mtRNA maturation pathway in human mitochondria. DOI: http://dx.doi.org/10.7554/eLife.27596.001

Neurospora crassa cpc-1 and Saccharomyces cerevisiae GCN4 are homologs specifying transcription activators that drive the transcriptional response to amino acid limitation. The cpc-1 mRNA contains two upstream open reading frames (uORFs) in its >700-nucleotide (nt) 5′ leader, and its expression is controlled at the level of translation in response to amino acid starvation. We used N. crassa cell extracts and obtained data indicating that cpc-1 uORF1 and uORF2 are functionally analogous to GCN4 uORF1 and uORF4, respectively, in controlling translation. We also found that the 5′ region upstream of the main coding sequence of the cpc-1 mRNA extends for more than 700 nucleotides without any in-frame stop codon. For 100 cpc-1 homologs from Pezizomycotina and from selected Basidiomycota, 5′ conserved extensions of the CPC1 reading frame are also observed. Multiple non-AUG near-cognate codons (NCCs) in the CPC1 reading frame upstream of uORF2, some deeply conserved, could potentially initiate translation. At least four NCCs initiated translation in vitro . In vivo data were consistent with initiation at NCCs to produce N-terminally extended N. crassa CPC1 isoforms. The pivotal role played by CPC1, combined with its translational regulation by uORFs and NCC utilization, underscores the emerging significance of noncanonical initiation events in controlling gene expression. IMPORTANCE There is a deepening and widening appreciation of the diverse roles of translation in controlling gene expression. A central fungal transcription factor, the best-studied example of which is Saccharomyces cerevisiae GCN4, is crucial for the response to amino acid limitation. Two upstream open reading frames (uORFs) in the GCN4 mRNA are critical for controlling GCN4 synthesis. We observed that two uORFs in the corresponding Neurospora crassa cpc-1 mRNA appear functionally analogous to the GCN4 uORFs. We also discovered that, surprisingly, unlike GCN4, the CPC1 coding sequence extends far upstream from the presumed AUG start codon with no other in-frame AUG codons. Similar extensions were seen in homologs from many filamentous fungi. We observed that multiple non-AUG near-cognate codons (NCCs) in this extended reading frame, some conserved, initiated translation to produce longer forms of CPC1, underscoring the significance of noncanonical initiation in controlling gene expression.

Programmed −1 ribosomal frameshifting is a mechanism of gene expression, whereby specific signals within messenger RNAs direct a proportion of translating ribosomes to shift −1 nt and continue translating in the new reading frame. Such frameshifting normally occurs at a set ratio and is utilized in the expression of many viral genes and a number of cellular genes. An open question is whether proteins might function as trans-acting switches to turn frameshifting on or off in response to cellular conditions. Here we show that frameshifting in a model RNA virus, encephalomyocarditis virus, is trans-activated by viral protein 2A. As a result, the frameshifting efficiency increases from 0 to 70% (one of the highest known in a mammalian system) over the course of infection, temporally regulating the expression levels of the viral structural and enzymatic proteins., Programmed −1 ribosomal frameshifting (−1 PRF) is a mechanism whereby specific signals within mRNAs direct ribosomes to shift into an alternative reading frame. Here the authors describe a mechanism of −1 PRF that is temporally regulated by a viral protein over the course of the virus replicative cycle.

Positioning test sequences between fused reporters permits monitoring of both translation levels and framing, before and after the test sequence. Many studies, including those on recoding such as productive ribosomal frameshifting and stop codon readthrough, use distinguishable luciferases or fluorescent proteins, as reporters. Occasional distortions, due to test sequence product interference with the individual reporter activities or stabilities, are here shown to be avoidable by the introduction of tandem StopGo sequences (2A) flanking the test sequence. Using this new vector system (pSGDluc), we provide evidence for the use of a 3’ stem loop stimulator for $\textit{ACP2}$ readthrough, but failed to detect the reported $\textit{VEGFA}$ readthrough.

We determined the complete genomic sequences of two previously discovered insect-specific flaviviruses, Marisma mosquito virus (MMV) and Nanay virus (NANV), using a combination of high-throughput sequencing, reverse transcription-polymerase chain reaction, 5' and 3' rapid amplification of cDNA ends and Sanger sequencing. Complete polyprotein amino acid sequence alignments revealed that the closest known relatives of MMV and NANV are Donggang virus (89% identity, 95% similarity) and Nounané virus (53% identity, 70% similarity), respectively. Phylogenetic inference is in agreement with these findings. Potential programmed -1 ribosomal frameshifting sites were bioinformatically identified in the genomes of both viruses.

We report the discovery of Nylanderia fulva virus 1 (NfV-1), the first virus identified and characterized from the ant, Nylanderia fulva. The NfV-1 genome (GenBank accession KX024775) is 10,881 nucleotides in length, encoding one large open reading frame (ORF). Helicase, protease, RNA-dependent RNA polymerase, and jelly-roll capsid protein domains were recognized within the polyprotein. Phylogenetic analysis placed NfV-1 in an unclassified clade of viruses. Electron microscopic examination of negatively stained samples revealed particles with icosahedral symmetry with a diameter of 28.7±1.1 nm. The virus was detected by RT-PCR in larval, pupal, worker and queen developmental stages. However, the replicative strand of NfV-1 was only detected in larvae. Vertical transmission did not appear to occur, but horizontal transmission was facile. The inter-colonial field prevalence of NfV-1 was 52±35% with some local infections reaching 100%. NfV-1 was not detected in limited samples of other Nylanderia species or closely related ant species., Graphical abstract fx1, Highlights • A new positive-strand RNA virus was discovered in the ant, Nylanderia fulva. • The Nylanderia fulva virus 1 genome was comprised of 10,881 nucleotides. • NfV-1 was detected in larval, pupal, queen and worker ants, but not eggs. • Replication of NfV-1 appeared to be limited to the larval stage.

Genetic decoding is not 'frozen' as was earlier thought, but dynamic. One facet of this is frame-shifting that often results in synthesis of a C-terminal region encoded by a new frame. Ribosomal frameshifting is utilized for the synthesis of additional products, for regulatory purposes and for translational 'correction' of problem or 'savior' indels. Utilization for synthesis of additional products occurs prominently in the decoding of mobile chromosomal element and viral genomes. One class of regulatory frameshifting of stable chromosomal genes governs cellular polyamine levels from yeasts to humans. In many cases of productively utilized frameshifting, the proportion of ribosomes that frameshift at a shift-prone site is enhanced by specific nascent peptide or mRNA context features. Such mRNA signals, which can be 5' or 3' of the shift site or both, can act by pairing with ribosomal RNA or as stem loops or pseudoknots even with one component being 4 kb 3' from the shift site. Transcriptional realignment at slippage-prone sequences also generates productively utilized products encoded trans-frame with respect to the genomic sequence. This too can be enhanced by nucleic acid structure. Together with dynamic codon redefinition, frameshifting is one of the forms of recoding that enriches gene expression.

Sequences corresponding to a putative, novel rhabdovirus [designated Merida virus (MERDV)] were initially detected in a pool of Culex quinquefasciatus collected in the Yucatan Peninsula of Mexico. The entire genome was sequenced, revealing 11 798 nt and five major ORFs, which encode the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). The deduced amino acid sequences of the N, G and L proteins have no more than 24, 38 and 43 % identity, respectively, to the corresponding sequences of all other known rhabdoviruses, whereas those of the P and M proteins have no significant identity with any sequences in GenBank and their identity is only suggested based on their genome position. Using specific reverse transcription-PCR assays established from the genome sequence, 27 571 C. quinquefasciatus which had been sorted in 728 pools were screened to assess the prevalence of MERDV in nature and 25 pools were found positive. The minimal infection rate (calculated as the number of positive mosquito pools per 1000 mosquitoes tested) was 0.9, and similar for both females and males. Screening another 140 pools of 5484 mosquitoes belonging to four other genera identified positive pools of Ochlerotatus spp. mosquitoes, indicating that the host range is not restricted to C. quinquefasciatus. Attempts to isolate MERDV in C6/36 and Vero cells were unsuccessful. In summary, we provide evidence that a previously undescribed rhabdovirus occurs in mosquitoes in Mexico.

Members of the family Coronaviridae have the largest genomes of all RNA viruses, typically in the region of 30 kilobases. Several coronaviruses, such as Severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV), are of medical importance, with high mortality rates and, in the case of SARS-CoV, significant pandemic potential. Other coronaviruses, such as Porcine epidemic diarrhea virus and Avian coronavirus, are important livestock pathogens. Ribosome profiling is a technique which exploits the capacity of the translating ribosome to protect around 30 nucleotides of mRNA from ribonuclease digestion. Ribosome-protected mRNA fragments are purified, subjected to deep sequencing and mapped back to the transcriptome to give a global “snap-shot” of translation. Parallel RNA sequencing allows normalization by transcript abundance. Here we apply ribosome profiling to cells infected with Murine coronavirus, mouse hepatitis virus, strain A59 (MHV-A59), a model coronavirus in the same genus as SARS-CoV and MERS-CoV. The data obtained allowed us to study the kinetics of virus transcription and translation with exquisite precision. We studied the timecourse of positive and negative-sense genomic and subgenomic viral RNA production and the relative translation efficiencies of the different virus ORFs. Virus mRNAs were not found to be translated more efficiently than host mRNAs; rather, virus translation dominates host translation at later time points due to high levels of virus transcripts. Triplet phasing of the profiling data allowed precise determination of translated reading frames and revealed several translated short open reading frames upstream of, or embedded within, known virus protein-coding regions. Ribosome pause sites were identified in the virus replicase polyprotein pp1a ORF and investigated experimentally. Contrary to expectations, ribosomes were not found to pause at the ribosomal frameshift site. To our knowledge this is the first application of ribosome profiling to an RNA virus., Author Summary Ribosome profiling is emerging as a powerful technique to monitor translation in living cells at sub-codon resolution. It has particular applicability to virology, with the capacity to identify viral mRNAs that are being translated during infection and to provide new insights into virus gene expression, regulation and host-virus interactions. In this work, we carried out the first ribosome profiling analysis of an RNA virus, using as a model system the murine coronavirus strain MHV-A59, a betacoronavirus in the same genus as the medically important SARS-CoV and MERS-CoV. Parallel ribosome profiling and RNA sequencing of infected-cell time points was performed during the course of MHV replication in mouse tissue culture cells and used to determine virus gene expression kinetics and the relative translational efficiencies of virus and host mRNAs. The sensitivity and precision of the approach permitted us to uncover several unanticipated features of coronavirus translation, giving insights into ribosomal frameshifting, ribosome pausing, and the utilisation of short, potentially regulatory, upstream open reading frames. We also identified some challenges associated with the technique that are of general relevance to the ribosome profiling technique and developed bioinformatic strategies to address these.

Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but uses duplex-specific nuclease (DSN) as a convenient, species-independent way to reduce rRNA contamination. We show that DSN-based depletion compares favorably with other commonly used rRNA depletion strategies and introduces little bias. The profiling protocol typically produces high levels of triplet periodicity, facilitating the detection of coding sequences, including upstream, downstream, and overlapping open reading frames (ORFs) and an alternative ribosome conformation evident during termination of protein synthesis. In addition, we provide a software package that presents a set of methods for parsing ribosomal profiling data from multiple samples, aligning reads to coding sequences, inferring alternative ORFs, and plotting average and transcript-specific aspects of the data. Methods are also provided for extracting the data in a form suitable for differential analysis of translation and translational efficiency.

Viruses in the family Luteoviridae have positive-sense RNA genomes of around 5.2 to 6.3 kb, and they are limited to the phloem in infected plants. The Luteovirus and Polerovirus genera include all but one virus in the Luteoviridae. They share a common gene block, which encodes the coat protein (ORF3), a movement protein (ORF4), and a carboxy-terminal extension to the coat protein (ORF5). These three proteins all have been reported to participate in the phloem-specific movement of the virus in plants. All three are translated from one subgenomic RNA, sgRNA1. Here, we report the discovery of a novel short ORF, termed ORF3a, encoded near the 5’ end of sgRNA1. Initially, this ORF was predicted by statistical analysis of sequence variation in large sets of aligned viral sequences. ORF3a is positioned upstream of ORF3 and its translation initiates at a non-AUG codon. Functional analysis of the ORF3a protein, P3a, was conducted with Turnip yellows virus (TuYV), a polerovirus, for which translation of ORF3a begins at an ACG codon. ORF3a was translated from a transcript corresponding to sgRNA1 in vitro, and immunodetection assays confirmed expression of P3a in infected protoplasts and in agroinoculated plants. Mutations that prevent expression of P3a, or which overexpress P3a, did not affect TuYV replication in protoplasts or inoculated Arabidopsis thaliana leaves, but prevented virus systemic infection (long-distance movement) in plants. Expression of P3a from a separate viral or plasmid vector complemented movement of a TuYV mutant lacking ORF3a. Subcellular localization studies with fluorescent protein fusions revealed that P3a is targeted to the Golgi apparatus and plasmodesmata, supporting an essential role for P3a in viral movement., Author Summary In order to maximize coding capacity, RNA viruses often encode overlapping genes and use unusual translational control mechanisms. Plant viruses express proteins required for movement of the virus through the plant, often from non-canonically translated open reading frames (ORFs). Viruses in the economically important Luteoviridae family are confined to the phloem (vascular) tissue, probably due to their specialized phloem-specific movement proteins. These proteins are translated from one viral mRNA, sgRNA1, via initiation at more than one AUG codon to express overlapping genes, and by ribosomal read-through of a stop codon. Here, we describe yet another gene translated from sgRNA1, ORF3a. Translation of ORF3a initiates at a non-standard (not AUG) start codon. We found that ORF3a is not required for viral genome replication, but is required for long-distance movement of the virus in the plant. The movement function could be restored in trans by providing the ORF3a product, P3a, from another viral or plasmid vector. P3a localizes in the Golgi apparatus and adjacent to the plasmodesmata, supporting a role in intercellular movement. In summary, we used a powerful bioinformatic tool to discover a cryptic gene whose product is required for movement of a phloem-specific plant virus, revealing multiple levels of translational control that regulate expression of four proteins from a single mRNA.

High-throughput somatic mutation screening using FFPE tissues is a major challenge because of a lack of established methods and validated variant calling algorithms. We aimed to develop a targeted sequencing protocol by Fluidigm multiplex PCR and Illumina sequencing and to establish a companion variant calling algorithm. The experimental protocol and variant calling algorithm were first developed and optimized against a series of somatic mutations (147 substitutions, 12 indels ranging from 1 to 33 bp) in seven genes, previously detected by Sanger sequencing of DNA from 163 FFPE lymphoma biopsy specimens. The optimized experimental protocol and variant calling algorithm were further ascertained in two separate experiments by including the seven genes as a part of larger gene panels (22 or 13 genes) using FFPE and high-molecular-weight lymphoma DNAs, respectively. We found that most false-positive variants were due to DNA degradation, deamination, and Taq polymerase errors, but they were nonreproducible and could be efficiently eliminated by duplicate experiments. A small fraction of false-positive variants appeared in duplicate, but they were at low alternative allele frequencies and could be separated from mutations when appropriate threshold value was used. In conclusion, we established a robust practical approach for high-throughput mutation screening using archival FFPE tissues.

Identification of the full complement of genes and other functional elements in any virus is crucial to fully understand its molecular biology and guide the development of effective control strategies. RNA viruses have compact multifunctional genomes that frequently contain overlapping genes and non-coding functional elements embedded within protein-coding sequences. Overlapping features often escape detection because it can be difficult to disentangle the multiple roles of the constituent nucleotides via mutational analyses, while high-throughput experimental techniques are often unable to distinguish functional elements from incidental features. However, RNA viruses evolve very rapidly so that, even within a single species, substitutions rapidly accumulate at neutral or near-neutral sites providing great potential for comparative genomics to distinguish the signature of purifying selection. Computationally identified features can then be efficiently targeted for experimental analysis. Here we analyze alignments of protein-coding virus sequences to identify regions where there is a statistically significant reduction in the degree of variability at synonymous sites, a characteristic signature of overlapping functional elements. Having previously tested this technique by experimental verification of discoveries in selected viruses, we now analyze sequence alignments for ∼700 RNA virus species to identify hundreds of such regions, many of which have not been previously described.

We investigated whether small RNA (sRNA) sequenced from field-collected mosquitoes and chironomids (Diptera) can be used as a proxy signature of viral prevalence within a range of species and viral groups, using sRNAs sequenced from wild-caught specimens, to inform total RNA deep sequencing of samples of particular interest. Using this strategy, we sequenced from adult Anopheles maculipennis s.l. mosquitoes the apparently nearly complete genome of one previously undescribed virus related to chronic bee paralysis virus, and, from a pool of Ochlerotatus caspius and Oc. detritus mosquitoes, a nearly complete entomobirnavirus genome. We also reconstructed long sequences (1503-6557 nt) related to at least nine other viruses. Crucially, several of the sequences detected were reconstructed from host organisms highly divergent from those in which related viruses have been previously isolated or discovered. It is clear that viral transmission and maintenance cycles in nature are likely to be significantly more complex and taxonomically diverse than previously expected. © 2013 Cook et al.

Background The genus Alphavirus includes several potentially lethal human viruses. Additionally, species such as Sindbis virus and Semliki Forest virus are important vectors for gene therapy, vaccination and cancer research, and important models for virion assembly and structural analyses. The genome encodes nine known proteins, including the small '6K' protein. 6K appears to be involved in envelope protein processing, membrane permeabilization, virion assembly and virus budding. In protein gels, 6K migrates as a doublet – a result that, to date, has been attributed to differing degrees of acylation. Nonetheless, despite many years of research, its role is still relatively poorly understood. Results We report that ribosomal -1 frameshifting, with an estimated efficiency of ~10–18%, occurs at a conserved UUUUUUA motif within the sequence encoding 6K, resulting in the synthesis of an additional protein, termed TF (TransFrame protein; ~8 kDa), in which the C-terminal amino acids are encoded by the -1 frame. The presence of TF in the Semliki Forest virion was confirmed by mass spectrometry. The expression patterns of TF and 6K were studied by pulse-chase labelling, immunoprecipitation and immunofluorescence, using both wild-type virus and a TF knockout mutant. We show that it is predominantly TF that is incorporated into the virion, not 6K as previously believed. Investigation of the 3' stimulatory signals responsible for efficient frameshifting at the UUUUUUA motif revealed a remarkable diversity of signals between different alphavirus species. Conclusion Our results provide a surprising new explanation for the 6K doublet, demand a fundamental reinterpretation of existing data on the alphavirus 6K protein, and open the way for future progress in the further characterization of the 6K and TF proteins. The results have implications for alphavirus biology, virion structure, viroporins, ribosomal frameshifting, and bioinformatic identification of novel frameshift-expressed genes, both in viruses and in cellular organisms.

BackgroundThe majority of introns in gene transcripts are found within the coding sequences (CDSs). A small but significant fraction of introns are also found to reside within the untranslated regions (5'UTRs and 3'UTRs) of expressed sequences. Alignment of the whole genome and expressed sequence tags (ESTs) of the model plantArabidopsis thalianahas identified introns residing in both coding and non-coding regions of the genome.ResultsA bioinformatic analysis revealed some interesting observations: (1) the density of introns in 5'UTRs is similar to that in CDSs but much higher than that in 3'UTRs; (2) the 5'UTR introns are preferentially located close to the initiating ATG codon; (3) introns in the 5'UTRs are, on average, longer than introns in the CDSs and 3'UTRs; and (4) 5'UTR introns have a different nucleotide composition to that of CDS and 3'UTR introns. Furthermore, we show that the 5'UTR intron of theA. thalianaEF1α-A3 gene affects the gene expression and the size of the 5'UTR intron influences the level of gene expression.ConclusionIntrons within the 5'UTR show specific features that distinguish them from introns that reside within the coding sequence and the 3'UTR. In the EF1α-A3 gene, the presence of a long intron in the 5'UTR is sufficient to enhance gene expression in plants in a size dependent manner.

Novel flaviviruses that are genetically related to pathogenic mosquito-borne flaviviruses (MBFV) have been isolated from mosquitoes in various geographical locations, including Finland. We isolated and characterized another novel virus of this group from Finnish mosquitoes collected in 2007, designated as Ilomantsi virus (ILOV). Unlike the MBFV that infect both vertebrates and mosquitoes, the MBFV-related viruses appear to be specific to mosquitoes similar to the insect-specific flaviviruses (ISFs). In this overview of MBFV-related viruses we conclude that they differ from the ISFs genetically and antigenically. Phylogenetic analyses separated the MBFV-related viruses isolated in Africa, the Middle East and South America from those isolated in Europe and Asia. Serological cross-reactions of MBFV-related viruses with other flaviviruses and their potential for vector-borne transmission require further characterization. The divergent MBFV-related viruses are probably significantly under sampled to date and provide new information on the variety, properties and evolution of vector-borne flaviviruses., Highlights • Mosquito-borne flavivirus-related viruses were isolated from Finnish mosquitoes. • Isolates were reactive with flavivirus antibodies but appeared mosquito-specific. • Sequence analysis identified related viruses from different parts of the world. • These viruses represent unique properties among the mosquito-borne flavivirus group.

In all biological systems, RNAs are associated with RNA-binding proteins (RBPs), forming complexes that control gene regulatory mechanisms, from RNA synthesis to decay. In mammalian mitochondria, post-transcriptional regulation of gene expression is conducted by mitochondrial RBPs (mt-RBPs) at various stages of mt-RNA metabolism, including polycistronic transcript production, its processing into individual transcripts, mt-RNA modifications, stability, translation and degradation. To date, only a handful of mt-RBPs have been characterized. Here, we describe a putative human mitochondrial protein, C6orf203, that contains an S4-like domain—an evolutionarily conserved RNA-binding domain previously identified in proteins involved in translation. Our data show C6orf203 to bind highly structured RNA in vitro and associate with the mitoribosomal large subunit in HEK293T cells. Knockout of C6orf203 leads to a decrease in mitochondrial translation and consequent OXPHOS deficiency, without affecting mitochondrial RNA levels. Although mitoribosome stability is not affected in C6orf203-depleted cells, mitoribosome profiling analysis revealed a global disruption of the association of mt-mRNAs with the mitoribosome, suggesting that C6orf203 may be required for the proper maturation and functioning of the mitoribosome. We therefore propose C6orf203 to be a novel RNA-binding protein involved in mitochondrial translation, expanding the repertoire of factors engaged in this process.

The substitution rates of viral polymerases have been studied extensively. However less is known about the tendency of these enzymes to 'slip' during RNA synthesis to produce progeny RNAs with nucleotide insertions or deletions. We recently described the functional utilization of programmed polymerase slippage in the family Potyviridae. This slippage results in either an insertion or a substitution, depending on whether the RNA duplex realigns following the insertion. In this study we investigated whether this phenomenon is a conserved feature of superfamily I viral RdRps, by inserting a range of potyvirus-derived slip-prone sequences into a picornavirus, Theiler's murine encephalomyelitis virus (TMEV). Deep-sequencing analysis of viral transcripts indicates that the TMEV polymerase 'slips' at the sequences U6-7 and A6-7 to insert additional nucleotides. Such sequences are under-represented within picornaviral genomes, suggesting that slip-prone sequences create a fitness cost. Nonetheless, the TMEV insertional and substitutional spectrum differed from that previously determined for the potyvirus polymerase.

Most viruses in the genus Flavivirus are horizontally transmitted between hematophagous arthropods and vertebrate hosts, but some are maintained in arthropod- or vertebrate-restricted transmission cycles. Flaviviruses maintained by vertebrate-only transmission are commonly referred to as no known vector (NKV) flaviviruses. Fourteen species and two subtypes of NKV flaviviruses are recognized by the International Committee on Taxonomy of Viruses (ICTV), and Tamana bat virus potentially belongs to this group. NKV flaviviruses have been isolated in nature almost exclusively from bats and rodents; exceptions are the two isolates of Dakar bat virus recovered from febrile humans and the recent isolations of Sokoluk virus from field-collected ticks, which raises questions as to whether it should remain classified as an NKV flavivirus. There is evidence to suggest that two other NKV flaviviruses, Entebbe bat virus and Yokose virus, may also infect arthropods in nature. The best characterized bat- and rodent-associated NKV flaviviruses are Rio Bravo and Modoc viruses, respectively, but both have received limited research attention compared to many of their arthropod-infecting counterparts. Herein, we provide a comprehensive review of NKV flaviviruses, placing a particular emphasis on their classification, host range, geographic distribution, replication kinetics, pathogenesis, transmissibility and molecular biology.