• CP: Molecular biology
• RNA structure
• cryo-EM
• reinitiation
• ribosome
• viral RNA

• RNA, Viral/metabolism
• RNA, Viral/genetics
• Ribosomes/metabolism
• Nucleic Acid Conformation
• Cryoelectron Microscopy
• Protein Biosynthesis
• Peptide Chain Initiation, Translational
• RNA, Messenger/metabolism
• RNA, Messenger/genetics
• Models, Molecular

• R35 GM118070 NIGMS NIH HHS
• T32 GM136444 NIGMS NIH HHS

• GFP
• RNA splicing
• bicistronic mRNA
• codon usage
• downstream ORF
• elongation
• eukaryote
• fluorescence
• main ORF
• polycistronic mRNA
• transcription
• translation
• upstream ORF

• Humans
• Open Reading Frames
• RNA, Messenger/genetics
• Protein Biosynthesis
• RNA Splicing
• Codon Usage
• HEK293 Cells

• Fondation pour la Recherche Médicale
• European Commission
• Fondation pour la Recherche Médicale
• European Commission

• R35 GM118070 NIGMS NIH HHS

• Caliciviridae
• Sanovirus
• Temminck’s stint calicivirus
• genome annotation
• phylogenetics

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.

Many RNA viruses employ programmed –1 ribosomal frameshifting (PRF) to expand their coding capacity and optimize production of viral proteins. Here, the authors report structural and biophysical analysis of protein 2A from a cardiovirus, with insights into the mechanism of its PRF-stimulatory function.

Leptospirosis is a zoonosis caused by the pathogenic bacteria of the genus Leptospira. The identification of conserved outer membrane proteins among pathogenic strains is a major research target in elucidating mechanisms of pathogenicity. Surface-exposed proteins are most probably the ones involved in the interaction of leptospires with the environment. Some spirochetes use outer membrane proteases as a way to penetrate host tissues. HtrA is a family of proteins found in various cell types, from prokaryotes to primates. They are a set of proteases usually composed of a serine protease and PDZ domains, and they are generally transported to the periplasm. Here, we identified four genes—annotated as HtrA, LIC11111, LIC20143, LIC20144 and LIC11037—and another one annotated as a serine protease, LIC11112. It is believed that the last forms a functional heterodimer with LIC11111, since they are organized in one operon. Our analyses showed that these proteins are highly conserved among pathogenic strains. LIC11112, LIC20143, and LIC11037 have the serine protease domain with the conserved catalytic triad His-Asp-Ser. This is the first bioinformatics analysis of HtrA proteins from Leptospira that suggests their proteolytic activity potential. Experimental studies are warranted to elucidate this possibility.

• HtrA protein
• Leptospira
• leptospirosis
• pathogenesis

• Saccharomyces cerevisiae
• BAX
• PDR1
• apoptosis
• attenuated expression
• eukaryotic translation coupling
• xenobiotic binding

• histone deacetylase 7
• peptide
• phosphorylation
• short open reading frame
• vascular progenitor cell

The genomes of many prokaryotes contain substantial fractions of gene pairs with overlapping stop and start codons (ATGA or TGATG). A potential benefit of overlapping gene pairs is translational coupling. In 720 genomes of archaea and bacteria representing all major phyla, we identify substantial, albeit highly variable, fractions of co-directed overlapping gene pairs. Various patterns are observed for the utilization of the SD motif for de novo initiation at upstream genes versus reinitiation at overlapping gene pairs. We experimentally test the predicted coupling in 9 gene pairs from the archaeon Haloferax volcanii and 5 gene pairs from the bacterium Escherichia coli. In 13 of 14 cases, translation of both genes is strictly coupled. Mutational analysis of SD motifs located upstream of the downstream genes indicate that the contribution of the SD to translational coupling widely varies from gene to gene. The nearly universal, abundant occurrence of overlapping gene pairs suggests that tight translational coupling is widespread in archaea and bacteria.

Archaea and bacteria often have gene pairs with overlapping stop and start codons, suggesting translational coupling. Here, Huber et al. analyse overlapping gene pairs from 720 genomes, and validate translational coupling via termination-reinitiation for 14 gene pairs in Haloferax volcanii and Escherichia coli.

Caliciviruses use a termination/reinitiation mechanism for translation of their minor capsid protein VP2. A sequence element of about 80 nucleotides denoted ‘termination upstream ribosomal binding site’ (TURBS) is crucial for reinitiation. RNA secondary structure probing and computer aided secondary structure prediction revealed a rather low degree of secondary structure determinants for the TURBS of the rabbit hermorrhagic disease virus. Mutation analysis showed that prevention of duplex formation had major impact on the VP2 expression levels. Restoration of complementarity of the respective sequences by reciprocal mutation at least partially restored reinitiating rates. Synthetic TURBS structures preserving only the secondary structure forming sequences and the known short motifs important for TURBS function were found to drive reinitiation when the altered sequence could be predicted to allow establishment of the crucial secondary structures of the TURBS.

The differences between high risk and low risk human papillomaviruses (HR-HPV and LR-HPV, respectively) that contribute to the tumorigenic potential of HR-HPV are not well understood but can be expected to involve the HPV oncoproteins, E6 and E7. We combine genome comparison and machine learning techniques to identify a previously unnoticed insert near the 3’-end of the E6 oncoprotein gene that is unique to HR-HPV. Analysis of the insert sequence suggests that it exerts a dual effect, by creating a PDZ domain-binding motif at the C-terminus of E6, as well as eliminating the overlap between the E6 and E7 coding regions in HR-HPV. We show that, as a result, the insert might enable coupled termination-reinitiation of the E6 and E7 genes, supported by motifs complementary to the human 18S rRNA. We hypothesize that the added functionality of E6 and positive regulation of E7 expression jointly account for the tumorigenic potential of HR-HPV.

• Papillomaviruses
• cervical cancer
• machine learning
• oncogenic risk
• translation terminatio-reinitiation

Viruses are genetically diverse, infect a wide range of tissues and host cells and follow unique processes for replicating themselves. All these processes were investigated and indexed in ViralZone knowledge base. To facilitate standardizing data, a simple ontology of viral life-cycle terms was developed to provide a common vocabulary for annotating data sets. New terminology was developed to address unique viral replication cycle processes, and existing terminology was modified and adapted. The virus life-cycle is classically described by schematic pictures. Using this ontology, it can be represented by a combination of successive terms: “entry”, “latency”, “transcription”, “replication” and “exit”. Each of these parts is broken down into discrete steps. For example Zika virus “entry” is broken down in successive steps: “Attachment”, “Apoptotic mimicry”, “Viral endocytosis/ macropinocytosis”, “Fusion with host endosomal membrane”, “Viral factory”. To demonstrate the utility of a standard ontology for virus biology, this work was completed by annotating virus data in the ViralZone, UniProtKB and Gene Ontology databases.

The unicellular green alga Chlamydomonas reinhardtii is a model organism that provides an opportunity to understand the evolution and functional biology of the lineage that includes the land plants, as well as aspects of the fundamental core biology conserved throughout the eukaryotic phylogeny. Although many tools are available to facilitate genetic, molecular biological, biochemical, and cell biological studies in Chlamydomonas, expression of unselected transgenes of interest (GOIs) has been challenging. In most methods used previously, the GOI and a selectable marker are expressed from two separate mRNAs, so that their concomitant expression is not guaranteed. In this study, we developed constructs that allow expression of an upstream GOI and downstream selectable marker from a single bicistronic mRNA. Although this approach in other systems has typically required a translation-enhancing element such as an internal ribosome entry site for the downstream marker, we found that a short stretch of unstructured junction sequence was sufficient to obtain adequate expression of the downstream gene, presumably through post-termination reinitiation. With this system, we obtained robust expression of both endogenous and heterologous GOIs, including fluorescent proteins and tagged fusion proteins, in the vast majority of transformants, thus eliminating the need for tedious secondary screening for GOI-expressing transformants. This improved efficiency should greatly facilitate a variety of genetic and cell-biological studies in Chlamydomonas and also enable new applications such as expression-based screens and large-scale production of foreign proteins.

• IRES
• algae
• bicistronic mRNA
• transgene expression
• translation reinitiation

• Homologous recombination
• Organelle genome editing
• Overlapping genes
• Rubisco LS mutation
• Translational coupling

• Chloroplasts/genetics
• Chloroplasts/metabolism
• Gene Editing/methods
• Genome, Chloroplast
• Plant Proteins/genetics
• Plant Proteins/metabolism
• Point Mutation
• Nicotiana/genetics
• Nicotiana/metabolism

• BB/I011552/1 Biotechnology and Biological Sciences Research Council
• University of Manchester

RNA-seq technologies have provided significant insight into the transcription networks of mycobacteria. However, such studies provide no definitive information on the translational landscape. Here, we use a combination of high-throughput transcriptome and proteome-profiling approaches to more rigorously understand protein expression in two mycobacterial species. RNA-seq and ribosome profiling in Mycobacterium smegmatis, and transcription start site (TSS) mapping and N-terminal peptide mass spectrometry in Mycobacterium tuberculosis, provide complementary, empirical datasets to examine the congruence of transcription and translation in the Mycobacterium genus. We find that nearly one-quarter of mycobacterial transcripts are leaderless, lacking a 5’ untranslated region (UTR) and Shine-Dalgarno ribosome-binding site. Our data indicate that leaderless translation is a major feature of mycobacterial genomes and is comparably robust to leadered initiation. Using translational reporters to systematically probe the cis-sequence requirements of leaderless translation initiation in mycobacteria, we find that an ATG or GTG at the mRNA 5’ end is both necessary and sufficient. This criterion, together with our ribosome occupancy data, suggests that mycobacteria encode hundreds of small, unannotated proteins at the 5’ ends of transcripts. The conservation of small proteins in both mycobacterial species tested suggests that some play important roles in mycobacterial physiology. Our translational-reporter system further indicates that mycobacterial leadered translation initiation requires a Shine Dalgarno site in the 5’ UTR and that ATG, GTG, TTG, and ATT codons can robustly initiate translation. Our combined approaches provide the first comprehensive view of mycobacterial gene structures and their non-canonical mechanisms of protein expression.

The current paradigm for bacterial translation is based on an mRNA that includes an untranslated leader sequence containing the ribosome-binding site upstream of the initiation codon. We applied genome-scale approaches to map the protein-coding regions in the genomes of Mycobacterium smegmatis and Mycobacterium tuberculosis. We found that nearly one-quarter of mycobacterial transcripts are leaderless in mycobacterial species, thus indicating that ribosomes must recognize these mRNAs by a novel mechanism and suggesting that there are alternative modes of bacterial translation beyond the Escherichia coli paradigm. Our translational profiling showed that many mycobacterial proteins are mis-annotated, and also found many new genes encoding small proteins that had been previously overlooked, which are likely to play novel roles in diverse cellular processes. We also developed a new reporter system that provides mechanistic insights into translation initiation through deep sequencing. Our data show that leaderless translation is a robust process that is conserved in mycobacteria, that leaderless translation only requires that the mRNA begin with a start codon, and predict that mycobacteria encode hundreds of small proteins. This work will help us understand gene structure, genome organization and protein expression in bacteria, and how the translational machinery differs in different organisms.

• Cap-independent translation
• Carmovirus
• IRES
• Leaky scanning
• Necrovirus
• Plant virus
• RNA structure
• RNA virus
• Readthrough
• Tombusviridae

There is a continuing need for novel antivirals to treat hepatitis B virus (HBV) infection, as it remains a major health problem worldwide. Ideally new classes of antivirals would target multiple steps in the viral lifecycle. In this review, we consider the steps in which HBV RNAs are processed, exported from the nucleus and translated. These are often overlooked steps in the HBV life-cycle. HBV, like retroviruses, incorporates a number of unusual steps in these processes, which use a combination of viral and host cellular machinery. Some of these unusual steps deserve a closer scrutiny. They may provide alternative targets to existing antiviral therapies, which are associated with increasing drug resistance. The RNA post-transcriptional regulatory element identified 20 years ago promotes nucleocytoplasmic export of all unspliced HBV RNAs. There is evidence that inhibition of this step is part of the antiviral action of interferon. Similarly, the structured RNA epsilon element situated at the 5’ end of the polycistronic HBV pregenomic RNA also performs key roles during HBV replication. The pregenomic RNA, which is the template for translation of both the viral core and polymerase proteins, is also encapsidated and used in replication. This complex process, regulated at the epsilon element, also presents an attractive antiviral target. These RNA elements that mediate and regulate gene expression are highly conserved and could be targeted using novel strategies employing RNAi, miRNAs or aptamers. Such approaches targeting these functionally constrained genomic regions should avoid escape mutations. Therefore understanding these regulatory elements, along with providing potential targets, may also facilitate the development of other new classes of antiviral drugs.

• Hepatitis B virus
• Translational control
• Antiviral
• Nuclear export
• Post-transcriptional control
• Nucleocytoplasmic export

• Animals
• Antiviral Agents/therapeutic use
• Drug Design
• Gene Expression Regulation, Viral/drug effects
• Hepatitis B/diagnosis
• Hepatitis B/drug therapy
• Hepatitis B/virology
• Hepatitis B virus/drug effects
• Hepatitis B virus/genetics
• Hepatitis B virus/growth & development
• Humans
• RNA Processing, Post-Transcriptional/drug effects
• RNA, Viral/genetics
• RNA, Viral/metabolism
• Viral Proteins/genetics
• Viral Proteins/metabolism

Expression of the minor virion structural protein VP2 of the calicivirus murine norovirus (MNV) is believed to occur by the unusual mechanism of termination codon-dependent reinitiation of translation. In this process, following translation of an upstream open reading frame (ORF) and termination at the stop codon, a proportion of 40S subunits remain associated with the mRNA and reinitiate at the AUG of a downstream ORF, which is typically in close proximity. Consistent with this, the VP2 start codon (AUG) of MNV overlaps the stop codon of the upstream VP1 ORF (UAA) in the pentanucleotide UAAUG.

Here, we confirm that MNV VP2 expression is regulated by termination-reinitiation and define the mRNA sequence requirements. Efficient reintiation is dependent upon 43 nt of RNA immediately upstream of the UAAUG site. Chemical and enzymatic probing revealed that the RNA in this region is not highly structured and includes an essential stretch of bases complementary to 18S rRNA helix 26 (Motif 1). The relative position of Motif 1 with respect to the UAAUG site impacts upon the efficiency of the process. Termination-reinitiation in MNV was also found to be relatively insensitive to the initiation inhibitor edeine.

The termination-reinitiation signal of MNV most closely resembles that of influenza BM2. Similar to other viruses that use this strategy, base-pairing between mRNA and rRNA is likely to play a role in tethering the 40S subunit to the mRNA following termination at the VP1 stop codon. Our data also indicate that accurate recognition of the VP2 ORF AUG is not a pre-requisite for efficient reinitiation of translation in this system.

• 5' Flanking Region/genetics
• Animals
• Base Sequence
• Codon, Initiator/genetics
• Codon, Terminator/genetics
• Edeine/pharmacology
• Luciferases/metabolism
• Mice
• Molecular Sequence Data
• Norovirus/drug effects
• Norovirus/metabolism
• Nucleic Acid Conformation
• Nucleotides/genetics
• Peptide Chain Initiation, Translational/drug effects
• Peptide Chain Termination, Translational/drug effects
• RNA, Complementary/genetics
• RNA, Messenger/genetics
• RNA, Ribosomal, 18S/genetics
• RNA, Viral/chemistry
• RNA, Viral/genetics
• Regulatory Sequences, Nucleic Acid/genetics
• Viral Proteins/metabolism

• BB/C007034/1 Biotechnology and Biological Sciences Research Council
• BB/D008190/1 Biotechnology and Biological Sciences Research Council
• RG41021 Biotechnology and Biological Sciences Research Council

Borna disease virus (BDV) is a nonsegmented, negative-strand RNA virus that employs several unique strategies for gene expression. The shortest transcript of BDV, X/P mRNA, encodes at least three open reading frames (ORFs): upstream ORF (uORF), X, and P in the 5′ to 3′ direction. The X is a negative regulator of viral polymerase activity, while the P phosphoprotein is a necessary cofactor of the polymerase complex, suggesting that the translation of X is controlled rigorously, depending on viral replication. However, the translation mechanism used by the X/P polycistronic mRNA has not been determined in detail. Here we demonstrate that the X/P mRNA autogenously regulates the translation of X via interaction with host factors. Transient transfection of cDNA clones corresponding to the X/P mRNA revealed that the X ORF is translated predominantly by uORF-termination-coupled reinitiation, the efficiency of which is upregulated by expression of P. We found that P may enhance ribosomal reinitiation at the X ORF by inhibition of the interaction of the DEAD-box RNA helicase DDX21 with the 5′ untranslated region of X/P mRNA, via interference with its phosphorylation. Our results not only demonstrate a unique translational control of viral regulatory protein, but also elucidate a previously unknown mechanism of regulation of polycistronic mRNA translation using RNA helicases.

All viruses rely on host cell factors to complete their life cycles. Therefore, the replication strategies of viruses may provide not only the understanding of virus pathogenesis but also useful models to disentangle the complex machinery of host cells. Translation regulation of viral mRNA is a good example of this. Borna disease virus (BDV) is a highly neurotropic RNA virus which is characterized by persistent infection. BDV expresses mRNAs as polycistronic coding transcripts. Among them, the 0.8 kb X/P mRNA encodes at least three open reading frames (ORFs), upstream ORF, X, and P. Although BDV X and P have opposing effects in terms of viral polymerase activity, the translational regulation of X/P polycistronic mRNA has not been elucidated. In this study, we show an ingenious strategy of translational control of viral regulatory protein using host factors. We demonstrate that host RNA helicases, mainly DDX21, can affect ribosomal reinitiation of X via interaction with the 5′ untranslated region (UTR) of X/P mRNA and that the downstream P protein autogenously controls the translation of X by interfering with the binding of DDX21 to the 5′ UTR. Our findings uncover not only a unique translational control of viral regulatory protein but also a previously unknown mechanism of translational regulation of polycistronic mRNA using RNA helicases.

• Borna disease virus/genetics
• Gene Expression Regulation, Viral
• Host-Pathogen Interactions
• RNA Helicases/metabolism
• RNA, Viral/metabolism
• Viral Regulatory and Accessory Proteins/genetics

• virus
• translation
• protein synthesis
• pkr
• perk
• upstream open reading frame
• ires
• frameshifting
• 4ebp
• reinitiation

• Animals
• Eukaryotic Initiation Factor-2/metabolism
• Host-Pathogen Interactions
• Humans
• Mammals/metabolism
• Mammals/virology
• Phosphorylation
• Protein Biosynthesis
• Viral Proteins/biosynthesis
• Viruses/metabolism