Alpha- and betacoronavirus cis-acting RNA elements.

Coronaviruses have exceptionally large RNA genomes and employ multiprotein replication/transcription complexes to orchestrate specific steps of viral RNA genome replication and expression. Most of these processes involve viral cis-acting RNA elements that are engaged in vital RNA-RNA and/or RNA-protein interactions. Over the past years, a large number of studies provided interesting new insight into the structures and, to a lesser extent, functions of specific RNA elements for representative coronaviruses, and there is evidence to suggest that (a majority of) these RNA elements are conserved across genetically divergent coronavirus genera. It is becoming increasingly clear that at least some of these elements do not function in isolation but operate through complex and highly dynamic RNA-RNA interactions. This article reviews structural and functional aspects of cis-acting RNA elements conserved in alpha- and betacoronavirus 5'- and 3'-terminal genome regions, focusing on their critical roles in viral RNA synthesis and gene expression.

Conserved Characteristics of NMPylation Activities of Alpha- and Betacoronavirus NiRAN Domains.

Coronavirus genome replication and expression are mediated by the viral replication-transcription complex (RTC) which is assembled from multiple nonstructural proteins (nsp). Among these, nsp12 represents the central functional subunit. It harbors the RNA-directed RNA polymerase (RdRp) domain and contains, at its N terminus, an additional domain called NiRAN which is widely conserved in coronaviruses and other nidoviruses. In this study, we produced bacterially expressed coronavirus nsp12s to investigate and compare NiRAN-mediated NMPylation activities from representative alpha- and betacoronaviruses. We found that the four coronavirus NiRAN domains characterized to date have a number of conserved properties, including (i) robust nsp9-specific NMPylation activities that appear to operate largely independently of the C-terminal RdRp domain, (ii) nucleotide substrate preference for UTP followed by ATP and other nucleotides, (iii) dependence on divalent metal ions, with Mn2+ being preferred over Mg2+, and (iv) a key role of N-terminal residues (particularly Asn2) of nsp9 for efficient formation of a covalent phosphoramidate bond between NMP and the N-terminal amino group of nsp9. In this context, a mutational analysis confirmed the conservation and critical role of Asn2 across different subfamilies of the family Coronaviridae, as shown by studies using chimeric coronavirus nsp9 variants in which six N-terminal residues were replaced with those from other corona-, pito- and letovirus nsp9 homologs. The combined data of this and previous studies reveal a remarkable degree of conservation among coronavirus NiRAN-mediated NMPylation activities, supporting a key role of this enzymatic activity in viral RNA synthesis and processing. IMPORTANCE There is strong evidence that coronaviruses and other large nidoviruses evolved a number of unique enzymatic activities, including an additional RdRp-associated NiRAN domain, that are conserved in nidoviruses but not in most other RNA viruses. Previous studies of the NiRAN domain mainly focused on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and suggested different functions for this domain, such as NMPylation/RNAylation of nsp9, RNA guanylyltransferase activities involved in canonical and/or unconventional RNA capping pathways, and other functions. To help resolve partly conflicting information on substrate specificities and metal ion requirements reported previously for the SARS-CoV-2 NiRAN NMPylation activity, we extended these earlier studies by characterizing representative alpha- and betacoronavirus NiRAN domains. The study revealed that key features of NiRAN-mediated NMPylation activities, such as protein and nucleotide specificity and metal ion requirements, are very well conserved among genetically divergent coronaviruses, suggesting potential avenues for future antiviral drug development targeting this essential viral enzyme.

Phosphorylation of the PA subunit of influenza polymerase at Y393 prevents binding of the 5'-termini of RNA and polymerase function.

The influenza A virus (IAV) polymerase is a multifunctional machine that can adopt alternative configurations to perform transcription and replication of the viral RNA genome in a temporally ordered manner. Although the structure of polymerase is well understood, our knowledge of its regulation by phosphorylation is still incomplete. The heterotrimeric polymerase can be regulated by posttranslational modifications, but the endogenously occurring phosphorylations at the PA and PB2 subunits of the IAV polymerase have not been studied. Mutation of phosphosites in PB2 and PA subunits revealed that PA mutants resembling constitutive phosphorylation have a partial (S395) or complete (Y393) defect in the ability to synthesize mRNA and cRNA. As PA phosphorylation at Y393 prevents binding of the 5' promoter of the genomic RNA, recombinant viruses harboring such a mutation could not be rescued. These data show the functional relevance of PA phosphorylations to control the activity of viral polymerase during the influenza infectious cycle.

Rocaglates as Antivirals: Comparing the Effects on Viral Resistance, Anti-Coronaviral Activity, RNA-Clamping on eIF4A and Immune Cell Toxicity.

Rocaglates are potent broad-spectrum antiviral compounds with a promising safety profile. They inhibit viral protein synthesis for different RNA viruses by clamping the 5'-UTRs of mRNAs onto the surface of the RNA helicase eIF4A. Apart from the natural rocaglate silvestrol, synthetic rocaglates like zotatifin or CR-1-31-B have been developed. Here, we compared the effects of rocaglates on viral 5'-UTR-mediated reporter gene expression and binding to an eIF4A-polypurine complex. Furthermore, we analyzed the cytotoxicity of rocaglates on several human immune cells and compared their antiviral activities in coronavirus-infected cells. Finally, the potential for developing viral resistance was evaluated by passaging human coronavirus 229E (HCoV-229E) in the presence of increasing concentrations of rocaglates in MRC-5 cells. Importantly, no decrease in rocaglate-sensitivity was observed, suggesting that virus escape mutants are unlikely to emerge if the host factor eIF4A is targeted. In summary, all three rocaglates are promising antivirals with differences in cytotoxicity against human immune cells, RNA-clamping efficiency, and antiviral activity. In detail, zotatifin showed reduced RNA-clamping efficiency and antiviral activity compared to silvestrol and CR-1-31-B, but was less cytotoxic for immune cells. Our results underline the potential of rocaglates as broad-spectrum antivirals with no indications for the emergence of escape mutations in HCoV-229E.

sRNA-mediated RNA processing regulates bacterial cell division.

Tight control of cell division is essential for survival of most organisms. For prokaryotes, the regulatory mechanisms involved in the control of cell division are mostly unknown. We show that the small non-coding sRNA StsR has an important role in controlling cell division and growth in the alpha-proteobacterium Rhodobacter sphaeroides. StsR is strongly induced by stress conditions and in stationary phase by the alternative sigma factors RpoHI/HII, thereby providing a regulatory link between cell division and environmental cues. Compared to the wild type, a mutant lacking StsR enters stationary phase later and more rapidly resumes growth after stationary phase. A target of StsR is UpsM, the most abundant sRNA in the exponential phase. It is derived from partial transcriptional termination within the 5' untranslated region of the mRNA of the division and cell wall (dcw) gene cluster. StsR binds to UpsM as well as to the 5' UTR of the dcw mRNA and the sRNA-sRNA and sRNA-mRNA interactions lead to a conformational change that triggers cleavage by the ribonuclease RNase E, affecting the level of dcw mRNAs and limiting growth. These findings provide interesting new insights into the role of sRNA-mediated regulation of cell division during the adaptation to environmental changes.

Hallmarks of Alpha- and Betacoronavirus non-structural protein 7+8 complexes.

Coronaviruses infect many different species including humans. The last two decades have seen three zoonotic coronaviruses, with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) causing a pandemic in 2020. Coronaviral non-structural proteins (nsps) form the replication-transcription complex (RTC). Nsp7 and nsp8 interact with and regulate the RNA-dependent RNA-polymerase and other enzymes in the RTC. However, the structural plasticity of nsp7+8 complexes has been under debate. Here, we present the framework of nsp7+8 complex stoichiometry and topology based on native mass spectrometry and complementary biophysical techniques of nsp7+8 complexes from seven coronaviruses in the genera Alpha- and Betacoronavirus including SARS-CoV-2. Their complexes cluster into three groups, which systematically form either heterotrimers or heterotetramers or both, exhibiting distinct topologies. Moreover, even at high protein concentrations, SARS-CoV-2 nsp7+8 consists primarily of heterotetramers. From these results, the different assembly paths can be pinpointed to specific residues and an assembly model proposed.

Coronavirus replication-transcription complex: Vital and selective NMPylation of a conserved site in nsp9 by the NiRAN-RdRp subunit.

RNA-dependent RNA polymerases (RdRps) of the Nidovirales (Coronaviridae, Arteriviridae, and 12 other families) are linked to an amino-terminal (N-terminal) domain, called NiRAN, in a nonstructural protein (nsp) that is released from polyprotein 1ab by the viral main protease (Mpro). Previously, self-GMPylation/UMPylation activities were reported for an arterivirus NiRAN-RdRp nsp and suggested to generate a transient state primed for transferring nucleoside monophosphate (NMP) to (currently unknown) viral and/or cellular biopolymers. Here, we show that the coronavirus (human coronavirus [HCoV]-229E and severe acute respiratory syndrome coronavirus 2) nsp12 (NiRAN-RdRp) has Mn2+-dependent NMPylation activity that catalyzes the transfer of a single NMP to the cognate nsp9 by forming a phosphoramidate bond with the primary amine at the nsp9 N terminus (N3825) following Mpro-mediated proteolytic release of nsp9 from N-terminally flanking nsps. Uridine triphosphate was the preferred nucleotide in this reaction, but also adenosine triphosphate, guanosine triphosphate, and cytidine triphosphate were suitable cosubstrates. Mutational studies using recombinant coronavirus nsp9 and nsp12 proteins and genetically engineered HCoV-229E mutants identified residues essential for NiRAN-mediated nsp9 NMPylation and virus replication in cell culture. The data corroborate predictions on NiRAN active-site residues and establish an essential role for the nsp9 N3826 residue in both nsp9 NMPylation in vitro and virus replication. This residue is part of a conserved N-terminal NNE tripeptide sequence and shown to be the only invariant residue in nsp9 and its homologs in viruses of the family Coronaviridae The study provides a solid basis for functional studies of other nidovirus NMPylation activities and suggests a possible target for antiviral drug development.

Structure-Activity Relationships of Benzamides and Isoindolines Designed as SARS-CoV Protease Inhibitors Effective against SARS-CoV-2.

Inhibition of coronavirus (CoV)-encoded papain-like cysteine proteases (PLpro ) represents an attractive strategy to treat infections by these important human pathogens. Herein we report on structure-activity relationships (SAR) of the noncovalent active-site directed inhibitor (R)-5-amino-2-methyl-N-(1-(naphthalen-1-yl)ethyl) benzamide (2 b), which is known to bind into the S3 and S4 pockets of the SARS-CoV PLpro . Moreover, we report the discovery of isoindolines as a new class of potent PLpro inhibitors. The studies also provide a deeper understanding of the binding modes of this inhibitor class. Importantly, the inhibitors were also confirmed to inhibit SARS-CoV-2 replication in cell culture suggesting that, due to the high structural similarities of the target proteases, inhibitors identified against SARS-CoV PLpro are valuable starting points for the development of new pan-coronaviral inhibitors.

Hallmarks of Alpha- and Betacoronavirus non-structural protein 7+8 complexes.

Coronaviruses infect many different species including humans. The last two decades have seen three zoonotic coronaviruses with SARS-CoV-2 causing a pandemic in 2020. Coronaviral non-structural proteins (nsp) built up the replication-transcription complex (RTC). Nsp7 and nsp8 interact with and regulate the RNA-dependent RNA-polymerase and other enzymes in the RTC. However, the structural plasticity of nsp7+8 complex has been under debate. Here, we present the framework of nsp7+8 complex stoichiometry and topology based on a native mass spectrometry and complementary biophysical techniques of nsp7+8 complexes from seven coronaviruses in the genera Alpha- and Betacoronavirus including SARS-CoV-2. Their complexes cluster into three groups, which systematically form either heterotrimers or heterotetramers or both, exhibiting distinct topologies. Moreover, even at high protein concentrations mainly heterotetramers are observed for SARS-CoV-2 nsp7+8. From these results, the different assembly paths can be pinpointed to specific residues and an assembly model is proposed.

Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis.

Sequence analyses of RNA virus genomes remain challenging owing to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called "quasispecies." Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (1) RNA virus genomes and (2) subgenome-length (sg) RNAs composed of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. By using DRS, we were able to map the longest (∼26-kb) contiguous read to the viral reference genome. By combining Illumina and Oxford Nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV)-229E genome (27.3 kb). Furthermore, by using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by showing the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that DRS may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.

Transcription attenuation-derived small RNA rnTrpL regulates tryptophan biosynthesis gene expression in trans.

Ribosome-mediated transcription attenuation is a basic posttranscriptional regulation mechanism in bacteria. Liberated attenuator RNAs arising in this process are generally considered nonfunctional. In Sinorhizobium meliloti, the tryptophan (Trp) biosynthesis genes are organized into three operons, trpE(G), ppiD-trpDC-moaC-moeA, and trpFBA-accD-folC, of which only the first one, trpE(G), contains a short ORF (trpL) in the 5'-UTR and is regulated by transcription attenuation. Under conditions of Trp sufficiency, transcription is terminated between trpL and trpE(G), and a small attenuator RNA, rnTrpL, is produced. Here, we show that rnTrpL base-pairs with trpD and destabilizes the polycistronic trpDC mRNA, indicating rnTrpL-mediated downregulation of the trpDC operon in trans. Although all three trp operons are regulated in response to Trp availability, only in the two operons trpE(G) and trpDC the Trp-mediated regulation is controlled by rnTrpL. Together, our data show that the trp attenuator coordinates trpE(G) and trpDC expression posttranscriptionally by two fundamentally different mechanisms: ribosome-mediated transcription attenuation in cis and base-pairing in trans. Also, we present evidence that rnTrpL-mediated regulation of trpDC genes expression in trans is conserved in Agrobacterium and Bradyrhizobium, suggesting that the small attenuator RNAs may have additional conserved functions in the control of bacterial gene expression.

Identification and Characterization of a Human Coronavirus 229E Nonstructural Protein 8-Associated RNA 3'-Terminal Adenylyltransferase Activity.

Coronavirus nonstructural protein 8 (nsp8) has been suggested to have diverse activities, including noncanonical template-dependent polymerase activities. Here, we characterized a recombinant form of the human coronavirus 229E (HCoV-229E) nsp8 and found that the protein has metal ion-dependent RNA 3'-terminal adenylyltransferase (TATase) activity, while other nucleotides were not (or very inefficiently) transferred to the 3' ends of single-stranded and (fully) double-stranded acceptor RNAs. Using partially double-stranded RNAs, very efficient TATase activity was observed if the opposite (template) strand contained a short 5' oligo(U) sequence, while very little (if any) activity was detected for substrates with other homopolymeric or heteropolymeric sequences in the 5' overhang. The oligo(U)-assisted/templated TATase activity on partial-duplex RNAs was confirmed for two other coronavirus nsp8 proteins, suggesting that the activity is conserved among coronaviruses. Replacement of a conserved Lys residue with Ala abolished the in vitro RNA-binding and TATase activities of nsp8 and caused a nonviable phenotype when the corresponding mutation was introduced into the HCoV-229E genome, confirming that these activities are mediated by nsp8 and critical for viral replication. In additional experiments, we obtained evidence that nsp8 has a pronounced specificity for adenylate and is unable to incorporate guanylate into RNA products, which strongly argues against the previously proposed template-dependent RNA polymerase activity of this protein. Given the presence of an oligo(U) stretch at the 5' end of coronavirus minus-strand RNAs, it is tempting to speculate (but remains to be confirmed) that the nsp8-mediated TATase activity is involved in the 3' polyadenylation of viral plus-strand RNAs.IMPORTANCE Previously, coronavirus nsp8 proteins were suggested to have template-dependent RNA polymerase activities resembling those of RNA primases or even canonical RNA-dependent RNA polymerases, while more recent studies have suggested an essential cofactor function of nsp8 (plus nsp7) for nsp12-mediated RNA-dependent RNA polymerase activity. In an effort to reconcile conflicting data from earlier studies, the study revisits coronavirus nsp8-associated activities using additional controls and proteins. The data obtained for three coronavirus nsp8 proteins provide evidence that the proteins share metal ion-dependent RNA 3' polyadenylation activities that are greatly stimulated by a short oligo(U) stretch in the template strand. In contrast, nsp8 was found to be unable to select and incorporate appropriate (matching) nucleotides to produce cRNA products from heteropolymeric and other homooligomeric templates. While confirming the critical role of nsp8 in coronavirus replication, the study amends the list of activities mediated by coronavirus nsp8 proteins in the absence of other proteins.

Characterization of a bafinivirus exoribonuclease activity.

White bream virus (WBV), a poorly characterized plus-strand RNA virus infecting freshwater fish of the Cyprinidae family, is the prototype species of the genus Bafinivirus in the subfamily Torovirinae (family Coronaviridae, order Nidovirales). In common with other nidoviruses featuring >20 kilobase genomes, bafiniviruses have been predicted to encode an exoribonuclease (ExoN) in their replicase gene. Here, we used information on the substrate specificity of bafinivirus 3C-like proteases to express WBV ExoN in an active form in Escherichia coli. The 374-residue protein displayed robust 3'-to-5' exoribonuclease activity in the presence of Mg2+ ions and, unlike its coronavirus homologues, did not require a protein cofactor for activity. Characterization of mutant forms of ExoN provided support for predictions on putative active-site and conserved zinc-binding residues. WBV ExoN was revealed to be most active on double-stranded RNA substrates containing one or two non-paired 3'-terminal nucleotides, supporting its presumed role in increasing the fidelity of the bafinivirus RNA-dependent RNA polymerase.

Broad-spectrum antiviral activity of the eIF4A inhibitor silvestrol against corona- and picornaviruses.

Coronaviruses (CoV) and picornaviruses are plus-strand RNA viruses that use 5' cap-dependent and cap-independent strategies, respectively, for viral mRNA translation initiation. Here, we analyzed the effects of the plant compound silvestrol, a specific inhibitor of the DEAD-box RNA helicase eIF4A, on viral translation using a dual luciferase assay and virus-infected primary cells. Silvestrol was recently shown to have potent antiviral activity in Ebola virus-infected human macrophages. We found that silvestrol is also a potent inhibitor of cap-dependent viral mRNA translation in CoV-infected human embryonic lung fibroblast (MRC-5) cells. EC50 values of 1.3 nM and 3 nM silvestrol were determined for MERS-CoV and HCoV-229E, respectively. For the highly pathogenic MERS-CoV, the potent antiviral activities of silvestrol were also confirmed using peripheral blood mononuclear cells (PBMCs) as a second type of human primary cells. Silvestrol strongly inhibits the expression of CoV structural and nonstructural proteins (N, nsp8) and the formation of viral replication/transcription complexes. Furthermore, potential antiviral effects against human rhinovirus (HRV) A1 and poliovirus type 1 (PV), representing different species in the genus Enterovirus (family Picornaviridae), were investigated. The two viruses employ an internal ribosomal entry site (IRES)-mediated translation initiation mechanism. For PV, which is known to require the activity of eIF4A, an EC50 value of 20 nM silvestrol was determined in MRC-5 cells. The higher EC50 value of 100 nM measured for HRV A1 indicates a less critical role of eIF4A activity in HRV A1 IRES-mediated translation initiation. Taken together, the data reveal a broad-spectrum antiviral activity of silvestrol in infected primary cells by inhibiting eIF4A-dependent viral mRNA translation.

Structural and functional conservation of cis-acting RNA elements in coronavirus 5'-terminal genome regions.

Structure predictions suggest a partial conservation of RNA structure elements in coronavirus terminal genome regions. Here, we determined the structures of stem-loops (SL) 1 and 2 of two alphacoronaviruses, human coronavirus (HCoV) 229E and NL63, by RNA structure probing and studied the functional relevance of these putative cis-acting elements. HCoV-229E SL1 and SL2 mutants generated by reverse genetics were used to study the effects on viral replication of single-nucleotide substitutions predicted to destabilize the SL1 and SL2 structures. The data provide conclusive evidence for the critical role of SL1 and SL2 in HCoV-229E replication and, in some cases, revealed parallels with previously characterized betacoronavirus SL1 and SL2 elements. Also, we were able to rescue viable HCoV-229E mutants carrying replacements of SL2 with equivalent betacoronavirus structural elements. The data obtained in this study reveal a remarkable degree of structural and functional conservation of 5'-terminal RNA structural elements across coronavirus genus boundaries.

RNase E and RNase J are needed for S-adenosylmethionine homeostasis in Sinorhizobium meliloti.

The ribonucleases (RNases) E and J play major roles in E. coli and Bacillus subtilis, respectively, and co-exist in Sinorhizobium meliloti. We analysed S. meliloti 2011 mutants with mini-Tn5 insertions in the corresponding genes rne and rnj and found many overlapping effects. We observed similar changes in mRNA levels, including lower mRNA levels of the motility and chemotaxis related genes flaA, flgB and cheR and higher levels of ndvA (important for glucan export). The acyl-homoserine lactone (AHL) levels were also higher during exponential growth in both RNase mutants, despite no increase in the expression of the sinI AHL synthase gene. Furthermore, several RNAs from both mutants migrated aberrantly in denaturing gels at 300 V but not under stronger denaturing conditions at 1300 V. The similarities between the two mutants could be explained by increased levels of the key methyl donor S-adenosylmethionine (SAM), since this may result in faster AHL synthesis leading to higher AHL accumulation as well as in uncontrolled methylation of macromolecules including RNA, which may strengthen RNA secondary structures. Indeed, we found that in both mutants the N6-methyladenosine content was increased almost threefold and the SAM level was increased at least sevenfold. Complementation by induced ectopic expression of the respective RNase restored the AHL and SAM levels in each of the mutants. In summary, our data show that both RNase E and RNase J are needed for SAM homeostasis in S. meliloti.

Identification of specific residues in avian influenza A virus NS1 that enhance viral replication and pathogenicity in mammalian systems.

Reassortment of their segmented genomes allows influenza A viruses (IAV) to gain new characteristics, which potentially enable them to cross the species barrier and infect new hosts. Improved replication was observed for reassortants of the strictly avian IAV A/FPV/Rostock/34 (FPV, H7N1) containing the NS segment from A/Goose/Guangdong/1/1996 (GD, H5N1), but not for reassortants containing the NS segment of A/Mallard/NL/12/2000 (MA, H7N3). The NS1 of GD and MA differ only in 8 aa positions. Here, we show that efficient replication of FPV-NSMA-derived mutants was linked to the presence of a single substitution (D74N) and more prominently to a triple substitution (P3S+R41K+D74N) in the NS1MA protein. The substitution(s) led to (i) increased virus titres, (ii) larger plaque sizes and (iii) increased levels and faster kinetics of viral mRNA and protein accumulation in mammalian cells. Interestingly, the NS1 substitutions did not affect viral growth characteristics in avian cells. Furthermore, we show that an FPV mutant with N74 in the NS1 (already possessing S3+K41) is able to replicate and cause disease in mice, demonstrating a key role of NS1 in the adaptation of avian IAV to mammalian hosts. Our data suggest that (i) adaptation to mammalian hosts does not necessarily compromise replication in the natural (avian) host and (ii) very few genetic changes may pave the way for zoonotic transmission. The study reinforces the need for close surveillance and characterization of circulating avian IAV to identify genetic signatures that indicate a potential risk for efficient transmission of avian strains to mammalian hosts.

RNA structure analysis of alphacoronavirus terminal genome regions.

Coronavirus genome replication is mediated by a multi-subunit protein complex that is comprised of more than a dozen virally encoded and several cellular proteins. Interactions of the viral replicase complex with cis-acting RNA elements located in the 5' and 3'-terminal genome regions ensure the specific replication of viral RNA. Over the past years, boundaries and structures of cis-acting RNA elements required for coronavirus genome replication have been extensively characterized in betacoronaviruses and, to a lesser extent, other coronavirus genera. Here, we review our current understanding of coronavirus cis-acting elements located in the terminal genome regions and use a combination of bioinformatic and RNA structure probing studies to identify and characterize putative cis-acting RNA elements in alphacoronaviruses. The study suggests significant RNA structure conservation among members of the genus Alphacoronavirus but also across genus boundaries. Overall, the conservation pattern identified for 5' and 3'-terminal RNA structural elements in the genomes of alpha- and betacoronaviruses is in agreement with the widely used replicase polyprotein-based classification of the Coronavirinae, suggesting co-evolution of the coronavirus replication machinery with cognate cis-acting RNA elements.

Detection of very long antisense transcripts by whole transcriptome RNA-Seq analysis of Listeria monocytogenes by semiconductor sequencing technology.

The Gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a severe food-borne infection characterised by abortion, septicaemia, or meningoencephalitis. L. monocytogenes causes outbreaks of febrile gastroenteritis and accounts for community-acquired bacterial meningitis in humans. Listeriosis has one of the highest mortality rates (up to 30%) of all food-borne infections. This human pathogenic bacterium is an important model organism for biomedical research to investigate cell-mediated immunity. L. monocytogenes is also one of the best characterised bacterial systems for the molecular analysis of intracellular parasitism. Recently several transcriptomic studies have also made the ubiquitous distributed bacterium as a model to understand mechanisms of gene regulation from the environment to the infected host on the level of mRNA and non-coding RNAs (ncRNAs). We have used semiconductor sequencing technology for RNA-seq to investigate the repertoire of listerial ncRNAs under extra- and intracellular growth conditions. Furthermore, we applied a new bioinformatic analysis pipeline for detection, comparative genomics and structural conservation to identify ncRNAs. With this work, in total, 741 ncRNA locations of potential ncRNA candidates are now known for L. monocytogenes, of which 611 ncRNA candidates were identified by RNA-seq. 441 transcribed ncRNAs have never been described before. Among these, we identified novel long non-coding antisense RNAs with a length of up to 5,400 nt e.g. opposite to genes coding for internalins, methylases or a high-affinity potassium uptake system, namely the kdpABC operon, which were confirmed by qRT-PCR analysis. RNA-seq, comparative genomics and structural conservation of L. monocytogenes ncRNAs illustrate that this human pathogen uses a large number and repertoire of ncRNA including novel long antisense RNAs, which could be important for intracellular survival within the infected eukaryotic host.

Ultra deep sequencing of Listeria monocytogenes sRNA transcriptome revealed new antisense RNAs.

Listeria monocytogenes, a gram-positive pathogen, and causative agent of listeriosis, has become a widely used model organism for intracellular infections. Recent studies have identified small non-coding RNAs (sRNAs) as important factors for regulating gene expression and pathogenicity of L. monocytogenes. Increased speed and reduced costs of high throughput sequencing (HTS) techniques have made RNA sequencing (RNA-Seq) the state-of-the-art method to study bacterial transcriptomes. We created a large transcriptome dataset of L. monocytogenes containing a total of 21 million reads, using the SOLiD sequencing technology. The dataset contained cDNA sequences generated from L. monocytogenes RNA collected under intracellular and extracellular condition and additionally was size fractioned into three different size ranges from <40 nt, 40-150 nt and >150 nt. We report here, the identification of nine new sRNAs candidates of L. monocytogenes and a reevaluation of known sRNAs of L. monocytogenes EGD-e. Automatic comparison to known sRNAs revealed a high recovery rate of 55%, which was increased to 90% by manual revision of the data. Moreover, thorough classification of known sRNAs shed further light on their possible biological functions. Interestingly among the newly identified sRNA candidates are antisense RNAs (asRNAs) associated to the housekeeping genes purA, fumC and pgi and potentially their regulation, emphasizing the significance of sRNAs for metabolic adaptation in L. monocytogenes.