Cryo-EM Structure of an Extended SARS-CoV-2 Replication and Transcription Complex Reveals an Intermediate State in Cap Synthesis.

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp82-nsp12-nsp132-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5' extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.

Transcript Buffering: A Balancing Act between mRNA Synthesis and mRNA Degradation.

Transcript buffering involves reciprocal adjustments between overall rates in mRNA synthesis and degradation to maintain similar cellular concentrations of mRNAs. This phenomenon was first discovered in yeast and encompasses coordination between the nuclear and cytoplasmic compartments. Transcript buffering was revealed by novel methods for pulse labeling of RNA to determine in vivo synthesis and degradation rates. In this Perspective, we discuss the current knowledge of transcript buffering. Emphasis is placed on the future challenges to determine the nature and directionality of the buffering signals, the generality of transcript buffering beyond yeast, and the molecular mechanisms responsible for this balancing.

Biochemical characterization of mRNA capping enzyme from Faustovirus.

The mammalian mRNA 5' cap structures play important roles in cellular processes such as nuclear export, efficient translation, and evading cellular innate immune surveillance and regulating 5'-mediated mRNA turnover. Hence, installation of the proper 5' cap is crucial in therapeutic applications of synthetic mRNA. The core 5' cap structure, Cap-0, is generated by three sequential enzymatic activities: RNA 5' triphosphatase, RNA guanylyltransferase, and cap N7-guanine methyltransferase. Vaccinia virus RNA capping enzyme (VCE) is a heterodimeric enzyme that has been widely used in synthetic mRNA research and manufacturing. The large subunit of VCE D1R exhibits a modular structure where each of the three structural domains possesses one of the three enzyme activities, whereas the small subunit D12L is required to activate the N7-guanine methyltransferase activity. Here, we report the characterization of a single-subunit RNA capping enzyme from an amoeba giant virus. Faustovirus RNA capping enzyme (FCE) exhibits a modular array of catalytic domains in common with VCE and is highly efficient in generating the Cap-0 structure without an activation subunit. Phylogenetic analysis suggests that FCE and VCE are descended from a common ancestral capping enzyme. We found that compared to VCE, FCE exhibits higher specific activity, higher activity toward RNA containing secondary structures and a free 5' end, and a broader temperature range, properties favorable for synthetic mRNA manufacturing workflows.

Reduction in gene expression noise by targeted increase in accessibility at gene loci.

Many eukaryotic genes are expressed in randomly initiated bursts that are punctuated by periods of quiescence. Here, we show that the intermittent access of the promoters to transcription factors through relatively impervious chromatin contributes to this "noisy" transcription. We tethered a nuclease-deficient Cas9 fused to a histone acetyl transferase at the promoters of two endogenous genes in HeLa cells. An assay for transposase-accessible chromatin using sequencing showed that the activity of the histone acetyl transferase altered the chromatin architecture locally without introducing global changes in the nucleus and rendered the targeted promoters constitutively accessible. We measured the gene expression variability from the gene loci by performing single-molecule fluorescence in situ hybridization against mature messenger RNAs (mRNAs) and by imaging nascent mRNA molecules present at active gene loci in single cells. Because of the increased accessibility of the promoter to transcription factors, the transcription from two genes became less noisy, even when the average levels of expression did not change. In addition to providing evidence for chromatin accessibility as a determinant of the noise in gene expression, our study offers a mechanism for controlling gene expression noise which is otherwise unavoidable.

E1B-55K Is a Phosphorylation-Dependent Transcriptional and Posttranscriptional Regulator of Viral Gene Expression in Human Adenovirus C5 Infection.

The multifunctional adenoviral E1B-55K phosphoprotein is a major regulator of viral replication and plays key roles in virus-mediated cell transformation. While much is known about its function in oncogenic cell transformation, the underlying features and exact mechanisms that implicate E1B-55K in the regulation of viral gene expression are less well understood. Therefore, this work aimed to unravel basic intranuclear principles of E1B-55K-regulated viral mRNA biogenesis using wild-type human adenovirus C5 (HAdV-C5) E1B-55K, a virus mutant with abrogated E1B-55K expression, and a mutant that expresses a phosphomimetic E1B-55K. By subnuclear fractionation, mRNA, DNA, and protein analyses as well as luciferase reporter assays, we show that (i) E1B-55K promotes the efficient release of viral late mRNAs from their site of synthesis in viral replication compartments (RCs) to the surrounding nucleoplasm, (ii) E1B-55K modulates the rate of viral gene transcription and splicing in RCs, (iii) E1B-55K participates in the temporal regulation of viral gene expression, (iv) E1B-55K can enhance or repress the expression of viral early and late promoters, and (v) the phosphorylation of E1B-55K regulates the temporal effect of the protein on each of these activities. Together, these data demonstrate that E1B-55K is a phosphorylation-dependent transcriptional and posttranscriptional regulator of viral genes during HAdV-C5 infection. IMPORTANCE Human adenoviruses are useful models to study basic aspects of gene expression and splicing. Moreover, they are one of the most commonly used viral vectors for clinical applications. However, key aspects of the activities of essential viral proteins that are commonly modified in adenoviral vectors have not been fully described. A prominent example is the multifunctional adenoviral oncoprotein E1B-55K that is known to promote efficient viral genome replication and expression while simultaneously repressing host gene expression and antiviral host responses. Our study combined different quantitative methods to study how E1B-55K promotes viral mRNA biogenesis. The data presented here propose a novel role for E1B-55K as a phosphorylation-dependent transcriptional and posttranscriptional regulator of viral genes.

Elongation factor 1 is a component of the Arabidopsis RNA polymerase II elongation complex and associates with a subset of transcribed genes.

Elongation factors modulate the efficiency of mRNA synthesis by RNA polymerase II (RNAPII) in the context of chromatin, thus contributing to implement proper gene expression programmes. The zinc-finger protein elongation factor 1 (ELF1) is a conserved transcript elongation factor (TEF), whose molecular function so far has not been studied in plants. Using biochemical approaches, we examined the interaction of Arabidopsis ELF1 with DNA and histones in vitro and with the RNAPII elongation complex in vivo. In addition, cytological assays demonstrated the nuclear localisation of the protein, and by means of double-mutant analyses, interplay with genes encoding other elongation factors was explored. The genome-wide distribution of ELF1 was addressed by chromatin immunoprecipitation. ELF1 isolated from Arabidopsis cells robustly copurified with RNAPII and various other elongation factors including SPT4-SPT5, SPT6, IWS1, FACT and PAF1C. Analysis of a CRISPR-Cas9-mediated gene editing mutant of ELF1 revealed distinct genetic interactions with mutants deficient in other elongation factors. Moreover, ELF1 associated with genomic regions actively transcribed by RNAPII. However, ELF1 occupied only c. 33% of the RNAPII transcribed loci with preference for inducible rather than constitutively expressed genes. Collectively, these results establish that Arabidopsis ELF1 shares several characteristic attributes with RNAPII TEFs.

Tactics targeting circular mRNA biosynthesis.

Faced with the development of mRNA technology in the field of medicine and vaccine, circular mRNA (circmRNA) becomes a strong alternative to mRNA for its circular secondary structure and higher stability. At present, the synthesis of circmRNAs has been realized by ligating linear mRNA precursors and is limited by poor efficiency. To solve this challenge, this study started with ribozyme catalysis and enzymatic reaction to explore different circmRNA biosynthesis strategies. In terms of ribozyme method, by screening different group I intron self-splicing system sequences, the sequence from thymidylate synthase (Td) gene of phage T4 showed the highest ligation efficiency. In terms of enzyme method, with the help of 20-bp homologous arm, T4 Rnl 2 was determined as the ligation method with the highest ligation efficiency. By comparing the two ligation methods, the expression level of circmRNA ligated by T4 Rnl 2 was 86% higher than that ligated by Td ribozyme. Based on these ligation methods, the screening results of internal ribosome entry site (IRES) sequences showed that mud crab dicistrovirus IRES was an IRES sequence with high ribosome binding ability and could be widely used in circmRNAs for efficient and stable translation in mammalian cells. These results should provide positive guidance for the industrial production of circmRNAs and the development of mRNA vaccines. Eventually, circmRNAs could widely function in the field of biomedicine.

Synthesis of low immunogenicity RNA with high-temperature in vitro transcription.

The use of synthetic RNA for therapeutics requires that the in vitro synthesis process be robust and efficient. The technology used for the synthesis of these in vitro-transcribed RNAs, predominantly using phage RNA polymerases (RNAPs), is well established. However, transcripts synthesized with RNAPs are known to display an immune-stimulatory activity in vivo that is often undesirable. Previous studies have identified double-stranded RNA (dsRNA), a major by-product of the in vitro transcription (IVT) process, as a trigger of cellular immune responses. Here we describe the characterization of a high-temperature IVT process using thermostable T7 RNAPs to synthesize functional mRNAs that demonstrate reduced immunogenicity without the need for a post-synthesis purification step. We identify features that drive the production of two kinds of dsRNA by-products-one arising from 3' extension of the run-off product and one formed by the production of antisense RNAs-and demonstrate that at a high temperature, T7 RNAP has reduced 3'-extension of the run-off product. We show that template-encoded poly(A) tailing does not affect 3'-extension but reduces the formation of the antisense RNA by-products. Combining high-temperature IVT with template-encoded poly(A) tailing prevents the formation of both kinds of dsRNA by-products generating functional mRNAs with reduced immunogenicity.

Nucleocapsid Protein Recruitment to Replication-Transcription Complexes Plays a Crucial Role in Coronaviral Life Cycle.

Coronavirus (CoV) nucleocapsid (N) proteins are key for incorporating genomic RNA into progeny viral particles. In infected cells, N proteins are present at the replication-transcription complexes (RTCs), the sites of CoV RNA synthesis. It has been shown that N proteins are important for viral replication and that the one of mouse hepatitis virus (MHV), a commonly used model CoV, interacts with nonstructural protein 3 (nsp3), a component of the RTCs. These two aspects of the CoV life cycle, however, have not been linked. We found that the MHV N protein binds exclusively to nsp3 and not other RTC components by using a systematic yeast two-hybrid approach, and we identified two distinct regions in the N protein that redundantly mediate this interaction. A selective N protein variant carrying point mutations in these two regions fails to bind nsp3 in vitro, resulting in inhibition of its recruitment to RTCs in vivo Furthermore, in contrast to the wild-type N protein, this N protein variant impairs the stimulation of genomic RNA and viral mRNA transcription in vivo and in vitro, which in turn leads to impairment of MHV replication and progeny production. Altogether, our results show that N protein recruitment to RTCs, via binding to nsp3, is an essential step in the CoV life cycle because it is critical for optimal viral RNA synthesis.IMPORTANCE CoVs have long been regarded as relatively harmless pathogens for humans. Severe respiratory tract infection outbreaks caused by severe acute respiratory syndrome CoV and Middle East respiratory syndrome CoV, however, have caused high pathogenicity and mortality rates in humans. These outbreaks highlighted the relevance of being able to control CoV infections. We used a model CoV, MHV, to investigate the importance of the recruitment of N protein, a central component of CoV virions, to intracellular platforms where CoVs replicate, transcribe, and translate their genomes. By identifying the principal binding partner at these intracellular platforms and generating a specific mutant, we found that N protein recruitment to these locations is crucial for promoting viral RNA synthesis. Moreover, blocking this recruitment strongly inhibits viral infection. Thus, our results explain an important aspect of the CoV life cycle and reveal an interaction of viral proteins that could be targeted in antiviral therapies.

Design, Assembly, Production, and Transfection of Synthetic Modified mRNA.

Proteins are drivers of cell functions and are targets of many therapies. Exogenous protein expression techniques, therefore, have been essential for research and medicine. The most common method for exogenous protein expression relies on DNA-based viral or non-viral vectors. However, DNA-based vectors have the potential to integrate into the host genome and cause permanent mutations. RNA-based vectors solve this shortcoming. In particular, synthetic modified mRNA provides non-viral, integration-free, zero-footprint method for expressing proteins. Modified mRNA can direct cell fate specification and cellular reprogramming faster and more efficiently than other methods. Furthermore, when simultaneously express multiple different proteins, mRNA vectors allow for greater flexibility and control over stoichiometric ratios, dose titrations, and complete silencing of expressions. Additionally, modified mRNAs have been shown to be viable and safe as therapeutic agents for gene therapy and vaccine, providing an alternative approach to address diseases. Despite these advantages, technical challenge, mRNA instability, and host immunogenicity have caused significant barriers to widespread use of this technology. The comprehensive method presented here addresses all of these shortcomings. This stepwise protocol describes every step necessary for the synthesis of modified mRNA from any coding DNA sequence of interest. The meticulously detailed protocol enables the users to make alterations to each component of modified mRNA for even more significant customization, allowing the researchers to apply this technology to a wide range of uses. This non-cytotoxic synthetic modified mRNA can be used for protein expression, regulation of cell reprogramming or differentiation, and drug delivery.

What do you mean by transcription rate?: the conceptual difference between nascent transcription rate and mRNA synthesis rate is essential for the proper understanding of transcriptomic analyses.

mRNA synthesis in all organisms is performed by RNA polymerases, which work as nanomachines on DNA templates. The rate at which their product is made is an important parameter in gene expression. Transcription rate encompasses two related, yet different, concepts: the nascent transcription rate, which measures the in situ mRNA production by RNA polymerase, and the rate of synthesis of mature mRNA, which measures the contribution of transcription to the mRNA concentration. Both parameters are useful for molecular biologists, but they are not interchangeable and they are expressed in different units. It is important to distinguish when and where each one should be used. We propose that for functional genomics the use of nascent transcription rates should be restricted to the evaluation of the transcriptional process itself, whereas mature mRNA synthesis rates should be employed to address the transcriptional input to mRNA concentration balance leading to variation of gene expression.

The emerging roles of eRNAs in transcriptional regulatory networks.

Following reports by ENCyclopedia Of DNA Elements (ENCODE; GENCODE) Consortium and others, it is now fairly evident that the majority (70-80%) of the mammalian genome has the potential to be transcribed into non-protein-coding RNAs (ncRNAs). Critical to our understanding of genetic processes is the mechanism by which ncRNAs exert their roles. Accordingly, ncRNAs are shown to regulate the expression of protein-coding loci (i.e., genes) at the transcriptional as well as post-transcriptional stages. We recently reported on a widespread transcription at the DNA enhancer elements in myogenic cells. In our study, we found certain enhancer RNAs (eRNAs) regulate chromatin accessibility of the transcriptional machinery at loci encoding master regulators of myogenesis (i.e., MyoD/MyoG), thus suggesting their significance and site-specific impact in cellular programming. Here, we examine recent discoveries pertinent to the proposed role(s) of eRNAs in regulating gene expression. We will highlight consistencies, discuss confounding observations, and consider a lack of critical information in a way to prioritize future objectives.

The mRNA vaccine, a swift warhead against a moving infectious disease target.

INTRODUCTION: The rapid development of mRNA vaccines against SARS-CoV-2 has revolutionized vaccinology, offering hope for swift responses to emerging infectious diseases. Initially met with skepticism, mRNA vaccines have proven effective and safe, reducing vaccine hesitancy amid the evolving COVID-19 pandemic. The COVID-19 pandemic has demonstrated that the time required to modify mRNA vaccines to counter new mutant strains is significantly shorter than the time it takes for pathogens to mutate and generate new variants that can thrive in vaccinated populations. This highlights the notion that mRNA vaccine technology appears to be outpacing viruses in the ongoing evolutionary race. AREAS COVERED: This review article offers valuable insights into several crucial aspects of mRNA vaccine development and deployment, including the fundamentals of mRNA vaccine design and synthesis, the utilization of delivery systems, considerations regarding vaccine safety, the longevity of the immune response, strategies for modifying the original mRNA vaccine to address emerging mutant strains, as well as addressing vaccine hesitancy and potential approaches to mitigate reluctance. EXPERT OPINION: Challenges such as stability, storage, manufacturing complexities, production capacity, allergic reactions, long-term effects, accessibility, and misinformation must be addressed. Despite these hurdles, mRNA vaccine technology holds promise for revolutionizing future vaccination strategies.

The zinc-finger transcription factor Sfp1 imprints specific classes of mRNAs and links their synthesis to cytoplasmic decay.

To function effectively as an integrated system, the transcriptional and post-transcriptional machineries must communicate through mechanisms that are still poorly understood. Here, we focus on the zinc-finger Sfp1, known to regulate transcription of proliferation-related genes. We show that Sfp1 can regulate transcription either by binding to promoters, like most known transcription activators, or by binding to the transcribed regions (gene bodies), probably via RNA polymerase II (Pol II). We further studied the first mode of Sfp1 activity and found that, following promoter binding, Sfp1 binds to gene bodies and affects Pol II configuration, manifested by dissociation or conformational change of its Rpb4 subunit and increased backtracking. Surprisingly, Sfp1 binds to a subset of mRNAs co-transcriptionally and stabilizes them. The interaction between Sfp1 and its client mRNAs is controlled by their respective promoters and coincides with Sfp1's dissociation from chromatin. Intriguingly, Sfp1 dissociation from the chromatin correlates with the extent of the backtracked Pol II. We propose that, following promoter recruitment, Sfp1 accompanies Pol II and regulates backtracking. The backtracked Pol II is more compatible with Sfp1's relocation to the nascent transcripts, whereupon Sfp1 accompanies these mRNAs to the cytoplasm and regulates their stability. Thus, Sfp1's co-transcriptional binding imprints the mRNA fate, serving as a paradigm for the cross-talk between the synthesis and decay of specific mRNAs, and a paradigm for the dual-role of some zinc-finger proteins. The interplay between Sfp1's two modes of transcription regulation remains to be examined.

The ability to fine-tune the production of proteins in a cell is essential for organisms to exist. An imbalance in protein levels can be the cause of various diseases. Messenger RNA molecules (mRNA) link the genetic information encoded in DNA and the produced proteins. Exactly how much protein is made mostly depends on the amount of mRNA in the cell's cytoplasm. This is controlled by two processes: the synthesis of mRNA (also known as transcription) and mRNA being actively degraded. Although much is known about mechanisms regulating transcription and degradation, how cells detect if they need to degrade mRNA based on the levels of its synthesis and vice versa is poorly understood. In 2013, researchers found that proteins known as 'RNA decay factors' responsible for mRNA degradation are actively moved from the cell's cytoplasm into its nucleus to instruct the transcription machinery to produce more mRNA. Kelbert, Jordán-Pla, de-Miguel-Jiménez et al. ­ including some of the researchers involved in the 2013 work ­ investigated how mRNA synthesis and degradation are coordinated to ensure a proper mRNA level. The researchers used advanced genome engineering methods to carefully manipulate and measure mRNA production and degradation in yeast cells. The experiments revealed that the protein Sfp1 ­ a well-characterized transcription factor for stimulating the synthesis of a specific class of mRNAs inside the nucleus ­ can also prevent the degradation of these mRNAs outside the nucleus. During transcription, Sfp1 bound directly to mRNA. The investigators could manipulate the co-transcriptional binding of Sfp1 to a certain mRNA, thereby changing the mRNA stability in the cytoplasm. This suggests that the ability of Sfp1 to regulate both the production and decay of mRNA is dependent on one another and that transcription can influence the fate of its transcripts. This combined activity can rapidly change mRNA levels in response to changes in the cell's environment. RNA plays a key role in ensuring correct levels of proteins. It can also function as an RNA molecule, independently of its coding capacity. Many cancers and developmental disorders are known to be caused by faulty interactions between transcription factors and nucleic acids. The finding that some transcription factors can directly regulate both mRNA synthesis and its destruction introduces new angles for studying and understanding these diseases.

Enhancing antitumor immunity and achieving tumor eradication with IL11RA mRNA immunotherapy.

Current methods for delivering genes to target tumors face significant challenges, including off-target effects and immune responses against delivery vectors. In this study, we developed a novel approach using messenger RNA (mRNA) to encode IL11RA for local immunotherapy, aiming to harness the immune system to combat tumors. Our research uncovered a compelling correlation between IL11RA expression and CD8 + T cell levels across multiple tumor types, with elevated IL11RA expression correlating with improved overall survival. Examination of the Pan-Cancer Atlas dataset showed a significant reduction in IL11RA expression in various cancer types compared to normal tissue, raising questions about its potential role in tumorigenesis. To achieve efficient in vivo expression of IL11RA, we synthesized two mRNA sequences mimicking the wild-type protein. These mRNA sequences were formulated and capped to ensure effective delivery, resulting in robust expression within tumor sites. Our investigation into IL11RA mRNA therapy demonstrated its effectiveness in controlling tumor growth when administered both intratumorally and intravenously in mouse models. Additionally, IL11RA mRNA treatment significantly stimulated the expansion of CD8 + T cells within tumors, draining lymph nodes, and the spleen. Transcriptome analysis revealed distinct transcriptional patterns associated with T cell functions. Using multiple deconvolution algorithms, we found substantial infiltration of CD8 + T cells following IL11RA mRNA treatment, highlighting its immunomodulatory effects within the tumor microenvironment. In conclusion, IL11RA mRNA therapy presents a promising strategy for tumor regression with potential immunomodulatory effects and clinical implications for improved survival outcomes.

Cold Exposure-Induced Up-Regulation of Hsp70 Positively Regulates PEDV mRNA Synthesis and Protein Expression In Vitro.

Porcine epidemic diarrhea (PED) is a highly contagious, intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV). PEDV as an emerging and re-emerging epizootic virus of swine causes substantial economic losses to the pig industry in China and other countries. In China, the occurrence of PED shows significant seasonal variations, usually outbreak during the winter season. The epidemic characteristics of PED may be highly correlated with the changes of ambient temperature. However, molecular mechanism on the seasonal occurrence of PED still remains unclear. It has been widely observed that low ambient temperature up-regulates the expression of host heat shock protein 70 (Hsp70). Here, we showed that nucleotide and protein levels of Hsp70 were up-regulated in the intestinal of cold exposed pig and cold exposed Vero E6 cells. We found that overexpression of Hsp70 could increase PEDV mRNA synthesis and protein expression in Vero E6 and IPEC-J2 cells, while the siRNAs mediated knockdown of Hsp70 and VER155008 mediated inhibition of Hsp70 resulted in inhibition of viral mRNA synthesis and protein expression in Vero E6 cells. These data suggested that Hsp70 positively regulated PEDV mRNA synthesis and protein expression, which being helpful for understanding the seasonality of PED epidemics and development of novel antiviral therapies in the future.

Optimizing Modified mRNA In Vitro Synthesis Protocol for Heart Gene Therapy.

Synthetic modified RNA (modRNA) is a novel vector for gene transfer to the heart and other organs. modRNA can mediate strong, transient protein expression with minimal induction of the innate immune response and risk for genome integration. modRNA is already being used in several human clinical trials, and its use in basic and translational science is growing. Due to the complexity of preparing modRNA and the high cost of its reagents, there is a need for an improved, cost-efficient protocol to make modRNA. Here we show that changing the ratio between anti-reverse cap analog (ARCA) and N1-methyl-pseudouridine (N1mΨ), favoring ARCA over N1mΨ, significantly increases the yield per reaction, improves modRNA translation, and reduces its immunogenicity in vitro. This protocol will make modRNA preparation more accessible and financially affordable for basic and translational research.

Consolidation of an aversive taste memory requires two rounds of transcriptional and epigenetic regulation in the insular cortex.

The current view of the neurobiology of learning and memory suggests that long-term memory (LTM) depends not only on the de novo protein synthesis but also on the synthesis of mRNA even hours after the acquisition of memory, as well as that the regulation of transcription through the histone acetylation is essential for the memory establishment. Our previous studies showed that protein synthesis inhibition around the time of training and 5-7 hours after acquisition in the insular cortex (IC) prevents the consolidation of conditioned taste aversion (CTA), a well-established learning and memory paradigm in which an animal learns to associate a novel taste with nausea. However, the participation of mRNA synthesis and the epigenetic regulation through histone acetylation in this process remains unexplored. In the present study we evaluated the effect of the inhibition of transcription as well as deacetylation of histones at two temporal windows on the consolidation of CTA. Thus, immediately or seven hours after CTA acquisition animals received a microinfusion of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) or MS-275 in the IC, respectively. The present results show that transcription inhibition immediately and 7 h after acquisition impairs the CTA memory consolidation, whereas the inhibition of histone deacetylation strengths this memory at those temporal windows. These findings reveal that CTA memory requires recurrent rounds of transcriptional modulation events in the IC in order to consolidate this memory trace, demonstrating that transcriptional and epigenetic modulation substantially contribute to memory-consolidation-related functions performed by a neocortical area even several hours after memory acquisition.

Nidovirus RNA polymerases: Complex enzymes handling exceptional RNA genomes.

Coronaviruses and arteriviruses are distantly related human and animal pathogens that belong to the order Nidovirales. Nidoviruses are characterized by their polycistronic plus-stranded RNA genome, the production of subgenomic mRNAs and the conservation of a specific array of replicase domains, including key RNA-synthesizing enzymes. Coronaviruses (26-34 kilobases) have the largest known RNA genomes and their replication presumably requires a processive RNA-dependent RNA polymerase (RdRp) and enzymatic functions that suppress the consequences of the typically high error rate of viral RdRps. The arteriviruses have significantly smaller genomes and form an intriguing package with the coronaviruses to analyse viral RdRp evolution and function. The RdRp domain of nidoviruses resides in a cleavage product of the replicase polyprotein named non-structural protein (nsp) 12 in coronaviruses and nsp9 in arteriviruses. In all nidoviruses, the C-terminal RdRp domain is linked to a conserved N-terminal domain, which has been coined NiRAN (nidovirus RdRp-associated nucleotidyl transferase). Although no structural information is available, the functional characterization of the nidovirus RdRp and the larger enzyme complex of which it is part, has progressed significantly over the past decade. In coronaviruses several smaller, non-enzymatic nsps were characterized that direct RdRp function, while a 3'-to-5' exoribonuclease activity in nsp14 was implicated in fidelity. In arteriviruses, the nsp1 subunit was found to maintain the balance between genome replication and subgenomic mRNA production. Understanding RdRp behaviour and interactions during RNA synthesis and subsequent processing will be key to rationalising the evolutionary success of nidoviruses and the development of antiviral strategies.