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We present a mild case of Cockayne syndrome that was referred to us with an extreme sunburn at the age of 3. In early teens, although her cutaneous symptoms alleviated without any medications, she developed tremor and dysarthria. Neurological examination and brain imaging suggested demyelination disorders. The patient's cells indicated a reduced recovery of RNA synthesis, which was partially restored by the introduction of CSB (Cockayne Syndrome B)-cDNA. In addition, her cells indicated a substantially reduced level of CSB protein. Despite the insidious progression of neurological symptoms, she gave birth to a child. Such mild cases of Cockayne syndrome may be misdiagnosed.

Zika virus (ZIKV), a mosquito-transmitted Flavivirus, emerged in the last decade causing serious diseases and affecting human health globally. Currently, no licensed vaccines or antivirals are available to combat ZIKV, although several vaccine candidates are in the pipeline. In recent years, the presence of non-canonical G-quadruplex (GQ) secondary structures in viral genomes has ignited significant attention as potential targets for antiviral strategy. In this study, we identified several novel conserved potential GQ structures by analyzing published ZIKV genome sequences using an in-house algorithm. Biophysical and biochemical analysis of the RNA sequences containing these potential GQ sequences suggested the existence of such structures in the ZIKV genomes. Studies with known GQ structure-binding and -stabilizing ligands such as Braco-19 and TMPyP4 provided support for this contention. The presence of these ligands in cell culture media led to significant inhibition of infectious ZIKV yield, as well as reduced viral genome replication and viral protein production. Overall, our results, for the first time, show that ZIKV replication can be inhibited by GQ structure-binding and -stabilizing compounds and suggest a new strategy against ZIKV infection mitigation and control., Graphical Abstract, G-quadruplex structures are non-canonical secondary structures present in nucleic acid. In this manuscript, we targeted the Zika GQ structures with GQ-binding ligands like Braco-19 and TMPyP4 to inhibit ZIKV genome replication and transcription. Overall, 100 μM Braco-19 treatment produced >80-fold (96 hpi) reduction in viral titer production in cell culture, having a potential anti-viral effect.

Synthesis of site-specifically modified oligonucleotides has become a major tool for RNA structure and function studies. Reporter groups or specific functional entities are required to be attached at a pre-defined site of the oligomer. An attractive strategy is the incorporation of suitably functionalized building blocks that allow post-synthetic conjugation of the desired moiety. A C8-alkynyl-modified adenosine derivative was synthesized, reviving an old synthetic pathway for iodination of purine nucleobases. Silylation of the C8-alkynyl-modified adenosine revealed unexpected selectivity of the two secondary sugar hydroxy groups, with the 3'-O-isomer being preferentially formed. Optimization of the protection scheme lead to a new and economic route to the desired C8-alkynylated building block and its incorporation in RNA.

Funding: This work was supported by Biotechnology and Biological Sciences Research Council (BBSRC) grants BB/L003988/1 and 1645891, and strategic funding to The Pirbright Institute, BBS/E/I/00007035, BBS/E/I/00007034, BBS/E/I/00007037 and BBS/E/I/00007039. Coronavirus sub-genomic mRNA (sgmRNA) synthesis occurs via a process of discontinuous transcription involving complementary transcription regulatory sequences (TRSs), one (TRS-L) encompassing the leader sequence of the 5' untranslated region (UTR), and the other upstream of each structural and accessory gene (TRS-B). Several coronaviruses have an ORF located between the N gene and the 3'-UTR, an area previously thought to be non-coding in the Gammacoronavirus infectious bronchitis virus (IBV) due to a lack of a canonical TRS-B. Here, we identify a non-canonical TRS-B allowing for a novel sgmRNA relating to this ORF to be produced in several strains of IBV: Beaudette, CR88, H120, D1466, Italy-02 and QX. Interestingly, the potential protein produced by this ORF is prematurely truncated in the Beaudette strain. A single nucleotide deletion was made in the Beaudette strain allowing for the generation of a recombinant IBV (rIBV) that had the potential to express a full-length protein. Assessment of this rIBV in vitro demonstrated that restoration of the full-length potential protein had no effect on viral replication. Further assessment of the Beaudette-derived RNA identified a second non-canonically transcribed sgmRNA located within gene 2. Deep sequencing analysis of allantoic fluid from Beaudette-infected embryonated eggs confirmed the presence of both the newly identified non-canonically transcribed sgmRNAs and highlighted the potential for further yet unidentified sgmRNAs. This HiSeq data, alongside the confirmation of non-canonically transcribed sgmRNAs, indicates the potential of the coronavirus genome to encode a larger repertoire of genes than has currently been identified. Publisher PDF

The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis. Here, we compared a variety of replication features of SARS-CoV-2 and SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the commonly used Vero E6 cell line. Compared to SARS-CoV, SARS-CoV-2 generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. Immunofluorescence microscopy of SARS-CoV-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of nonstructural proteins, membrane and nucleocapsid protein of SARS-CoV. Electron microscopy revealed that the ultrastructural changes induced by the two SARS viruses are very similar and occur within comparable time frames after infection. Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (Remdesivir, Alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. An important difference between the two viruses is the fact that - upon passaging in Vero E6 cells - SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. These mutations change or delete a putative ‘furin-like cleavage site’ in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays.

Influenza A virus (IAV) utilizes cap-snatching to obtain host capped small RNAs for priming viral mRNA synthesis, generating capped hybrid mRNAs for translation. Previous studies have been focusing on canonical cap-snatching, which occurs at the very 5′ end of viral mRNAs. Here we discovered noncanonical cap-snatching, which generates capped hybrid mRNAs/noncoding RNAs mapped to the region ∼300 nucleotides (nt) upstream of each mRNA 3′ end, and to the 5′ region, primarily starting at the second nt, of each virion RNAs (vRNA). Like canonical cap-snatching, noncanonical cap-snatching utilizes a base-pairing between the last nt G of host capped RNAs and a nt C of template RNAs to prime RNA synthesis. However, the nt upstream of this template C is usually A/U rather than just U; prime-realignment occurs less frequently. We also demonstrate that IAV can snatch capped IAV RNAs in addition to host RNAs. Noncanonical cap-snatching likely generates novel mRNAs with start AUG encoded in viral or host RNAs. These findings expand our understanding of cap-snatching mechanisms and suggest that IAV may utilize noncanonical cap-snatching to diversify its mRNAs/ncRNAs.

The stochasticity of transcription can be quantified by mRNA distribution \begin{document}$ P_m(t) $\end{document} , the probability that there are \begin{document}$ m $\end{document} mRNA molecules for the gene at time \begin{document}$ t $\end{document} in one cell. However, it still lacks of a standard method to calculate \begin{document}$ P_m(t) $\end{document} in a transparent formula. Here, we employ an infinite series method to express \begin{document}$ P_m(t) $\end{document} based on the classical two-state model. Intriguingly, we observe that a unimodal distribution of mRNA numbers at steady-state could be transformed from a dynamical bimodal distribution. This indicates that "bet hedging" strategy can be still achieved for the gene that generates phenotypic homogeneity of the cell population. Moreover, the formation and duration of such bimodality are tightly correlated with mRNA synthesis rate, reinforcing the modulation scenario of some inducible genes that manipulates mRNA synthesis rate in response to environmental change. More generally, the method presented here may be implemented to the other stochastic transcription models with constant rates.

[Simple Summary] Hepatocellular carcinoma (HCC) is the fourth most frequent cause of cancer-related mortality worldwide. While ablation, resection and orthotopic liver transplantation are indicated at an early stage of the disease, Sorafenib (Sfb) is the current most administrated first-line treatment for advanced HCC, even though its therapeutic benefit is limited due to the appearance of resistance. Deep knowledge on the molecular consequences of Sfb-treatment is essentially required for optimizing novel therapeutic strategies to improve the outcomes for patients with advanced HCC. In this study, we analyzed differential gene expression changes in two well characterized liver cancer cell lines upon a Sfb-treatment, demonstrating that both lines responded similarly to the treatment. Our results provide valuable information on the molecular action of Sfb on diverse cellular fundamental processes such as DNA repair, translation and proteostasis and identify rationalization issues that could provide a different therapeutic perspective to Sfb., Hepatocellular carcinoma (HCC) is one of the most frequent and essentially incurable cancers in its advanced stages. The tyrosine kinase inhibitor Sorafenib (Sfb) remains the globally accepted treatment for advanced HCC. However, the extent of its therapeutic benefit is limited. Sfb exerts antitumor activity through its cytotoxic, anti-proliferative and pro-apoptotic roles in HCC cells. To better understand the molecular mechanisms underlying these effects, we used RNA sequencing to generate comprehensive transcriptome profiles of HepG2 and SNU423, hepatoblastoma- (HB) and HCC-derived cell lines, respectively, following a Sfb treatment at a pharmacological dose. This resulted in similar alterations of gene expression in both cell lines. Genes functionally related to membrane trafficking, stress-responsible and unfolded protein responses, circadian clock and activation of apoptosis were predominantly upregulated, while genes involved in cell growth and cycle, DNA replication and repair, ribosome biogenesis, translation initiation and proteostasis were downregulated. Our results suggest that Sfb causes primary effects on cellular stress that lead to upregulation of selective responses to compensate for its negative effect and restore homeostasis. No significant differences were found specifically affecting each cell line, indicating the robustness of the Sfb mechanism of action despite the heterogeneity of liver cancer. We discuss our results on terms of providing rationalization for possible strategies to improve Sfb clinical outcomes., This publication is part of the project I+D+I PID2019-103850-GB-I00, funded by MCIN/AEI/10.13039/501100011033 to J.d.l.C. We also acknowledge the Andalusian Regional Government (BIO-271). We thank the Institute of Health Carlos III (ISCiii) co-financed by the European Regional Development Fund “A way to achieve Europe” (ERDF) (PI19/01266), the Andalusian Ministry of Health (PIP-0215-2020 and PI-0216-2020), as well as the Biomedical Research Network Center for Liver and Digestive Diseases (CIBERehd) founded by the ISCiii and cofinanced by the ERDF for their financial support to J.M. L.C. is a recipient of an FPU contract from the Spanish Ministry of Education and Vocational Training (FPU16/05127). A.R.-G. is funded by CIBERONC (CB16/12/00480) and the University of Seville, with funds from the Andalusian Regional Government and the ERDF (US-1380874).

Circular RNA (circRNA) is a unique type of noncoding RNA molecule. Compared with traditional linear RNA, circRNA is a covalently closed circle produced by a process called backsplicing. CircRNA is abundant in many cells and has rich functions in cells, such as acting as miRNA sponge, protein sponge, protein scaffold, and mRNA regulator. With the continuous development of circRNA study, circRNA has also played an important role in medical applications, including circRNA vaccines and gene therapy. In this review, we illustrate the synthesis of circRNAs in vitro. We focus on biological ligation methods, such as enzymatic ligation from the bacteriophage T4 and ribozyme method. In addition, we summarize the current challenges in the design, synthesis, application, and production of circRNAs, and propose possible solutions in the future. CircRNA is expected to play an essential role in basic research and medical applications.

The coronavirus nucleocapsid (N) protein comprises two RNA-binding domains connected by a central spacer, which contains a serine- and arginine-rich (SR) region. The SR region engages the largest subunit of the viral replicase-transcriptase, nonstructural protein 3 (nsp3), in an interaction that is essential for efficient initiation of infection by genomic RNA. We carried out an extensive genetic analysis of the SR region of the N protein of mouse hepatitis virus in order to more precisely define its role in RNA synthesis. We further examined the N-nsp3 interaction through construction of nsp3 mutants and by creation of an interspecies N protein chimera. Our results indicate a role for the central spacer as an interaction hub of the N molecule that is partially regulated by phosphorylation. These findings are discussed in relation to the recent discovery that nsp3 forms a molecular pore in the double-membrane vesicles that sequester the coronavirus replicase-transcriptase., Graphical abstract Image 1

The MIND-IHOP study was funded by the IHOP grant (NIH R01 HL090335), Lung MicroCHIP grant (NIH U01 HL098964), and K24 grant (NIH K24 HL087713). These sources provided the funding to support participant enrollment and specimen collection. Emmanuel Musisi was supported by a scholarship from the Pulmonary Complications of AIDS Research Training Program (NIH D43 TW009607). N.D.W., R.M.S., J.L.D., and P.N. acknowledge funding from the U.S. National Institutes of Health (1R01AI127300-01A1). N.D.W. and M.I.V. acknowledge funding from the U.S. National Institutes of Health (1R21AI135652-01). N.D.W. acknowledges funding from Veterans Affairs (1IK2CX000914-01A1 and 1I01BX004527-01A1) and from the Doris Duke Charitable Foundation Clinical Scientist Development Award. There is a critical need for improved pharmacodynamic markers for use in human tuberculosis (TB) drug trials. Pharmacodynamic monitoring in TB has conventionally used culture or molecular methods to enumerate the burden of Mycobacterium tuberculosis organisms in sputum. A recently proposed assay called the rRNA synthesis (RS) ratio measures a fundamentally novel property, how drugs impact ongoing bacterial rRNA synthesis. Here, we evaluated RS ratio as a potential pharmacodynamic monitoring tool by testing pretreatment sputa from 38 Ugandan adults with drug-susceptible pulmonary TB. We quantified the RS ratio in paired pretreatment sputa and evaluated the relationship between the RS ratio and microbiologic and molecular markers of M. tuberculosis burden. We found that the RS ratio was highly repeatable and reproducible in sputum samples. The RS ratio was independent of M. tuberculosis burden, confirming that it measures a distinct new property. In contrast, markers of M. tuberculosis burden were strongly associated with each other. These results indicate that the RS ratio is repeatable and reproducible and provides a distinct type of information from markers of M. tuberculosis burden. Importance This study takes a major next step toward practical application of a novel pharmacodynamic marker that we believe will have transformative implications for tuberculosis. This article follows our recent report in Nature Communications that an assay called the rRNA synthesis (RS) ratio indicates the treatment-shortening of drugs and regimens. Distinct from traditional measures of bacterial burden, the RS ratio measures a fundamentally novel property, how drugs impact ongoing bacterial rRNA synthesis. Publisher PDF

Current opinion in biotechnology 74, 8-14 (2022). doi:10.1016/j.copbio.2021.10.015 special issue: "Tissue, Cell and Pathway Engineering (2022) / Edited by Lorenzo Moroni, Martijn van Griensven", Published by Elsevier Science, Amsterdam [u.a.]

N6-methyladenosine (m6A) is the most abundant internal modification described in eukaryotic mRNA and several viral RNA including human respiratory syncytial virus (HRSV). Here, we evaluated the impact of m6A writers, erasers and readers on HRSV genomic RNA accumulation and inclusion bodies assembly during viral replication. We observed that the METTL3/METTL14 m6A writer complex plays a negative role in HRSV protein synthesis and viral titers, while m6A erasers FTO and ALKBH5 had the opposite effect. We also observed that m6A readers YTHDF1-3 bind to the viral genomic RNA inducing a decrease in its intracellular levels and thus, inhibiting viral replication. Finally, we observed that overexpression of YTHDFs proteins caused a decrease in the size of inclusion bodies (IBs), accompanied by an increase in their number. METTL3 knockdown cells showed an opposite effect indicating that the dynamics of IBs assembly and coalescence are strongly affected by m6A readers in a mechanism dependent on m6A writers. Taken together, our results demonstrated that the m6A modification negatively affects HRSV replication, possibly through a mechanism involving the assembly of inclusion bodies, the main factories of viral genomic RNA synthesis.

The catalytic subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp) Nsp12 has a unique nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain that transfers nucleoside monophosphates to the Nsp9 protein and the nascent RNA. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites, and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation in bacterial cells) Nsp12 exists in an inactive state in which NiRAN-RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or nucleoside triphosphates (NTPs) partially rescue RdRp activity. Our data show that adenosine and remdesivir triphosphates promote the synthesis of A-less RNAs, as does ppGpp, while amino acid substitutions at the NiRAN-RdRp interface augment activation, suggesting that ligand binding to the NiRAN catalytic site modulates RdRp activity. The existence of allosterically linked nucleotidyl transferase sites that utilize the same substrates has important implications for understanding the mechanism of SARS-CoV-2 replication and the design of its inhibitors. IMPORTANCE In vitro interrogations of the central replicative complex of SARS-CoV-2, RNA-dependent RNA polymerase (RdRp), by structural, biochemical, and biophysical methods yielded an unprecedented windfall of information that, in turn, instructs drug development and administration, genomic surveillance, and other aspects of the evolving pandemic response. They also illuminated the vast disparity in the methods used to produce RdRp for experimental work and the hidden impact that this has on enzyme activity and research outcomes. In this report, we elucidate the positive and negative effects of codon optimization on the activity and folding of the recombinant RdRp and detail the design of a highly sensitive in vitro assay of RdRp-dependent RNA synthesis. Using this assay, we demonstrate that RdRp is allosterically activated by nontemplating phosphorylated nucleotides, including naturally occurring alarmone ppGpp and synthetic remdesivir triphosphate.

Rotavirus is a well-studied RNA virus that causes severe gastroenteritis in children. During viral entry, the outer layer of the virion is shed, creating a double-layered particle (DLP) that is competent to perform viral transcription (i.e., mRNA synthesis) and launch infection. While inactive forms of rotavirus DLPs have been structurally characterized in detail, information about the transcriptionally-active DLP remains limited. Here, we used cryo-Electron Microscopy (cryo-EM) and 3D image reconstructions to compare the structures of internal protein components in transcriptionally-active versus inactive DLPs. Our findings showed that transcriptionally-active DLPs gained internal order as mRNA synthesis unfolded, while inactive DLPs remained dynamically disordered. Regions of viral protein/RNA constituents were analyzed across two different axes of symmetry to provide a more comprehensive view of moving components. Taken together, our results bring forth a new view of active DLPs, which may enable future pharmacological strategies aimed at obliterating rotavirus transcription as a therapeutic approach., Graphical Abstract Unlabelled Image

As one of many nonstructural proteins of porcine reproductive and respiratory syndrome virus (PRRSV), nonstructural protein 12 (Nsp12) has received relatively little attention, and its role in virus replication, if any, is essentially unknown. By the application of reverse genetic manipulation of an infectious PRRSV clone, the current study is the first to demonstrate that Nsp12 is a key component of PRRSV replication. In addition, the biochemical properties of Nsp12 were evaluated, revealing that Nsp12 forms dimers when exposed to oxidative conditions. Furthermore, we systemically analyzed the function of Nsp12 in PRRSV RNA synthesis using a strand-specific PCR method. To our surprise, Nsp12 was not found to be involved in minus-strand genomic RNA (-gRNA) synthesis; importantly, our results indicate that Nsp12 is involved in the synthesis of both plus- and minus-strand subgenomic mRNAs (+sgmRNA and -sgmRNA). Finally, we found that the combination of cysteine 35 and cysteine 79 in Nsp12 is required for sgmRNA synthesis. To our knowledge, we are the first to report the biological role of Nsp12 in the PRRSV lifecycle, and we conclude that Nsp12 is involved in the synthesis of both + sgRNA and -sgRNA.

At the focus of abiotic chemical reactions is the synthesis of ribose. No satisfactory explanation was provided as to the missing link between the prebiotic synthesis of ribose and prebiotic RNA (preRNA). Hydrogen cyanide (HCN) is assumed to have been the principal precursor in the prebiotic formation of aldopentoses in the formose reaction and in the synthesis of ribose. Ribose as the best fitting aldopentose became the exclusive sugar component of RNA. The elevated yield of ribose synthesis at higher temperatures and its protection from decomposition could have driven the polymerization of the ribose-phosphate backbone and the coupling of nucleobases to the backbone. RNA could have come into being without the involvement of nucleotide precursors. The first nucleoside monophosphate is likely to have appeared upon the hydrolysis of preRNA contributed by the presence of reactive 2′-OH moieties in the preRNA chain. As a result of phosphorylation, nucleoside monophosphates became nucleoside triphosphates, substrates for the selective synthesis of genRNA.

SUMMARYThe catalytic subunit of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), Nsp12, has a unique NiRAN domain that transfers nucleoside monophosphates to the Nsp9 protein. The NiRAN and RdRp modules form a dynamic interface distant from their catalytic sites and both activities are essential for viral replication. We report that codon-optimized (for the pause-free translation) Nsp12 exists in inactive state in which NiRAN/RdRp interactions are broken, whereas translation by slow ribosomes and incubation with accessory Nsp7/8 subunits or NTPs partially rescue RdRp activity. Our data show that adenosine and remdesivir triphosphates promote synthesis of A-less RNAs, as does ppGpp, while amino acid substitutions at the NiRAN/RdRp interface augment activation, suggesting that ligand binding to the NiRAN catalytic site modulates RdRp activity. The existence of allosterically-linked nucleotidyl transferase sites that utilize the same substrates has important implications for understanding the mechanism of SARS-CoV-2 replication and design of its inhibitors.HighlightsCodon-optimization of Nsp12 triggers misfolding and activity lossSlow translation, accessory Nsp7 and Nsp8 subunits, and NTPs rescue Nsp12Non-substrate nucleotides activate RNA chain synthesis, likely via NiRAN domainCrosstalk between two Nsp12 active sites that bind the same ligands

Infections with respiratory viruses constitute a huge burden on our health and economy. Antivirals against some respiratory viruses are available, but further options are urgently needed. Enisamium iodide (laboratory code FAV00A, trade name Amizon) is an antiviral marketed in countries of the Commonwealth of Independent States for the treatment of viral respiratory infections, but its clinical efficacy and mode of action are not well understood., Infections with respiratory viruses constitute a huge burden on our health and economy. Antivirals against some respiratory viruses are available, but further options are urgently needed. Enisamium iodide (laboratory code FAV00A, trade name Amizon) is an antiviral, marketed in countries of the Commonwealth of Independent States for the treatment of viral respiratory infections, but its clinical efficacy and mode of action are not well understood. In this study, we investigated the efficacy of enisamium in patients aged between 18 and 60 years with confirmed influenza virus and other viral respiratory infections. Enisamium treatment resulted in reduced influenza virus shedding (at day 3, 71.2% in the enisamium group tested negative versus 25.0% in placebo group [P

Despite gene expression programs being notoriously complex, RNA abundance is usually assumed as a proxy for transcriptional activity. Recently developed approaches, able to disentangle transcriptional and post-transcriptional regulatory processes, have revealed a more complex scenario. It is now possible to work out how synthesis, processing and degradation kinetic rates collectively determine the abundance of each gene’s RNA. It has become clear that the same transcriptional output can correspond to different combinations of the kinetic rates. This underscores the fact that markedly different modes of gene expression regulation exist, each with profound effects on a gene’s ability to modulate its own expression. This review describes the development of the experimental and computational approaches, including RNA metabolic labeling and mathematical modeling, that have been disclosing the mechanisms underlying complex transcriptional programs. Current limitations and future perspectives in the field are also discussed.

Morbilliviruses, such as measles virus (MeV) and canine distemper virus (CDV), are highly infectious members of the paramyxovirus family. MeV is responsible for major morbidity and mortality in non-vaccinated populations. ERDRP-0519, a pan-morbillivirus small molecule inhibitor for the treatment of measles, targets the morbillivirus RNA-dependent RNA-polymerase (RdRP) complex and displayed unparalleled oral efficacy against lethal infection of ferrets with CDV, an established surrogate model for human measles. Resistance profiling identified the L subunit of the RdRP, which harbors all enzymatic activity of the polymerase complex, as the molecular target of inhibition. Here, we examined binding characteristics, physical docking site, and the molecular mechanism of action of ERDRP-0519 through label-free biolayer interferometry, photoaffinity cross-linking, and in vitro RdRP assays using purified MeV RdRP complexes and synthetic templates. Results demonstrate that unlike all other mononegavirus small molecule inhibitors identified to date, ERDRP-0519 inhibits all phosphodiester bond formation in both de novo initiation of RNA synthesis at the promoter and RNA elongation by a committed polymerase complex. Photocrosslinking and resistance profiling-informed ligand docking revealed that this unprecedented mechanism of action of ERDRP-0519 is due to simultaneous engagement of the L protein polyribonucleotidyl transferase (PRNTase)-like domain and the flexible intrusion loop by the compound, pharmacologically locking the polymerase in pre-initiation conformation. This study informs selection of ERDRP-0519 as clinical candidate for measles therapy and identifies a previously unrecognized druggable site in mononegavirus L polymerase proteins that can silence all synthesis of viral RNA., Author summary The mononegavirus order contains major established and recently emerged human pathogens. Despite the threat to human health, antiviral therapeutics directed against this order remain understudied. The mononegavirus polymerase complex represents a promising drug target due to its central importance for both virus replication and viral mitigation of the innate host antiviral response. In this study, we have mechanistically characterized a clinical candidate small-molecule MeV polymerase inhibitor. The compound blocked all phosphodiester bond formation activity, a unique mechanism of action unlike all other known mononegavirus polymerase inhibitors. Photocrosslinking-based target site mapping demonstrated that this class-defining prototype inhibitor stabilizes a pre-initiation conformation of the viral polymerase complex that sterically cannot accommodate template RNA. Function-equivalent druggable sites exist in all mononegavirus polymerases. In addition to its direct anti-MeV impact, the insight gained in this study can therefore serve as a blueprint for indication spectrum expansion through structure-informed scaffold engineering or targeted drug discovery.

The Gammacoronavirus infectious bronchitis virus (IBV) is a highly contagious economically important respiratory pathogen of domestic fowl. Reverse genetics allows for the molecular study of pathogenic determinants to enable rational vaccine design. The recombinant IBV (rIBV) Beau-R, a molecular clone of the apathogenic Beaudette strain, has previously been investigated as a vaccine platform. To determine tissues in which Beau-R could effectively deliver antigenic genes, an in vivo study in chickens, the natural host, was used to compare the pattern of viral dissemination of Beau-R to the pathogenic strain M41-CK. Replication of Beau-R was found to be restricted to soft tissue within the beak, whereas M41-CK was detected in beak tissue, trachea and eyelid up to seven days post infection. In vitro assays further identified that, unlike M41-CK, Beau-R could not replicate at 41 &deg, C, the core body temperature of a chicken, but is able to replicate a 37 &deg, C, a temperature relatable to the very upper respiratory tract. Using a panel of rIBVs with defined mutations in the structural and accessory genes, viral replication at permissive and non-permissive temperatures was investigated, identifying that the Beau-R replicase gene was a determinant of temperature sensitivity and that sub-genomic mRNA synthesis had been affected. The identification of temperature sensitive allelic lesions within the Beau-R replicase gene opens up the possibility of using this method of attenuation in other IBV strains for future vaccine development as well as a method to investigate the functions of the IBV replicase proteins.

Transcription activation by cyclic AMP (cAMP) receptor protein (CAP) is the classic paradigm of transcription regulation in bacteria. CAP was suggested to activate transcription on class-II promoters via a recruitment and isomerization mechanism. However, whether and how it modifies RNA polymerase (RNAP) to initiate transcription remains unclear. Here, we report cryo–electron microscopy (cryo-EM) structures of an intact Escherichia coli class-II CAP-dependent transcription activation complex (CAP-TAC) with and without de novo RNA transcript. The structures reveal two distinct architectures of TAC and raise the possibility that CAP binding may induce substantial conformational changes in all the subunits of RNAP and transiently widen the main cleft of RNAP to facilitate DNA promoter entering and formation of the initiation open complex. These structural changes vanish during further RNA transcript synthesis. The observations in this study may reveal a possible on-pathway intermediate and suggest a possibility that CAP activates transcription by inducing intermediate state, in addition to the previously proposed stabilization mechanism., Cryo-EM structures of transcription activation complexes comprising cyclic AMP receptor protein (CAP) bound to a class-II promoter reveal two distinct architectures and suggest a possibility that CAP activates transcription by inducing an intermediate state, an important supplement to the previously proposed stabilization mechanism.

Simple Summary Replication studies are frequently based on viral production, which provides limited information to understand certain processes. Therefore, to discover which failures in the viral haemorrhagic septicaemia virus (VHSV) replication cycle might be involved in the differences in its virulence on fish, a different approach has been taken. Our results have demonstrated that adsorption and morphogenesis are the steps most involved in the modulation of virulence, although failures in the synthesis step were also observed. As a potential application of our results, we believe that this kind of knowledge relating in vivo virulence to in vitro markers could help reduce the need for experimental infections in animals, representing a step forward in ethical issues. Abstract The viral haemorrhagic septicaemia virus (VHSV), a single-stranded negative-sense RNA novirhabdovirus affecting a wide range of marine and freshwater fish species, is a main concern for European rainbow trout (Oncorhynchus mykiss) fish farmers. Its genome is constituted by six genes, codifying five structural and one nonstructural proteins. Many studies have been carried out to determine the participation of each gene in the VHSV virulence, most of them based on genome sequence analysis and/or reverse genetics to construct specific mutants and to evaluate their virulence phenotype. In the present study, we have used a different approach with a similar aim: hypothesizing that a failure in any step of the replication cycle can reduce the virulence in vivo, we studied in depth the in vitro replication of VHSV in different cell lines, using sets of strains from different origins, with high, low and moderate levels of virulence for fish. The results demonstrated that several steps in the viral replication cycle could affect VHSV virulence in fish, including adsorption, RNA synthesis and morphogenesis (including viral release). Notably, differences among strains in any step of the replication cycle were mostly strain-specific and reflected only in part the in vivo phenotype (high and low virulent). Our data, therefore, support the need for further studies aimed to construct completely avirulent VHSV recombinants targeting a combination of genes rather than a single one in order to study the mechanisms of genes interplay and their effect on viral phenotype in vitro and in vivo.

The sudden emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at the end of 2019 from the Chinese province of Hubei and its subsequent pandemic spread highlight the importance of understanding the full molecular details of coronavirus infection and pathogenesis. Here, we compared a variety of replication features of SARS-CoV-2 and SARS-CoV and analysed the cytopathology caused by the two closely related viruses in the commonly used Vero E6 cell line. Compared to SARS-CoV, SARS-CoV-2 generated higher levels of intracellular viral RNA, but strikingly about 50-fold less infectious viral progeny was recovered from the culture medium. lmmunofluorescence microscopy of SARS-CoV-2-infected cells established extensive cross-reactivity of antisera previously raised against a variety of non-structural proteins, membrane and nucleocapsid protein of SARS-CoV. Electron microscopy revealed that the ultrastructural changes induced by the two SARS viruses are very similar and occur within comparable time frames after infection. Furthermore, we determined that the sensitivity of the two viruses to three established inhibitors of coronavirus replication (remdesivir, alisporivir and chloroquine) is very similar, but that SARS-CoV-2 infection was substantially more sensitive to pre-treatment of cells with pegylated interferon alpha. An important difference between the two viruses is the fact that - upon passaging in Vero E6 cells - SARS-CoV-2 apparently is under strong selection pressure to acquire adaptive mutations in its spike protein gene. These mutations change or delete a putative furin-like cleavage site in the region connecting the S1 and S2 domains and result in a very prominent phenotypic change in plaque assays.

Eight-segmented, negative-sense, single-stranded genomic RNAs of influenza A virus are terminated with 5′ and 3′ untranslated regions (UTRs). All segments have highly conserved extremities of 13 and 12 nucleotides at the 5′ and 3′ UTRs, respectively, constructing the viral RNA (vRNA) promoter. Adjacent to the duplex stem of 3 base pairs (bps) between the two conserved strands, additional 1–4 bps are existing in a segment-specific manner. We investigated the roles of the matrix (M) segment-specific base pair between the 14th nucleotide uridine (U14′) of the 5′ UTR and the 13th nucleotide adenosine (A13) of the 3′ UTR by preparing possible vRNA promoters, named vXY, as well as cRNA promoters, named cYX. We analysed their RNA-dependent RNA replication efficiency using the minigenome replicon system and an enzyme assay system in vitro with synthetic RNA promoters. Notably, in contrast to vAC(s) that is a synthetic vRNA promoter with A14′ and C13, base-pair disruption at the complementary RNA (cRNA) promoter in cAC(s), which has A13′ and C14, not only reduced viral RNA replication in cells but also impaired de novo initiation of unprimed vRNA synthesis. Reverse genetics experiments confirmatively exhibited that this breakage in the cRNA promoter affected the rescue of infectious virus. The present study suggests that the first segment-specific base pair plays an essential role in generating infectious viruses by regulating the promoter activity of cRNA rather than vRNA. It could provide insights into the role of the segment-specific nucleotides in viral genome replication for sustainable infection.

The bifunctional nsp14 subunit of the coronavirus replicase contains 3′-to-5′ exoribonuclease (ExoN) and guanine-N7-methyltransferase domains. For the betacoronaviruses MHV and SARS-CoV, ExoN was reported to promote the fidelity of genome replication, presumably by mediating a form of proofreading. For these viruses, ExoN knockout mutants are viable while displaying an increased mutation frequency. Strikingly, we have now established that the equivalent ExoN knockout mutants of two other betacoronaviruses, MERS-CoV and SARS-CoV-2, are nonviable, suggesting an additional and critical ExoN function in their replication. This is remarkable in light of the very limited genetic distance between SARS-CoV and SARS-CoV-2, which is highlighted, for example, by 95% amino acid sequence identity in their nsp14 sequences. For (recombinant) MERS-CoV nsp14, both its enzymatic activities were evaluated using newly developed in vitro assays that can be used to characterize these key replicative enzymes in more detail and explore their potential as target for antiviral drug development., Coronaviruses (CoVs) stand out for their large RNA genome and complex RNA-synthesizing machinery comprising 16 nonstructural proteins (nsps). The bifunctional nsp14 contains 3′-to-5′ exoribonuclease (ExoN) and guanine-N7-methyltransferase (N7-MTase) domains. While the latter presumably supports mRNA capping, ExoN is thought to mediate proofreading during genome replication. In line with such a role, ExoN knockout mutants of mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus (SARS-CoV) were previously reported to have crippled but viable hypermutation phenotypes. Remarkably, using reverse genetics, a large set of corresponding ExoN knockout mutations has now been found to be lethal for another betacoronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV). For 13 mutants, viral progeny could not be recovered, unless—as happened occasionally—reversion had first occurred. Only a single mutant was viable, likely because its E191D substitution is highly conservative. Remarkably, a SARS-CoV-2 ExoN knockout mutant was found to be unable to replicate, resembling observations previously made for alpha- and gammacoronaviruses, but starkly contrasting with the documented phenotype of ExoN knockout mutants of the closely related SARS-CoV. Subsequently, we established in vitro assays with purified recombinant MERS-CoV nsp14 to monitor its ExoN and N7-MTase activities. All ExoN knockout mutations that proved lethal in reverse genetics were found to severely decrease ExoN activity while not affecting N7-MTase activity. Our study strongly suggests that CoV nsp14 ExoN has an additional function, which apparently is critical for primary viral RNA synthesis and thus differs from the proofreading function that, based on previous MHV and SARS-CoV studies, was proposed to boost longer-term replication fidelity. IMPORTANCE The bifunctional nsp14 subunit of the coronavirus replicase contains 3′-to-5′ exoribonuclease (ExoN) and guanine-N7-methyltransferase domains. For the betacoronaviruses MHV and SARS-CoV, ExoN was reported to promote the fidelity of genome replication, presumably by mediating a form of proofreading. For these viruses, ExoN knockout mutants are viable while displaying an increased mutation frequency. Strikingly, we have now established that the equivalent ExoN knockout mutants of two other betacoronaviruses, MERS-CoV and SARS-CoV-2, are nonviable, suggesting an additional and critical ExoN function in their replication. This is remarkable in light of the very limited genetic distance between SARS-CoV and SARS-CoV-2, which is highlighted, for example, by 95% amino acid sequence identity in their nsp14 sequences. For (recombinant) MERS-CoV nsp14, both its enzymatic activities were evaluated using newly developed in vitro assays that can be used to characterize these key replicative enzymes in more detail and explore their potential as target for antiviral drug development.

In recent years, it has become clear that RNA molecules are involved in almost all vital cellular processes and pathogenesis of human disorders. The functional diversity of RNA comes from its structural richness. Although composed of only four nucleotides, RNA molecules present a plethora of secondary and tertiary structures critical for intra and intermolecular contacts with other RNAs and ligands (proteins, small metabolites, etc.). In order to fully understand RNA function it is necessary to define its spatial structure. Crystallography, nuclear magnetic resonance and cryogenic electron microscopy have demonstrated considerable success in determining the structures of biologically important RNA molecules. However, these powerful methods require large amounts of sample. Despite their limitations, chemical synthesis and in vitro transcription are usually employed to obtain milligram quantities of RNA for structural studies, delivering simple and effective methods for large-scale production of homogenous samples. The aim of this paper is to provide an overview of methods for large-scale RNA synthesis with emphasis on chemical synthesis and in vitro transcription. We also present our own results of testing the efficiency of these approaches in order to adapt the material acquisition strategy depending on the desired RNA construct.

International audience; 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.

The literature devoted to changes in the expression of the renin-angiotensin system (RAS) proteins of cancer cells was analyzed. The dynamics of RAS protein expression in malignant tumors and the possible role of epigenetic mechanisms in these processes are briefly reviewed. Through research of the epigenetic mechanisms in cancer, principally new techniques for their correction based on the use of selective regulatory systems of covalent modification of histone proteins (for example, deacetylase inhibitor) and microRNA synthesis technologies have been developed. Literature data show promising pharmacological correction of epigenetic modification of chromatin in the treatment of cancer., Analizirana je literatura posvećena promjenama u izraženosti RAS proteina stanica raka. Daje se kratak pregled dinamike izraženosti RAS proteina kod zloćudnih tumora i moguće uloge epigenetskih mehanizama u ovim procesima. Istraživanje epigenetskih mehanizama kod raka rezultiralo je razvojem bitno novih tehnika za njihovo ispravljanje, utemeljenih na primjeni selektivnih regulatornih sustava kovalentne modifikacije histona (npr. inhibitor deacetilaze) i tehnologije sinteze mikroRNA. Literaturni podaci pokazuju obećavajuće farmakološko ispravljanje epigenetske modifikacije kromatina u liječenju raka.

Baloxavir marboxil (Xofluza™; baloxavir) is an oral cap-dependent endonuclease inhibitor that has been developed by Roche and Shionogi. The drug blocks influenza virus proliferation by inhibiting the initiation of mRNA synthesis. In February 2018, baloxavir received its first global approval in Japan for the treatment of influenza A or B virus infections. Phase III development is underway in the USA, EU and other countries for this indication. This article summarized the milestones in the development of baloxavir leading to this first global approval for influenza A or B virus infections.

It is generally thought that the promoters of non-segmented, negative strand RNA viruses (nsNSVs) direct the polymerase to initiate RNA synthesis exclusively opposite the 3´ terminal nucleotide of the genome RNA by a de novo (primer independent) initiation mechanism. However, recent studies have revealed that there is diversity between different nsNSVs with pneumovirus promoters directing the polymerase to initiate at positions 1 and 3 of the genome, and ebolavirus polymerases being able to initiate at position 2 on the template. Studies with other RNA viruses have shown that polymerases that engage in de novo initiation opposite position 1 typically have structural features to stabilize the initiation complex and ensure efficient and accurate initiation. This raised the question of whether different nsNSV polymerases have evolved fundamentally different structural properties to facilitate initiation at different sites on their promoters. Here we examined the functional properties of polymerases of respiratory syncytial virus (RSV), a pneumovirus, human parainfluenza virus type 3 (PIV-3), a paramyxovirus, and Marburg virus (MARV), a filovirus, both on their cognate promoters and on promoters of other viruses. We found that in contrast to the RSV polymerase, which initiated at positions 1 and 3 of its promoter, the PIV-3 and MARV polymerases initiated exclusively at position 1 on their cognate promoters. However, all three polymerases could recognize and initiate from heterologous promoters, with the promoter sequence playing a key role in determining initiation site selection. In addition to examining de novo initiation, we also compared the ability of the RSV and PIV-3 polymerases to engage in back-priming, an activity in which the promoter template is folded into a secondary structure and nucleotides are added to the template 3´ end. This analysis showed that whereas the RSV polymerase was promiscuous in back-priming activity, the PIV-3 polymerase generated barely detectable levels of back-primed product, irrespective of promoter template sequence. Overall, this study shows that the polymerases from these three nsNSV families are fundamentally similar in their initiation properties, but have differences in their abilities to engage in back-priming., Author summary The non-segmented negative strand RNA viruses are a large group of viruses that includes a number of significant human pathogens. Their viral genome is transcribed and replicated by a virally-encoded polymerase, which is considered to be a good target for intervention with antiviral drugs. Defining the similarities and differences between the polymerases of different viruses could be helpful for developing inhibitors with potency against a broad spectrum of related viruses. In this study, we examined the RNA synthesis initiation properties of polymerases of viruses from three different non-segmented negative strand RNA virus families and showed that they are fundamentally similar in the ways that they can initiate RNA synthesis. However, we also found that polymerases from two different virus families differed significantly in their ability to perform another activity, referred to as back-priming, which suggests that there might be structural differences between them. Thus, this study identifies similarities and differences between the polymerases in this group of viruses.

To suppress serious influenza infections in persons showing insufficient protection from the vaccines, antiviral drugs are of vital importance. There is a need for novel agents with broad activity against influenza A (IAV) and B (IBV) viruses and with targets that differ from those of the current antivirals. We here report a new small molecule influenza virus inhibitor referred to as CPD A (chemical name: N-(pyridin-3-yl)thiophene-2-carboxamide). In an influenza virus minigenome assay, this non-nucleoside compound inhibited RNA synthesis of IAV and IBV with EC50 values of 2.3 μM and 2.6 μM, respectively. Robust in vitro activity was noted against a broad panel of IAV (H1N1 and H3N2) and IBV strains, with a median EC50 value of 0.20 μM, which is 185-fold below the 50% cytotoxic concentration. The action point in the viral replication cycle was located between 1 and 5 h p.i., showing a similar profile as ribavirin. Like this nucleoside analogue, CPD A was shown to cause strong depletion of the cellular GTP pool and, accordingly, its antiviral activity was antagonized when this pool was restored with exogenous guanosine. This aligns with the observed inhibition in a cell-based IMP dehydrogenase (IMPDH) assay, which seems to require metabolic activation of CPD A since no direct inhibition was seen in an enzymatic IMPDH assay. The combination of CPD A with ribavirin, another IMPDH inhibitor, proved strongly synergistic. To conclude, we established CPD A as a new inhibitor of influenza A and B virus replication and RNA synthesis, and support the potential of IMPDH inhibitors for influenza therapy with acceptable safety profile. ispartof: ANTIVIRAL RESEARCH vol:196 ispartof: location:Netherlands status: published

Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the “backtracked” state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.

We have characterized the single subunit RNA polymerase from Klebsiella phage KP34. The enzyme is unique among known bacteriophage RNA polymerases in that it recognizes two unrelated promoter sequences, which provided clues for the evolution of phage single-subunit RNA polymerases. As the first representative enzyme from the “phiKMV-like viruses” cluster, its use in run-off RNA synthesis was investigated. RNA-Seq analysis revealed that the KP34 RNA polymerase does not possess the undesired self-templated RNA terminus extension known for T7 RNA polymerase and is suitable to synthesize RNAs with structured 3′ termini such as sgRNAs. A KP34 RNA polymerase Y603F mutant is engineered to incorporate deoxy- and 2′-fluoro ribonucleotide into RNA.

The Red clover necrotic mosaic virus (RCNMV) genome consists of two plus-strand RNA genome segments, RNA1 and RNA2. RNA2 contains a multifunctional RNA structure known as the trans-activator (TA) that (i) promotes subgenomic mRNA transcription from RNA1, (ii) facilitates replication of RNA2, and (iii) mediates particle assembly and copackaging of genome segments. The TA has long been considered a unique RNA element in RCNMV. However, by examining results from RCNMV genome analyses in the ViRAD virus (re-)annotation database, a putative functional RNA element in the polymerase-coding region of RNA1 was identified. Structural and functional analyses revealed that the novel RNA element adopts a TA-like structure (TALS) and, similar to the requirement of the TA for RNA2 replication, the TALS is necessary for the replication of RNA1. Both the TA and TALS possess near-identical asymmetrical internal loops that are critical for efficient replication of their corresponding genome segments, and these structural motifs were found to be functionally interchangeable. Moreover, replacement of the TA in RNA2 with a stabilized form of the TALS directed both RNA2 replication and packaging of both genome segments. Based on their comparable properties and considering evolutionary factors, we propose that the TALS appeared de novo in RNA1 first and, subsequently, the TA arose de novo in RNA2 as a functional mimic of the TALS. This and other related information were used to formulate a plausible evolutionary pathway to describe the genesis of the bi-segmented RCNMV genome. The resulting scenario provides an evolutionary framework to further explore and test possible origins of this segmented RNA plant virus., Author summary We have identified a novel RNA element in RNA1 of the segmented positive-strand RNA genome of Red clover necrotic mosaic virus (RCNMV). Its similarity in structure to a known important RNA element in RNA2 suggests that the two RNA structures perform similar viral functions in their respective genome segments, and this was confirmed experimentally. Based on evolutionary considerations, the RNA element in RNA1 is proposed to have appeared earlier in RCNMV genome genesis, with the corresponding element in RNA2 arising subsequently as a functional mimic of the former. This information was integrated into a plausible pathway for the emergence and evolution of this segmented viral genome, thereby providing novel insights into the puzzling history of RCNMV.

How homochirality concerning biopolymers (DNA/RNA/proteins) could have originally occurred (i.e., arisen from a non-life chemical world, which tended to be chirality-symmetric) is a long-standing scientific puzzle. For many years, people have focused on exploring plausible physic-chemical mechanisms that may have led to prebiotic environments biased to one chiral type of monomers (e.g., D-nucleotides against L-nucleotides; L-amino-acids against D-amino-acids)–which should have then assembled into corresponding polymers with homochirality, but as yet have achieved no convincing advance. Here we show, by computer simulation–with a model based on the RNA world scenario, that the biased-chirality may have been established at polymer level instead, just deriving from a racemic mixture of monomers (i.e., equally with the two chiral types). In other words, the results suggest that the homochirality may have originated along with the advent of biopolymers during the origin of life, rather than somehow at the level of monomers before the origin of life., Author summary People have long been curious about the fact that central molecules in the living world (biopolymers), i.e., nucleic acids and proteins, are asymmetric in chirality (handedness), but as the relevant background, the chemical world is symmetric in chirality. Now that life should have originated from a prebiotic non-life background, how could this dissymmetry have occurred? Previous studies in this area focused their efforts on how the chirality-symmetry may have been broken at the monomer level (i.e., nucleotides or amino acids), but have achieved little advance over decades of years. Here we demonstrate, by in silico simulation, that instead, the required chirality-deviation may have been established along with the emergence of biopolymers at the beginning stage in the origin of life–just deriving from a chirality-symmetric monomer pool. The process is actually not only an issue of chemistry but also an issue involving evolution–thus previously difficult to reveal by pure lab work in this area. By modelling the evolutionary process, the present computer simulation study provides significant clues for experiments in future.

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.

The nuclear organelle the nucleolus and the transcription factor nuclear factor of kappa-light-chain-enhancer of activated B cells (NF-kappa B) are both central to the control of cellular homeostasis, dysregulated in common diseases and implicated in the ageing process. Until recently, it was believed that they acted independently to regulate homeostasis in health and disease. However, there is an emerging body of evidence suggesting that nucleoli and NF-kappa B signalling converge at multiple levels. Here we will review current understanding of this crosstalk. We will discuss activation of the NF-kappa B pathway by nucleolar stress and induction of apoptosis by nucleolar sequestration of NF-kappa B/RelA. We will also discuss the role of TIF-IA, COMMD1, and nucleophosmin, which are key players in this crosstalk, and the therapeutic relevance, particularly with respect to the antitumour effects of aspirin.

DNA microarrays have become commonplace in the last two decades, but the synthesis of other nucleic acids biochips, most importantly RNA, has only recently been developed to a similar extent. RNA microarrays can be seen as organized surfaces displaying a potentially very large number of unique sequences and are of invaluable help in understanding the complexity of RNA structure and function as they allow the probing and treatment of each of the many different sequences simultaneously. Three approaches have emerged for the fabrication of RNA microarrays. The earliest examples used a direct, manual or mechanical, deposition of pre-synthesized, purified RNA oligonucleotides onto the surface in a process called spotting. In a second approach, pre-spotted or in situ-synthesized DNA microarrays are employed as templates for the transcription of RNA, subsequently or immediately captured on the surface. Finally, a third approach attempts to mirror the phosphoramidite-based protocols for in situ synthesis of high-density DNA arrays in order to produce in situ synthesized RNA microarrays. In this mini-review, we describe the chemistry and the engineering behind the fabrications methods, underlining the advantages and shortcomings of each, and illustrate how versatile these platforms can be by presenting some of their applications.

Rotaviruses (RVs) are medically significant gastrointestinal pathogens and are a leading cause of childhood mortality in many countries. The RV RNA-dependent RNA polymerase, VP1, synthesizes RNA during viral replication only in the presence of another RV protein, VP2, which comprises the innermost core shell layer of the virion. Though these VP1-VP2 interactions are essential for RV replication, the mechanism by which the core shell regulates polymerase activity remains incompletely understood. Here, we sought to identify and characterize specific regions of both VP1 and VP2 that are required for core shell dependent polymerase activity. First, we used bioinformatics approaches to analyze VP1 and VP2 sequence diversity across many RV strains and identify positional locations of critical amino acid changes within the context of known structural domains and motifs. We next tested how the identified sequence differences influenced VP2-dependent VP1 activity in vitro. These data revealed that VP1 and VP2 protein diversity correlates with functional differences between avian and mammalian RV strains. Then, we used these sequential and functional incompatibilities to map key regions of VP1 important for mediating RNA synthesis. To pinpoint critical interacting regions of VP1 and VP2, we used site directed mutagenesis to engineer several modified VP1 and VP2 proteins. Then, we employed an in vitro RNA synthesis assay to test how the introduced mutations influenced VP2-dependent VP1 activity. Altogether, our results revealed several functionally important VP1 residues critical for in vitro VP2-dependent VP1 activity, either individually or in combination with neighboring residues, including E265/L267, R614, and D971/S978/I980. Structural analyses show VP2 interactions at these surface-exposed VP1 sites, which altogether supports a direct contact model of core shell dependent RV polymerase activity. Moreover, recombinant VP1 proteins containing multiple mutations at buried residues were incapable of facilitating RNA synthesis in vitro under the assay conditions, indicating that an extensive intramolecular signaling network exists to mediate VP1 activity. Taken together, these results suggest that VP2 binding at the VP1 surface may induce intramolecular interactions critical for VP1 activity. Overall, results from these studies provide important insight into VP1-VP2 binding interface(s) that are necessary for RV replication. Ph. D. Rotaviruses (RVs) are clinically-significant gastrointestinal pathogens that cause severe diarrhea and dehydration in children. RVs encode a specialized polymerase enzyme, called VP1, which functions to synthesize RNA during viral replication. RNA synthesis activities of VP1 are tightly regulated by another RV protein, VP2, which comprises the innermost core shell layer of the virion. Though these VP1-VP2 interactions are essential for viral replication, the mechanism by which the core shell supports polymerase activity remains poorly understood. Here, we sought to identify and characterize specific regions of both VP1 and VP2 that are essential for polymerase activity in a test tube (i.e., in vitro). First, we analyzed VP1 and VP2 sequence diversity across many RV strains. Then, we tested how the identified sequence differences influenced VP2-dependent VP1 activation in vitro. To pinpoint critical regions of VP1 and VP2, we next engineered and assayed several mutant proteins. Altogether, our results revealed several functionally important residues of VP1 and VP2, which raises new ideas about VP1-VP2 binding interface(s) that are important for viral replication. Moreover, results from these studies may provide a scientific platform for the rational design of next-generation RV vaccines or antiviral therapeutics.

Accumulation of DNA lesions causing transcription stress is associated with natural and accelerated aging and culminates with profound metabolic alterations. Our understanding of the mechanisms governing metabolic redesign upon genomic instability, however, is highly rudimentary. Using Ercc1-defective mice and Xpg knock-out mice, we demonstrate that combined defects in transcription-coupled DNA repair (TCR) and in nucleotide excision repair (NER) directly affect bioenergetics due to declined transcription, leading to increased ATP levels. This in turn inhibits glycolysis allosterically and favors glucose rerouting through the pentose phosphate shunt, eventually enhancing production of NADPH-reducing equivalents. In NER/TCR-defective mutants, augmented NADPH is not counterbalanced by increased production of pro-oxidants and thus pentose phosphate potentiation culminates in an over-reduced redox state. Skin fibroblasts from the TCR disease Cockayne syndrome confirm results in animal models. Overall, these findings unravel a mechanism connecting DNA damage and transcriptional stress to metabolic redesign and protective antioxidant defenses., ERCC1 is involved in a number of DNA repair pathways including nucleotide excision repair. Here the authors showed that reduced transcription in Ercc1-deficient mouse livers and cells increases ATP levels, suppressing glycolysis and rerouting glucose into the pentose phosphate shunt that generates reductive stress.

RNA synthesis in respiratory syncytial virus (RSV), a negative-sense (−) nonsegmented RNA virus, consists of viral gene transcription and genome replication. Gene transcription includes the positive-sense (+) viral mRNA synthesis, 5′-RNA capping and methylation, and 3′ end polyadenylation. Genome replication includes (+) RNA antigenome and (−) RNA genome synthesis. RSV executes the viral RNA synthesis using an RNA synthesis ribonucleoprotein (RNP) complex, comprising four proteins, the nucleoprotein (N), the large protein (L), the phosphoprotein (P), and the M2-1 protein. We provide an overview of the RSV RNA synthesis and the structural insights into the RSV gene transcription and genome replication process. We propose a model of how the essential four proteins coordinate their activities in different RNA synthesis processes.

Ribosome biogenesis is a complex process that is responsible for the formation of ribosomes and ultimately global protein synthesis. The first step in this process is the synthesis of the ribosomal RNA in the nucleolus, transcribed by RNA Polymerase I. Historically, abnormal nucleolar structure is indicative of poor cancer prognoses. In recent years, it has been shown that ribosome biogenesis, and rDNA transcription in particular, is dysregulated in cancer cells. Coupled with advancements in screening technology that allowed for the discovery of novel drugs targeting RNA Polymerase I, this transcriptional machinery is an increasingly viable target for cancer therapies. In this review, we discuss ribosome biogenesis in breast cancer and the different cellular pathways involved. Moreover, we discuss current therapeutics that have been found to affect rDNA transcription and more novel drugs that target rDNA transcription machinery as a promising avenue for breast cancer treatment.

All cellular genetic information is transcribed into RNA by multisubunit RNA polymerases (RNAPs). The basal transcription initiation factors of cellular RNAPs stimulate the initial RNA synthesis via poorly understood mechanisms. Here, we explored the mechanism employed by the bacterial factor σ in promoter-independent initial transcription. We found that the RNAP holoenzyme lacking the promoter-binding domain σ4 is ineffective in de novo transcription initiation and displays high propensity to pausing upon extension of RNAs 3 to 7 nucleotides in length. The nucleotide at the RNA 3' end determines the pause lifetime. The σ4 domain stabilizes short RNA:DNA hybrids and suppresses pausing by stimulating RNAP active-center translocation. The anti-pausing activity of σ4 is modulated by its interaction with the β subunit flap domain and by the σ remodeling factors AsiA and RbpA. Our results suggest that the presence of σ4 within the RNA exit channel compensates for the intrinsic instability of short RNA:DNA hybrids by increasing RNAP processivity, thus favoring productive transcription initiation. This "RNAP boosting" activity of the initiation factor is shaped by the thermodynamics of RNA:DNA interactions and thus, should be relevant for any factor-dependent RNAP.

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., Graphical Abstract, Highlights • Structure of SARS-CoV-2 elongation complex with nsp9 has been determined • Nsp9 binds to the catalytic center of the nsp12 (RdRp) NiRAN domain • The nsp12 NiRAN domain catalyzes the formation of the cap core structure (GpppA) • The structure reveals an intermediate state for RTC in cap synthesis, Yan et al. present a cryo-EM structure of the SARS-CoV-2 replication and transcription complex that includes the single RNA-binding protein nsp9. The structural snapshot provides insight into how the viral machinery catalyzes a crucial step in viral mRNA cap synthesis, which is important for virus survival.

The CRISPR-Cas9 system has become the most popular and efficient method for genome engineering in mammalian cells. The Streptococcus pyogenes Cas9 nuclease can function with two types of guide RNAs: the native dual crRNA and tracrRNA (crRNA:tracrRNA) or a chimeric single guide RNA (sgRNA). Although sgRNAs expressed from a DNA vector are predominant in the literature, guide RNAs can be rapidly generated by chemical synthesis and provide equivalent functionality in gene editing experiments. This review highlights the attributes and advantages of chemically synthesized guide RNAs including the incorporation of chemical modifications to enhance gene editing efficiencies in certain applications. The use of synthetic guide RNAs is also uniquely suited to genome-scale high throughput arrayed screening, particularly when using complex phenotypic assays for functional genomics studies. Finally, the use of synthetic guide RNAs along with DNA-free sources of Cas9 (mRNA or protein) allows for transient CRISPR-Cas9 presence in the cell, thereby resulting in a decreased probability of off-target events.

The term proteogenomics is basically the integration of proteomics with genomics and transcriptomics. Today, proteogenomics is developing on the way to combined understanding about overall cellular functions. At present globally, structure of genes, expression of genes (in the form of mRNA synthesis), synthesis of proteins (translation of mRNA) and post-translational modification (structural modification of proteins) have turn out to be technically practicable and act as a novel viewpoint to molecular procedures. Current research has proved the importance of proteogenomics technology in cancer for studying molecular signature of tumors particularly in human beings, and its treatment and prevention. Proteogenomics is not restricted to oncology but it also plays a vital role in other areas of life sciences and biomedicines and anticipation can make up these areas. Here in this minireview, we will discuss the latest progress made in recent years, challenges and viewpoints about proteogenomics technology.

Previous studies have demonstrated that the nuclear localization of ras homolog family member A (RhoA), with prominent concentration in the nucleolus, is a common feature in human cancer tissues and cancer cell lines. Although a previous study has demonstrated that the nuclear translocation of RhoA occurs via active transport, a process that occurs through importin α in a nuclear factor-κB-dependent manner, the mechanism, biological function and pathological meaning of the nucleolar residency of RhoA remain to be elucidated. As the cell nucleolus is the site of ribosome biosynthesis, the aim of the present study was to investigate the association between RNA synthesis and the nucleolar localization of RhoA, as well as the molecular mechanisms underlying the residency of RhoA in the nucleolus of HEp-2 (human larynx epithelial carcinoma) cells. Indirect immunofluorescence microscopy was used to evaluate the subcellular distribution of nuclear RhoA, and immunoblotting analysis was used to determine the total cellular protein level of RhoA. Consistent with the results of previous studies, untreated HEp-2 cells exhibited bright nucleolar staining, indicating an increased concentration of RhoA in the nucleoli. Treatment with actinomycin D for the inhibition of RNA synthesis caused a redistribution of RhoA from the nucleoli to the nucleoplasm with a speckled staining pattern. Immunoblotting revealed that neither the total cellular amount of RhoA nor the integrity of RhoA was affected by treatment with actinomycin D. In cells that were treated at a decreased concentration (0.05 mg/l) of actinomycin D, the redistribution of RhoA was reversible following the removal of the drug from the culture medium. However, this reversal was not observed at an increased drug concentration (1 mg/l). Overall, to the best of our knowledge, the results of the present study provide the first in situ evidence that the inhibition of RNA synthesis induces a redistribution of nucleolar RhoA to the nucleoplasm, and additionally suggest that the nucleolar residency of RhoA in HEp-2 cells may be associated with active RNA synthesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic novel coronavirus that has caused a worldwide outbreak.Here we describe a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that uses a portable device for efficient detection of SARS-CoV-2. This RT-LAMP assay specifically detected SARS-CoV-2 without cross-reacting with the most closely related human coronavirus, SARS-CoV. Clinical evaluation of nasal swab samples from suspected SARS-CoV-2 pneumonia (COVID-19) patients showed that the assay could detect over 23.7 copies within 15 min with a 100% probability. Since the RT-LAMP assay can be performed with a portable battery-supported device, it is a rapid, simple, and sensitive diagnostic assay for COVID-19 that can be available at point-of-care. We also developed the RT-LAMP assay without the RNA extraction step-Direct RT-LAMP, which could detect more than 1.43 x 103 copies within 15 min with a 100% probability in clinical evaluation test. Although the Direct RT-LAMP assay was less sensitive than the standard RT-LAMP, the Direct RT-LAMP assay can be available as the rapid first screening of COVID-19 in poorly equipped areas, such as rural areas in developing countries., PLoS neglected tropical diseases, 14(11), art.no.e0008855; 2020