Quercetin may reduce the risk of developing the symptoms of COVID-19.

Objective: Recent evidence reported that some dietary compounds like quercetin and apigenin as the most well-known flavonoids with anti-inflammatory effects may inhibit SARS-CoV-2 main protease. The hypothesis of the promising effects and possible mechanisms of action of quercetin against COVID-19 were assessed in this article. Materials and Methods: Related papers on the inhibitory effects of quercetin against COVID-19 were collected using the following search strategy: "corona or coronavirus or COVID or COVID-19 or viral or virus" AND "nutrient or flavonoid or Quercetin". Results: The findings indicated that quercetin can be considered an effective agent against COVID-19 because of its SARS-CoV-2 main protease and RNA-dependent RNA polymerase inhibitory effects. In addition, quercetin may attenuate angiotensin-converting enzyme-2 (ACE-2) receptors leading to a reduction of SARS-CoV-2 ability to enter host cells. Moreover, the antiviral, anti-inflammatory, and immunomodulatory activities of quercetin have been frequently reported. Conclusion: Quercetin may be an effective agent for managing the complications of COVID-19. Further longitudinal human studies are warranted.

Investigating the Potential of 2-Ethylbutyl-(Phenoxy)phosphoryl-D-Alaninate Against RNA-Dependent RNA Polymerase (RdRp) of SARS-CoV-2.

There was an emergency call globally when COVID-19 was detected in December 2019. The SARS-CoV-2 virus, a modified virus, is what causes this contagious disease. Although research is being conducted throughout the world, the main target is still to find the promising candidate to target RNA-dependent RNA polymerase (RdRp) to provide possible drug against COVID-19. Aim of this work is to find a molecule to inhibit the translational process of viral protein synthesis. Density Functional Theory calculations revealed information about the formation of the desired ligand (RD). Molecular docking of RD with RdRp was performed and compared with some reported molecules and the data revealed that RD had the best docking score with RdRp (-6.7 kcal/mol). Further, molecular dynamics (MD) simulations of RD with RdRp of SARS-CoV-2 revealed the formation of stable complex with a maximum number of seven hydrogen bonds. Root mean square deviations values are in acceptable range and root mean square fluctuations has less fluctuation indicate stable complex formation. Further, based on MM-GBSA calculation, RD formed a stable complex with RdRp of nCoV with ΔG° of -12.28 kcal·mol-1.

How does the polymerase of non-segmented negative strand RNA viruses commit to transcription or genome replication?

The Mononegavirales, or non-segmented negative-sense RNA viruses (nsNSVs), includes significant human pathogens, such as respiratory syncytial virus, parainfluenza virus, measles virus, Ebola virus, and rabies virus. Although these viruses differ widely in their pathogenic properties, they are united by each having a genome consisting of a single strand of negative-sense RNA. Consistent with their shared genome structure, the nsNSVs have evolved similar ways to transcribe their genome into mRNAs and replicate it to produce new genomes. Importantly, both mRNA transcription and genome replication are performed by a single virus-encoded polymerase. A fundamental and intriguing question is: how does the nsNSV polymerase commit to being either an mRNA transcriptase or a replicase? The polymerase must become committed to one process or the other either before it interacts with the genome template or in its initial interactions with the promoter sequence at the 3´ end of the genomic RNA. This review examines the biochemical, molecular biology, and structural biology data regarding the first steps of transcription and RNA replication that have been gathered over several decades for different families of nsNSVs. These findings are discussed in relation to possible models that could explain how an nsNSV polymerase initiates and commits to either transcription or genome replication.

Japanese encephalitis virus NS5 protein interacts with nucleolin to enhance the virus replication.

Japanese encephalitis virus (JEV) is an arthropod-borne, plus-strand flavivirus causing viral encephalitis in humans with a high case fatality rate. The JEV non-structural protein 5 (NS5) with the RNA-dependent RNA polymerase activity interacts with the viral and host proteins to constitute the replication complex. We have identified the multifunctional protein Nucleolin (NCL) as one of the several NS5-interacting host proteins. We demonstrate the interaction and colocalization of JEV NS5 with NCL in the virus-infected HeLa cells. The siRNA-mediated knockdown of NCL indicated that it was required for efficient viral replication. Importantly, JEV grew to higher titers in cells over-expressing exogenous NCL, demonstrating its pro-viral role. We demonstrated that NS5 interacted with the RRM and GAR domains of NCL. We show that the NCL-binding aptamer AS1411 containing the G-quadruplex (GQ) structure and the GQ ligand BRACO-19 caused significant inhibition of JEV replication. The antiviral effect of AS1411 and BRACO-19 could be overcome in HeLa cells by the overexpression of exogenous NCL. We demonstrated that the synthetic RNAs derived from the 3'-NCR of JEV genomic RNA containing the GQ sequence could bind NCL in vitro. The replication complex binding to the 3'-NCR is required for the viral RNA synthesis. It is likely that NCL present in the replication complex destabilizes the GQ structures in the genomic RNA, thus facilitating the movement of the replication complex resulting in efficient virus replication.IMPORTANCEJapanese encephalitis virus (JEV) is endemic in most parts of South-East Asia and the Western Pacific region, causing epidemics of encephalitis with a high case fatality rate. While a tissue culture-derived JEV vaccine is available, no antiviral therapy exists. The JEV NS5 protein has RNA-dependent RNA polymerase activity. Together with several host and viral proteins, it constitutes the replication complex necessary for virus replication. Understanding the interaction of NS5 with the host proteins could help design novel antivirals. We identified Nucleolin (NCL) as a crucial host protein interactor of JEV NS5 having a pro-viral role in virus replication. The NS5-interacting NCL binds to the G-quadruplex (GQ) structure sequence in the 3'-NCR of JEV RNA. This may smoothen the movement of the replication complex along the genomic RNA, thereby facilitating the virus replication. This study is the first report on how NCL, a host protein, helps in JEV replication through GQ-binding.

Inhibition of early RNA replication in Chikungunya and Dengue virus by lycorine: In vitro and in silico studies.

Arboviruses such as chikungunya virus (CHIKV) and dengue virus (DENV) collectively afflict millions of individuals worldwide particularly in endemic countries like India, leading to substantial morbidity and mortality. With the lack of effective vaccines for both CHIKV and DENV in India, the search for antiviral compounds becomes paramount to control these viral infections. In line with this, our investigation was focused on screening natural compounds for their potential antiviral activity against CHIKV and DENV. Using different assays, including plaque assay, immunofluorescence, and reverse transcription-quantitative real-time PCR (qRT-PCR), out of 109 natural compounds tested, we confirmed lycorine's in vitro antiviral activity against CHIKV and DENV at low micromolar concentrations in different cell types. Time of addition assays indicated that lycorine does not impede viral entry. Additionally, qRT-PCR results along with time of addition assay suggested that lycorine interferes with the synthesis of negative strand viral RNA. Molecular docking analysis was done to understand the mode of inhibition of viral replication. The results revealed that the most likely binding site with the highest binding affinity of lycorine, was at the palm and finger domains, in the vicinity of the catalytic site of CHIKV and DENV RNA-dependent RNA polymerase (RdRp). Collectively, our data underscores the potential of lycorine to be developed as a direct acting inhibitor for DENV and CHIKV, addressing the critical need of requirement of an antiviral in regions where these viruses pose significant public health threats.

Point mutations at specific sites of the nsp12-nsp8 interface dramatically affect the RNA polymerization activity of SARS-CoV-2.

In a recent characterization of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variability present in 30 diagnostic samples from patients of the first COVID-19 pandemic wave, 41 amino acid substitutions were documented in the RNA-dependent RNA polymerase (RdRp) nsp12. Eight substitutions were selected in this work to determine whether they had an impact on the RdRp activity of the SARS-CoV-2 nsp12-nsp8-nsp7 replication complex. Three of these substitutions were found around the polymerase central cavity, in the template entry channel (D499G and M668V), and within the motif B (V560A), and they showed polymerization rates similar to the wild type RdRp. The remaining five mutations (P323L, L372F, L372P, V373A, and L527H) were placed near the nsp12-nsp8F contact surface; residues L372, V373, and L527 participated in a large hydrophobic cluster involving contacts between two helices in the nsp12 fingers and the long α-helix of nsp8F. The presence of any of these five amino acid substitutions resulted in important alterations in the RNA polymerization activity. Comparative primer elongation assays showed different behavior depending on the hydrophobicity of their side chains. The substitution of L by the bulkier F side chain at position 372 slightly promoted RdRp activity. However, this activity was dramatically reduced with the L372P, and L527H mutations, and to a lesser extent with V373A, all of which weaken the hydrophobic interactions within the cluster. Additional mutations, specifically designed to disrupt the nsp12-nsp8F interactions (nsp12-V330S, nsp12-V341S, and nsp8-R111A/D112A), also resulted in an impaired RdRp activity, further illustrating the importance of this contact interface in the regulation of RNA synthesis.

[Research Advances in the Association Between Alzheimer's Disease and Double-Stranded RNA-Dependent Protein Kinase].

Alzheimer's disease (AD) is a severe threat to human health and one of the three major causes of human death.Double-stranded RNA-dependent protein kinase (PKR) is an interferon-induced protein kinase involved in innate immunity.In the occurrence and development of AD,PKR is upregulated and continuously activated.On the one hand,the activation of PKR triggers an integrated stress response in brain cells.On the other hand,it indirectly upregulates the expression of ß-site amyloid precursor protein cleaving enzyme 1 and facilitates the accumulation of amyloid-ß protein (Aß),which could activate PKR activator to further activate PKR,thus forming a sustained accumulation cycle of Aß.In addition,PKR can promote Tau phosphorylation,thereby reducing microtubule stability in nerve cells.Inflammation in brain tissue,neurotoxicity resulted from Aß accumulation,and disruption of microtubule stability led to the progression of AD and the declines of memory and cognitive function.Therefore,PKR is a key molecule in the development and progression of AD.Effective PKR detection can aid in the diagnosis and prediction of AD progression and provide opportunities for clinical treatment.The inhibitors targeting PKR are expected to control the activity of PKR,thereby controlling the progression of AD.Therefore,PKR could be a target for the development of therapeutic drugs for AD.

SARS-CoV-2 replication and drug discovery.

The coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed millions of people and continues to wreak havoc across the globe. This sudden and deadly pandemic emphasizes the necessity for anti-viral drug development that can be rapidly administered to reduce morbidity, mortality, and virus propagation. Thus, lacking efficient anti-COVID-19 treatment, and especially given the lengthy drug development process as well as the critical death tool that has been associated with SARS-CoV-2 since its outbreak, drug repurposing (or repositioning) constitutes so far, the ideal and ready-to-go best approach in mitigating viral spread, containing the infection, and reducing the COVID-19-associated death rate. Indeed, based on the molecular similarity approach of SARS-CoV-2 with previous coronaviruses (CoVs), repurposed drugs have been reported to hamper SARS-CoV-2 replication. Therefore, understanding the inhibition mechanisms of viral replication by repurposed anti-viral drugs and chemicals known to block CoV and SARS-CoV-2 multiplication is crucial, and it opens the way for particular treatment options and COVID-19 therapeutics. In this review, we highlighted molecular basics underlying drug-repurposing strategies against SARS-CoV-2. Notably, we discussed inhibition mechanisms of viral replication, involving and including inhibition of SARS-CoV-2 proteases (3C-like protease, 3CLpro or Papain-like protease, PLpro) by protease inhibitors such as Carmofur, Ebselen, and GRL017, polymerases (RNA-dependent RNA-polymerase, RdRp) by drugs like Suramin, Remdesivir, or Favipiravir, and proteins/peptides inhibiting virus-cell fusion and host cell replication pathways, such as Disulfiram, GC376, and Molnupiravir. When applicable, comparisons with SARS-CoV inhibitors approved for clinical use were made to provide further insights to understand molecular basics in inhibiting SARS-CoV-2 replication and draw conclusions for future drug discovery research.

Design of novel broad-spectrum antiviral nucleoside analogues using natural bases ring-opening strategy.

The global prevalence of RNA virus infections has presented significant challenges to public health in recent years, necessitating the expansion of its alternative therapeutic library. Due to its evolutional conservation, RNA-dependent RNA polymerase (RdRp) has emerged as a potential target for broad-spectrum antiviral nucleoside analogues. However, after over half a century of structural modification, exploring unclaimed chemical space using frequently-used structural substitution methods to design new nucleoside analogues is challenging. In this study, we explore the use of the "ring-opening" strategy to design new base mimics, thereby using these base mimics to design new nucleoside analogues with broad-spectrum antiviral activities. A total of 29 compounds were synthesized. Their activity against viral RdRp was initially screened using an influenza A virus RdRp high-throughput screening model. Then, the antiviral activity of 38a was verified against influenza virus strain A/PR/8/34 (H1N1), demonstrating a 50% inhibitory concentration (IC50) value of 9.95 µM, which was superior to that of ribavirin (the positive control, IC50 = 11.43 µM). Moreover, 38a also has inhibitory activity against coronavirus 229E with an IC50 of 30.82 µM. In addition, compounds 42 and 46f exhibit an 82% inhibition rate against vesicular stomatitis virus at a concentration of 20 µM and hardly induce cytotoxicity in host cells. This work demonstrates the feasibility of designing nucleoside analogues with "ring-opening" bases and suggests the "ring-opening" nucleosides may have greater polarity, and designing prodrugs is an important aspect of optimizing their antiviral activity. Future research should focus on enhancing the conformational restriction of open-loop bases to mimic Watson-Crick base pairing better and improve antiviral activity.

Adenosine modifications impede SARS-CoV-2 RNA-dependent RNA transcription.

SARS-CoV-2, the causative virus of the COVID-19 pandemic, follows SARS and MERS as recent zoonotic coronaviruses causing severe respiratory illness and death in humans. The recurrent impact of zoonotic coronaviruses demands a better understanding of their fundamental molecular biochemistry. Nucleoside modifications, which modulate many steps of the RNA lifecycle, have been found in SARS-CoV-2 RNA, although whether they confer a pro- or anti-viral effect is unknown. Regardless, the viral RNA-dependent RNA polymerase will encounter these modifications as it transcribes through the viral genomic RNA. We investigated the functional consequences of nucleoside modification on the pre-steady state kinetics of SARS-CoV-2 RNA-dependent RNA transcription using an in vitro reconstituted transcription system with modified RNA templates. Our findings show that N6-methyladenosine and 2'O-methyladenosine modifications slow the rate of viral transcription at magnitudes specific to each modification, which has the potential to impact SARS-CoV-2 genome maintenance.

Screening for SARS-CoV-2 and Other Coronaviruses in Urban Pigeons (Columbiformes) from the North of Spain under a 'One Health' Perspective.

Coronaviruses have a major impact on human and animal health. The SARS-CoV-2, a beta coronavirus responsible for the COVID-19 pandemic, is a clear example. It continues circulating and causes human deaths, and its high replication rate results in numerous variants. Coronaviruses adapt to birds and mammals and constitute a serious threat, and new viruses are likely to emerge. Urban pigeons (Columbiformes) are synanthropic birds of great interest from a 'One Health' perspective, due to their interaction with humans and other animals. Aware that they may act as viral reservoirs and contribute to their spread, we aimed to investigate the possible presence of SARS-CoV-2 and other coronaviruses in Columbiformes in the city of Logroño, Spain. Oropharyngeal and cloacal swabs were tested using real-time (N1 and E genes from SARS-CoV-2) and conventional PCR assays (RdRp gene from all coronaviruses). SARS-CoV-2 was not detected. A total of 13.3% of pigeons harbored coronaviruses closely related to Gamma coronavirus (Igacovirus) from Columbiformes in Finland, Poland and China. Monitoring the emergence of a new variant of SARS-CoV-2 capable of infecting Columbiformes should continue. SARS-CoV-2 is still circulating, the viral RNA of this virus has been detected in avian species (Phasianidae and Anatidae), and other coronaviruses are associated with animals that are in close contact with humans. The presence of Gamma coronavirus in urban pigeons must be considered for the risk of surveillance of human infections.

Genetic detection and analysis of porcine norovirus in pigs farmed in north Vietnam.

Norovirus (NoV) causing gastroenteritis symptoms, which has been reported in several hosts, including humans, pigs, and rats. This study was conducted to identify porcine viral infection and to characterize NoV strains from pigs in some provinces in north Vietnam. Totally, 102 fecal samples from diarrheal pigs on farms in six cities and provinces in northern Vietnam during July 2022 to March 2023 were collected. Polymerase chain reaction was used to identify the viral genome. Positive samples were used for nucleotide sequencing of the partial RNA-dependent RNA polymerase gene sequence. Five (4.9 %) positive stool samples were detected from animals farmed in five different farms, with one positive animal identified in each farm. Genetic analysis indicated that nucleotide identity was in the range 97.77-99.62 % among the 5 NoVs in this study. Phylogenetic analysis pointed out that the five NoVs were Genotype II.19 viruses. Genetically, these strains were closely related to porcine NoV strains that were reported in China in 2009.

Mechanism and spectrum of inhibition of a 4´-cyano modified nucleotide analog against diverse RNA polymerases of prototypic respiratory RNA viruses.

The development of safe and effective broad-spectrum antivirals that target the replication machinery of respiratory viruses is of high priority in pandemic preparedness programs. Here, we studied the mechanism of action of a newly discovered nucleotide analog against diverse RNA-dependent RNA polymerases (RdRp) of prototypic respiratory viruses. GS-646939 is the active 5'-triphosphate (TP) metabolite of a 4'-cyano modified C-adenosine analog phosphoramidate prodrug GS-7682. Enzyme kinetics show that the RdRps of human rhinovirus type 16 (HRV-16) and enterovirus 71 (EV-71) incorporate GS-646939 with unprecedented selectivity; GS-646939 is incorporated 20-50-fold more efficiently than its natural ATP counterpart. The RdRp complex of respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) incorporate GS-646939 and ATP with similar efficiency. In contrast, influenza B RdRp shows a clear preference for ATP and human mitochondrial RNA polymerase (h-mtRNAP) does not show significant incorporation of GS-646939. Once incorporated into the nascent RNA strand, GS-646939 acts as a chain-terminator although higher NTP concentrations can partially overcome inhibition for some polymerases. Modeling and biochemical data suggest that the 4'-modification inhibits RdRp translocation. Comparative studies with GS-443902, the active triphosphate form of the 1'-cyano modified prodrugs remdesivir and obeldesivir, reveal not only different mechanisms of inhibition, but also differences in the spectrum of inhibition of viral polymerases. In conclusion, 1'-cyano and 4'-cyano modifications of nucleotide analogs provide complementary strategies to target the polymerase of several families of respiratory RNA viruses.

Exploring the source of TYLCV resistance in Nicotiana benthamiana.

Tomato Yellow Leaf Curl Virus (TYLCV) is one of the most devastating pathogens of tomato, worldwide. It is vectored by the globally prevalent whitefly, Bemisia tabaci, and is asymptomatic in a wide range of plant species that act as a virus reservoir. The most successful crop protection for tomato in the field has been from resistance genes, of which five loci have been introgressed fromwild relatives. Of these, the Ty-1/Ty-3 locus, which encodes an RNA-dependent RNA polymerase 3 (RDR3), has been the most effective. Nevertheless, several TYLCV strains that break this resistance are beginning to emerge, increasing the need for new sources of resistance. Here we use segregation analysis and CRISPR-mediated gene dysfunctionalisation to dissect the differential response of two isolates of Nicotiana benthamiana to TYLCV infection. Our study indicates the presence of a novel non-RDR3, but yet to be identified, TYLCV resistance gene in a wild accession of N. benthamiana. This gene has the potential to be incorporated into tomatoes.

HSP90 is part of a protein complex with the L polymerase of Rift Valley fever phlebovirus and prevents its degradation by the proteasome during the viral genome replication/transcription stage.

The mosquito-borne Rift Valley fever virus (RVFV) from the Phenuiviridae family is a single-stranded RNA virus that causes the re-emerging zoonotic disease Rift Valley fever (RVF). Classified as a Category A agent by the NIH, RVFV infection can cause debilitating disease or death in humans and lead to devastating economic impacts by causing abortion storms in pregnant cattle. In a previous study, we showed that the host chaperone protein HSP90 is an RVFV-associated host factor that plays a critical role post viral entry, during the active phase of viral genome replication/transcription. In this study, we have elucidated the molecular mechanisms behind the regulatory effect of HSP90 during infection with RVFV. Our results demonstrate that during the early infection phase, host HSP90 associates with the viral RNA-dependent RNA polymerase (L protein) and prevents its degradation through the proteasome, resulting in increased viral replication.

RNA-dependent RNA polymerases regulate ascospore discharge through the exonic-sRNA-mediated RNAi pathway.

Ascospores, forcibly released into the air from perithecia, are the primary inoculum for Fusarium head blight. In Fusarium graminearum, the biological functions of four RNA-dependent RNA polymerases (RdRPs) (Fgrdrp1-4) have been reported, but their regulatory mechanisms are poorly understood and the function of Fgrdrp5 is still unknown. In this study, we found that in addition to Fgrdrp1 and Fgrdrp2, Fgrdrp5 also plays an important role in ascospore discharge, and they all participate in the generation of turgor pressure in a polyol-dependent manner. Moreover, these three genes all affect the maturation of ascospores. Deep sequencing and co-analysis of small RNA and mRNA certified that Fgrdrp1, Fgrdrp2, and Fgrdrp5 partly share their functions in the biogenesis and accumulation of exonic small interference RNA (ex-siRNA), and these three RdRPs negatively regulate the expression levels of ex-siRNA corresponding genes, including certain genes associated with ascospore development or discharge. Furthermore, the differentially expressed genes of deletion mutants, those involved in lipid and sugar metabolism or transport as well as sexual development-related transcription factors, may also contribute to the defects in ascospore maturation or ascospore discharge. In conclusion, our study suggested that the components of the dicer-dependent ex-siRNA-mediated RNA interference pathway include at least Fgrdrp1, Fgrdrp2, and Fgrdrp5. IMPORTANCE: We found that in addition to Fgrdrp1 and Fgrdrp2, Fgrdrp5 also plays important roles in ascospore maturation and ascospore discharge of Fusarium graminearum. These three RNA-dependent RNA polymerases participate in the biogenesis and accumulation of exonic small interference RNA and then regulate ascospore discharge.

Safety of RNA-Dependent RNA Polymerase Inhibitors, Molnupiravir and VV116, for Oral Treatment of COVID-19: A Meta-Analysis.

Background: The RNA-dependent RNA polymerase (RdRp) inhibitors, molnupiravir and VV116, have the potential to maximize clinical benefits in the oral treatment of COVID-19. Subjects who consume these drugs may experience an increased incidence of adverse events. This study aimed to evaluate the safety profile of molnupiravir and VV116. Methods: A comprehensive search of scientific and medical databases, such as PubMed Central/Medline, Embase, Web of Science, and Cochrane Library, was conducted to find relevant articles in English from January 2020 to June 2023. Any kind of adverse events reported in the study were pooled and analyzed in the drug group versus the control group. Estimates of risk effects were summarized through the random effects model using Review Manager version 5.2, and sensitivity analysis was performed by Stata 17.0 software. Results: Fifteen studies involving 32,796 subjects were included. Eleven studies were placebo-controlled, and four were Paxlovid-controlled. Twelve studies reported adverse events for molnupiravir, and three studies described adverse events for VV116. The total odds ratio (OR) for adverse events in the RdRp inhibitor versus the placebo-controlled group was 1.01 (95% CI=0.84-1.22; I2=26%), P=0.88. The total OR for adverse events in the RdRp inhibitor versus the Paxlovid-controlled group was 0.32 (95% CI=0.16-0.65; I2=87%), P=0.002. Individual drug subgroup analysis in the placebo-controlled study showed that compared with the placebo group, a total OR for adverse events was 0.97 (95% CI, 0.85-1.10; I2=0%) in the molnupiravir group and 3.77 (95% CI=0.08-175.77; I2=85%) in the VV116 group. Conclusion: The RdRp inhibitors molnupiravir and VV116 are safe for oral treatment of COVID-19. Further evidence is necessary that RdRp inhibitors have a higher safety profile than Paxlovid.

Novel analogues of a nonnucleoside SARS-CoV-2 RdRp inhibitor as potential antivirotics.

The RNA-dependent RNA polymerase (RdRp) represents a prominent target in the discovery and development of new antivirotics against RNA viruses, inhibiting the replication process. One of the most targeted RNA viruses of the last years is, without doubt, SARS-CoV-2, the cause of the recent COVID-19 pandemic. HeE1-2Tyr, a known inhibitor of flaviviral RdRp, has been discovered to also have antiviral potency against this coronavirus. In this study, we report three distinct modifications of HeE1-2Tyr: conversion of the core from a benzothiazole to a benzoxazole moiety and two different scaffold simplifications, respectively. We provide a novel synthetic approach and, in addition, evaluate the final molecules in an in vitro polymerase assay for biological activity.

The myriad roles of RNA structure in the flavivirus life cycle.

As positive-sense RNA viruses, the genomes of flaviviruses serve as the template for all stages of the viral life cycle, including translation, replication, and infectious particle production. Yet, they encode just 10 proteins, suggesting that the structure and dynamics of the viral RNA itself helps shepherd the viral genome through these stages. Herein, we highlight advances in our understanding of flavivirus RNA structural elements through the lens of their impact on the viral life cycle. We highlight how RNA structures impact translation, the switch from translation to replication, negative- and positive-strand RNA synthesis, and virion assembly. Consequently, we describe three major themes regarding the roles of RNA structure in flavivirus infections: 1) providing a layer of specificity; 2) increasing the functional capacity; and 3) providing a mechanism to support genome compaction. While the interactions described herein are specific to flaviviruses, these themes appear to extend more broadly across RNA viruses.

A Semiquantitative Protein-Fragment Complementation Assay to Study Protein-Protein Interactions of the Polymerase Complex in Cellula.

Protein-fragment complementation assays (PCAs) are powerful tools to investigate protein-protein interactions in a cellular context. These are especially useful to study unstable proteins and weak interactions that may not resist protein isolation or purification. The PCA based on the reconstitution of the Gaussia princeps luciferase (split-luc) is a sensitive approach allowing the mapping of protein-protein interactions and the semiquantitative measurement of binding affinity. Here, we describe the split-luc protocol we used to map the viral interactome of measles virus polymerase complex.