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Host polymerases mediate off-target toxicity of nucleotide analogs and the active metabolite of sofosbuvir was found to not be efficiently incorporated by host polymerases including the mitochondrial RNA polymerase (POLRMT). Knowledge from studying inhibitors of HCV RdRp serves to advance antiviral drug discovery for other emerging RNA viruses including the discovery of remdesivir as an inhibitor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2), the virus that causes COVID-19.Nucleotide analogs are the cornerstone of direct acting antivirals used to control infection by RNA viruses. Here we review what is known about existing nucleotide/nucleoside analogs and the kinetics and mechanisms of RNA and DNA replication, with emphasis on the SARS-CoV-2 RNA dependent RNA polymerase (RdRp) in comparison to HIV reverse transcriptase and Hepatitis C RdRp. We demonstrate how accurate kinetic analysis reveals surprising results to explain the effectiveness of antiviral nucleoside analogs providing guidelines for the design of new inhibitors.The treatment of viral infections remains challenging, in particular in the face of emerging pathogens. Broad-spectrum antiviral drugs could potentially be used as a first line of defense. The RNA-dependent RNA polymerase (RdRp) of RNA viruses serves as a logical target for drug discovery and development efforts. Herein we discuss compounds that target RdRp of poliovirus, hepatitis C virus, influenza viruses, respiratory syncytial virus, and the growing data on coronaviruses. We focus on nucleotide analogs and mechanisms of action and resistance.RNA polymerase (RNAP) is the central enzyme of gene expression, which transcribes DNA to RNA. All cellular organisms synthesize RNA with highly conserved multi-subunit DNA-dependent RNAPs, except mitochondrial RNA transcription, which is carried out by a single-subunit RNAP. Over 60 years of extensive research has elucidated the structures and functions of cellular RNAPs. In this review, we introduce a brief structural feature of bacterial RNAP, the most well characterized model enzyme, and a novel experimental approach known as "Time-dependent soak-trigger-freeze X-ray crystallography" which can be used to observe the RNA synthesis reaction at atomic resolution in real time. This principle methodology can be used for elucidating fundamental mechanisms of cellular RNAP transcription.Flaviviruses such as dengue, Japanese encephalitis, West Nile, Yellow Fever and Zika virus, cause viral hemorrhagic fever and encephalitis in humans. However, antiviral therapeutics to treat or prevent flavivirus infections are not yet available. Thus, there is pressing need to develop therapeutics and vaccines that target flavivirus infections. All flaviviruses carry a positive-sense single-stranded RNA genome, which encodes ten proteins; three structural proteins form the virus shell, and seven nonstructural (NS) proteins are involved in replication of the viral genome. While all NS proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) are part of a functional membrane-bound replication complex, enzymatic activities required for flaviviral replication reside in only two NS proteins, NS3 and NS5. NS3 functions as a protease, helicase, and triphosphatase, and NS5 as a capping enzyme, methyltransferase, and RNA-dependent RNA polymerase. In this chapter, we provide an overview of viral replication focusing on the structure and function of NS3 and NS5 replicases. We further describe strategies and examples of current efforts to identify potential flavivirus inhibitors against NS3 and NS5 enzymatic activities that can be developed as therapeutic agents to combat flavivirus infections.The 2C proteins of Picornaviridae are unique members of AAA+ protein family. Although picornavirus 2C shares many conserved motifs with Super Family 3 DNA helicases, duplex unwinding activity of many 2C proteins remains undetected, and high-resolution structures of 2C hexamers are unavailable. All characterized 2C proteins exhibit ATPase activity, but the purpose of ATP hydrolysis is not fully understood. 2C is highly conserved among picornaviruses and plays crucial roles in nearly all steps of the virus lifecycle. It is therefore considered as an effective target for broad-spectrum antiviral drug development. Crystallographic investigation of enterovirus 2C proteins provide structural details important for the elucidation of 2C function and development of antiviral drugs. This chapter summarizes not only the findings of enzymatic activities, biochemical and structural characterizations of the 2C proteins, but also their role in virus replication, immune evasion and morphogenesis. The linkage between structure and function of the 2C proteins is discussed in detail. Inhibitors targeting the 2C proteins are also summarized to provide an overview of drug development. Finally, we raise several key questions to be addressed in this field and provide future research perspective on this unique class of ATPases.RNA-dependent RNA polymerases (RdRPs) encoded by RNA viruses represent a unique class of processive nucleic acid polymerases, carrying out DNA-independent replication/transcription processes. Although viral RdRPs have versatile global structures, they do share a structurally highly conserved active site comprising catalytic motifs A-G. In spite of different initiation modes, the nucleotide addition cycle (NAC) in the RdRP elongation phase probably follows consistent mechanisms. In this chapter, representative structures of picornavirus RdRP elongation complexes are used to illustrate RdRP NAC mechanisms. In the pre-chemistry part of the NAC, RdRPs utilize a unique palm domain-based active site closure that can be further decomposed into two sequential steps. In the post-chemistry part of the NAC, the translocation process is stringently controlled by the RdRP-specific motif G, resulting in asymmetric movements of the template-product RNA. Future efforts to elucidate regulation/intervention mechanisms by mismatched NTPs or nucleotide analog antivirals are necessary to achieve comprehensive understandings of viral RdRP NAC.Stochastic outcomes of viral infections are attributed in large part to multiple layers of intrinsic and extrinsic heterogeneity that exist within a population of cells and viruses. Traditional methods in virology often lack the ability to demonstrate cell-to-cell variability in response to the invasion of viruses, and to decipher the sources of heterogeneities that are reflected in the variable infection dynamics. To overcome this challenge, the field of single-cell virology emerged less than a decade ago, enabling researchers to reveal the behavior of single, isolated, infected cells that has been masked in population-based assays. The use of microfluidics in single-cell virology, in particular, has resulted in the development of high-throughput devices that are capable of capturing, isolating, and monitoring single infected cells over the duration of an infection. Results from the studies of viral infection dynamics presented in this chapter indicate how single-cell data provide a more accurate prediction of the start time, replication rate, duration, and yield of infection when compared to population-based data. Additionally, single-cell analysis reveals striking differences between genetically distinct viruses that are almost indistinguishable in population methods. Importantly, both the efficacy and distinct mechanisms of action of antiviral compounds can be elucidated by using single-cell analysis.All RNA viruses encode an RNA-dependent RNA polymerase (RdRp) responsible for genome replication. It is now recognized that enzymes in general, and RdRps specifically, are dynamic macromolecular machines such that their moving parts, including active site loops, play direct functional roles. While X-ray crystallography has provided deep insight into structural elements important for RdRp function, this methodology generally provides only static snapshots, and so is limited in its ability to report on dynamic fluctuations away from the lowest energy conformation. Nuclear magnetic resonance (NMR), molecular dynamics (MD) simulations and other biophysical techniques have brought new insight into RdRp function by their ability to characterize the trajectories, kinetics and thermodynamics of conformational motions. In particular, these methodologies have identified coordinated motions among conserved structural motifs necessary for nucleotide selection and incorporation. Disruption of these motions through amino acid substitutions or inhibitor binding impairs RdRp function. Understanding and re-engineering these motions thus provides exciting new avenues for anti-viral strategies. This chapter outlines the basics of these methodologies, summarizes the dynamic motions observed in different RdRps important for nucleotide selection and incorporation, and illustrates how this information can be leveraged towards rational vaccine strain development and anti-viral drug design.Faithfull replication of genomic information relies on the coordinated activity of the multi-protein machinery known as the replisome. Several constituents of the replisome operate as molecular motors that couple thermal and chemical energy to a mechanical task. Over the last few decades, in vitro single-molecule manipulation techniques have been used to monitor and manipulate mechanically the activities of individual molecular motors involved in DNA replication with nanometer, millisecond, and picoNewton resolutions. These studies have uncovered the real-time kinetics of operation of these biological systems, the nature of their transient intermediates, and the processes by which they convert energy to work (mechano-chemistry), ultimately providing new insights into their inner workings of operation not accessible by ensemble assays. In this chapter, we describe two of the most widely used single-molecule manipulation techniques for the study of DNA replication, optical and magnetic tweezers, and their application in the study of the activities of proteins involved in viral DNA replication.The ongoing Covid-19 pandemic has spurred research in the biology of the nidovirus severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Much focus has been on the viral RNA synthesis machinery due to its fundamental role in viral propagation. The central and essential enzyme of the RNA synthesis process, the RNA-dependent RNA polymerase (RdRp), functions in conjunction with a coterie of viral-encoded enzymes that mediate crucial nucleic acid transactions. Some of these enzymes share common features with other RNA viruses, while others play roles unique to nidoviruses or CoVs. The RdRps are proven targets for viral pathogens, and many of the other nucleic acid processing enzymes are promising targets. The purpose of this review is to summarize recent advances in our understanding of the mechanisms of RNA synthesis in CoVs. By reflecting on these studies, we hope to emphasize the remaining gaps in our knowledge. The recent onslaught of structural information related to SARS-CoV-2 RNA synthesis, in combination with previous structural, genetic and biochemical studies, have vastly improved our understanding of how CoVs replicate and process their genomic RNA. Structural biology not only provides a blueprint for understanding the function of the enzymes and cofactors in molecular detail, but also provides a basis for drug design and optimization. Venetoclax The concerted efforts of researchers around the world, in combination with the renewed urgency toward understanding this deadly family of viruses, may eventually yield new and improved antivirals that provide relief to the current global devastation.
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