Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bunyaviridae family is the largest and most diverse family of RNA viruses. It has more than 350 members divided into five genera: Orthobunyavirus, Phlebovirus, Nairovirus, Hantavirus, and Tospovirus. They are present in the five continents, causing recurrent epidemics, epizootics, and considerable agricultural loss. The genome of bunyaviruses is divided into three segments of negative single-stranded RNA according to their relative size: L (Large), M (Medium) and S (Small) segment. Bunyaviridae RNA-dependent RNA polymerase (RdRp) is encoded by the L segment, and is in charge of the replication and transcription of the viral RNA in the cytoplasm of the infected cell. Viral RdRps share a characteristic right hand-like structure with three subdomains: finger, palm, and thumb subdomains that define the formation of the catalytic cavity. In addition to the N-terminal endonuclease domain, eight conserved motifs (A-H) have been identified in the RdRp of Bunyaviridae. In this review, we have summarized the recent insights from the structural and functional studies of RdRp to understand the roles of different motifs shared by RdRps, the mechanism of viral RNA replication, genome segment packaging by the nucleoprotein, cap-snatching, mRNA transcription, and other RNA mechanisms of bunyaviruses.
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PMID:Bunyaviridae RdRps: structure, motifs, and RNA synthesis machinery. 2841 34

Influenza polymerase is a heterotrimer protein with both endonuclease and RNA-dependent RNA polymerase (RdRp) activity. It plays a critical role in viral RNA replication and transcription and has been targeted for antiviral drug development. In this study, we characterized the activity of recombinant RdRp purified at 1:1:1 ratio in both ApG-primed RNA replication and mRNA-initiated RNA transcription. The heterotrimer complex showed comparable activity profiles to that of viral particle derived crude replication complex, and in contrast to the crude replication complex, was suitable for detailed mechanistic studies of nucleotide incorporation. The recombinant RdRp was further used to examine distinct modes of inhibition observed with five different nucleotide analog inhibitors, and the apparent steady-state binding affinity Kapp was measured for selected analogs to correlate antiviral activity and enzymatic inhibition with substrate efficiency.
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PMID:Biochemical characterization of recombinant influenza A polymerase heterotrimer complex: Polymerase activity and mechanisms of action of nucleotide analogs. 2902 Jan

The influenza RNA-dependent RNA polymerase (RdRP) is conserved among different types of influenza virus, playing an important part in transcription and replication. In this regard, influenza RdRP is an attractive target for novel anti-influenza drug discovery. Herein, we will introduce the structural and functional information of influenza polymerase; and an overview of inhibitors targeting the PA endonuclease and PB2 cap-binding site is provided, along with the approaches utilized for identification of these inhibitors. The protein-protein interactions (PPIs) of the three polymerase subunits: PA, PB1 and PB2, are described based on the published crystal structures, and inhibitors targeting the PA-PB1 interaction are introduced briefly.
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PMID:Influenza A virus polymerase: an attractive target for next-generation anti-influenza therapeutics. 2933 7

The Arenaviridae family, together with the Bunyaviridae and Orthomyxoviridae families, is one of the three negative-stranded RNA viral families that encode an endonuclease in their genome. The endonuclease domain is at the N-terminus of the L protein, a multifunctional protein that includes the RNA-dependent RNA polymerase. The synthesis of mRNA in arenaviruses is a process that is primed by capped nucleotides that are 'stolen' from the cellular mRNA by the endonuclease domain in cooperation with other domains of the L protein. This molecular mechanism has been demonstrated previously for the endonuclease of the prototype Lymphocytic choriomeningitis virus (LCMV). However, the mode of action of this enzyme is not fully understood as the original structure did not contain catalytic metal ions. The pivotal role played by the cap-snatching process in the life cycle of the virus and the highly conserved nature of the endonuclease domain make it a target of choice for the development of novel antiviral therapies. Here, the binding affinities of two diketo-acid (DKA) compounds (DPBA and L-742,001) for the endonuclease domain of LCMV were evaluated using biophysical methods. X-ray structures of the LCMV endonuclease domain with catalytic ions in complex with these two compounds were determined, and their efficacies were assessed in an in vitro endonuclease-activity assay. Based on these data and computational simulation, two new DKAs were synthesized. The LCMV endonuclease domain exhibits a good affinity for these DKAs, making them a good starting point for the design of arenavirus endonuclease inhibitors. In addition to providing the first example of an X-ray structure of an arenavirus endonuclease incorporating a ligand, this study provides a proof of concept that the design of optimized inhibitors against the arenavirus endonuclease is possible.
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PMID:Crystal structures of Lymphocytic choriomeningitis virus endonuclease domain complexed with diketo-acid ligands. 2976 12

The heterotrimeric influenza A virus RNA-dependent RNA polymerase complex, composed of PB1, PB2, and PA subunits, is responsible for transcribing and replicating the viral RNA genome. The N-terminal endonuclease domain of the PA subunit performs endonucleolytic cleavage of capped host RNAs to generate capped RNA primers for viral transcription. A surface-exposed flexible loop (PA^51-72-loop) in the PA endonuclease domain has been shown to be dispensable for endonuclease activity. Interestingly, the PA^51-72-loop was found to form different intramolecular interactions depending on the conformational arrangement of the polymerase. In this study, we show that a PA subunit lacking the PA^51-72-loop assembles into a heterotrimeric polymerase with PB1 and PB2. We demonstrate that in a cellular context, the PA^51-72-loop is required for RNA replication but not transcription by the viral polymerase. In agreement, recombinant viral polymerase lacking the PA^51-72-loop is able to carry out cap-dependent transcription but is inhibited in de novo replication initiation in vitro Furthermore, viral RNA (vRNA) synthesis is also restricted during ApG-primed extension, indicating that the PA^51-72-loop is required not only for replication initiation but also for elongation on a cRNA template. We propose that the PA^51-72-loop plays a role in the stabilization of the replicase conformation of the polymerase. Together, these results further our understanding of influenza virus RNA genome replication in general and highlight a role of the PA endonuclease domain in polymerase function in particular.IMPORTANCE Influenza A viruses are a major global health threat, not only causing significant morbidity and mortality every year but also having the potential to cause severe pandemic outbreaks like the 1918 influenza pandemic. The viral polymerase is a protein complex which is responsible for transcription and replication of the viral genome and therefore is an attractive target for antiviral drug development. For that purpose it is important to understand the mechanisms of how the virus replicates its genome and how the viral polymerase works on a molecular level. In this report, we characterize the role of the flexible surface-exposed PA^51-72-loop in polymerase function and offer new insights into the replication mechanism of influenza A viruses.
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PMID:The Surface-Exposed PA^51-72-Loop of the Influenza A Virus Polymerase Is Required for Viral Genome Replication. 2987 49

The hantavirus RNA-dependent RNA polymerase (RdRp) snatches 5' capped mRNA fragments from the host cell transcripts and uses them as primers to initiate transcription and replication of the viral genome in the cytoplasm of infected cells. Hantavirus nucleocapsid protein (N protein) binds to the 5' caps of host cell mRNA and protects them from the attack of cellular decapping machinery. N protein rescues long capped mRNA fragments in cellular P bodies that are later processed by an unknown mechanism to generate 10- to 14-nucleotide-long capped RNA primers with a 3' G residue. Hantavirus RdRp has an N-terminal endonuclease domain and a C-terminal uncharacterized domain that harbors a binding site for the N protein. The purified endonuclease domain of RdRp nonspecifically degraded RNA in vitro It is puzzling how such nonspecific endonuclease activity generates primers of appropriate length and specificity during cap snatching. We fused the N-terminal endonuclease domain with the C-terminal uncharacterized domain of the RdRp. The resulting NC mutant, with the assistance of N protein, generated capped primers of appropriate length and specificity from a test mRNA in cells. Bacterially expressed and purified NC mutant and N protein required further incubation with the lysates of human umbilical vein endothelial cells (HUVECs) for the specific endonucleolytic cleavage of a test mRNA to generate capped primers of appropriate length and defined 3' terminus in vitro Our results suggest that an unknown host cell factor facilitates the interaction between N protein and NC mutant and brings the N protein-bound capped RNA fragments in close proximity to the endonuclease domain of the RdRp for specific cleavage at a precise length from the 5' cap. These studies provide critical insights into the cap-snatching mechanism of cytoplasmic viruses and have revealed potential new targets for their therapeutic intervention.IMPORTANCE Humans acquire hantavirus infection by the inhalation of aerosolized excreta of infected rodent hosts. Hantavirus infections cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), with mortality rates of 15% and 50%, respectively (1). Annually 150,000 to 200,000 cases of hantavirus infections are reported worldwide, for which there is no treatment at present. Cap snatching is an early event in the initiation of virus replication in infected hosts. Interruption in cap snatching will inhibit virus replication and will likely improve the prognosis of the hantavirus disease. Our studies provide mechanistic insight into the cap-snatching mechanism and demonstrate the requirement of a host cell factor for successful cap snatching. Identification of this host cell factor will reveal a novel therapeutic target for combating this viral illness.
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PMID:Hantavirus RdRp Requires a Host Cell Factor for Cap Snatching. 3054 36

In eukaryotes with multiple small RNA pathways, the mechanisms that channel RNAs within specific pathways are unclear. Here, we reveal the reactions that account for channeling in the small interfering RNA (siRNA) biogenesis phase of the Arabidopsis RNA-directed DNA methylation pathway. The process begins with template DNA transcription by NUCLEAR RNA POLYMERASE IV (Pol IV), whose atypical termination mechanism, induced by nontemplate DNA base-pairing, channels transcripts to the associated RNA-dependent RNA polymerase RDR2. RDR2 converts Pol IV transcripts into double-stranded RNAs and then typically adds an extra untemplated 3' terminal nucleotide to the second strands. The dicer endonuclease DCL3 cuts resulting duplexes to generate 24- and 23-nt siRNAs. The 23-nt RNAs bear the untemplated terminal nucleotide of the RDR2 strand and are underrepresented among ARGONAUTE4-associated siRNAs. Collectively, our results provide mechanistic insights into Pol IV termination, Pol IV-RDR2 coupling, and RNA channeling, from template DNA transcription to siRNA strand discrimination.
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PMID:Reaction Mechanisms of Pol IV, RDR2, and DCL3 Drive RNA Channeling in the siRNA-Directed DNA Methylation Pathway. 3139 24

Significant efforts have been reported on the development of influenza antivirals including inhibitors of the RNA-dependent RNA polymerase PA N-terminal (PA_N) endonuclease. Based on recently identified, highly active metal-binding pharmacophores (MBPs) for PA_N endonuclease inhibition, a fragment-based drug development campaign was pursued. Guided by coordination chemistry and structure-based drug design, MBP scaffolds were elaborated to improve activity and selectivity. Structure-activity relationships were established and used to generate inhibitors of influenza endonuclease with tight-binding affinities. The activity of these inhibitors was analyzed using a fluorescence-quenching-based nuclease activity assay, and binding was validated using differential scanning fluorometry. Lead compounds were found to be highly selective for PA_N endonuclease against several related dinuclear and mononuclear metalloenzymes. Combining principles of bioinorganic and medicinal chemistry in this study has resulted in some of the most active in vitro influenza PA_N endonuclease inhibitors with high ligand efficiencies.
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PMID:SAR Exploration of Tight-Binding Inhibitors of Influenza Virus PA Endonuclease. 3153 40

Tomato spotted wilt virus (TSWV; genus Orthotospovirus, family Tospoviridae) has a huge impact on a large range of plants worldwide. In this study, we determined the sequence of the large (L) RNA segment that encodes the RNA-dependent RNA polymerase (RdRp) from a TSWV isolate (LYE51) collected in the south of France. Analysis of the phylogenetic relationships of TSWV-LYE51 with other TSWV isolates shows that it is closely related to other European isolates. A 3D model of TSWV-LYE51 RdRp was built by homology with the RdRp structure of the La Crosse virus (genus Orthobunyavirus, family Peribunyaviridae). Finally, an analysis of positive and negative selection was carried out on 30 TSWV full-length RNA L sequences and compared with the phylogeny and the protein structure data. We showed that the seven codons that are under positive selection are distributed all along the RdRp gene. By contrast, the codons associated with negative selection are especially concentrated in three highly constrained domains: the endonuclease in charge of the cap-snatching mechanism, the thumb domain and the mid domain. Those three domains could constitute good candidates to look for host targets on which genetic resistance by loss of susceptibility could be developed.
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PMID:Analysis of tomato spotted wilt virus RNA-dependent RNA polymerase adaptative evolution and constrained domains using homology protein structure modelling. 3195 51

Influenza virus RNA-dependent RNA polymerase (vRdRp) does not have capping activity and relies on the capped RNAs produced by the host RNA polymerase II (RNAPII). The viral polymerases process the capped RNAs to produce short capped RNA fragments that are used as primers to initiate the transcription of viral mRNAs. This process, known as cap-snatching, can be targeted by antiviral therapeutics. Here, anthralin was identified as an inhibitor against influenza a virus (IAV) infection by targeting the cap-snatching activity of the viral polymerase. Anthralin, an FDA-approved drug used in the treatment of psoriasis, shows antiviral activity against IAV infection in vitro and in vivo. Importantly, anthralin significantly reduces weight loss, lung injury, and mortality caused by IAV infection in mice. The mechanism of action study revealed that anthralin inhibits the cap-binding function of PB2 subunit and endonuclease activity of PA. As a result, viral mRNA transcription is blocked, leading to the decreases in viral RNA replication and viral protein expression. In conclusion, anthralin has been demonstrated to have the potential of an alternative antiviral against influenza virus infection. Also, targeting the captive pocket structure that includes the N-terminus of PA endonuclease domain and the C-terminal of PB2 cap-binding domain of IAV RdRp may be an excellent strategy for developing anti-influenza drugs.
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PMID:Anthralin Suppresses the Proliferation of Influenza Virus by Inhibiting the Cap-Binding and Endonuclease Activity of Viral RNA Polymerase. 3213 85


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