RESUMEN
The influenza RNA-dependent RNA polymerase (RdRp) both replicates the flu's RNA genome and transcribes its mRNA. Replication occurs de novo; however, initiation of transcription requires a 7-methylguanosine 5'-capped primer that is "snatched" from host mRNA via endonuclease and cap binding functions of the influenza polymerase. A key question is how the virus regulates the relative amounts of transcription and replication. We found that the concentration of a capped cellular mRNA, the concentration of the 5' end of the viral RNA, and the concentration of RdRp all regulate the relative amounts of replication versus transcription. The host mRNA, from which the RdRp snatches its capped primer, acts to upregulate transcription and repress replication. Elevated concentrations of the RdRp itself switch the influenza polymerase toward replication, likely through an oligomerization of the polymerase. The 5' end of the vRNA template both activates replication and inhibits transcription of the vRNA template, thereby indicating that RdRp contains an allosteric binding site for the 5' end of the vRNA template. These data provide insights into the regulation of RdRp throughout the viral life cycle and how it synthesizes the appropriate amounts of viral mRNA and replication products (vRNA and cRNA).
Asunto(s)
Orthomyxoviridae/enzimología , Caperuzas de ARN/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Transcripción Genética , Replicación Viral , Sitio Alostérico/fisiología , ARN Complementario/genética , ARN Mensajero/metabolismo , ARN Viral/metabolismoRESUMEN
A novel assay using a hybridization-based method was developed for real-time monitoring of RNA synthesis. In this work, a "broken beacon" in which the fluor and quencher were located on separate but complementary oligonucleotides was used to quantify the amount of RNA production by T7 polymerase. The relative lengths of the fluor-oligo and quencher-oligo, and their relative concentrations were optimized. The experimentally determined limit-of-detection was approximately 45 nM. The new assay was compared to the "gold-standard" radiolabel ([(32)P]NTP incorporation) assay for RNA quantification. While the broken beacon assay exhibited a higher limit of detection, it provided an accurate measure of RNA production rates. However, the broken beacon assay provided the significant analytical advantages of (i) a real-time and continuous measurement, (ii) no requirement for the use of radiolabels or gel-based analysis, and (iii) substantial time and labor savings.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Sondas de Oligonucleótidos/genética , Carbocianinas/química , Cinética , Técnicas de Amplificación de Ácido Nucleico/métodos , Hibridación de Ácido Nucleico/métodos , Sondas de Oligonucleótidos/química , Radioisótopos de Fósforo/química , ARN/metabolismo , Reproducibilidad de los ResultadosRESUMEN
The antibiotic oxytetracycline (OTC) has shown immense promise for combating the causative agent of Withering syndrome (WS), a Rickettsia-like procaryote (WS-RLP) that has severely impacted California abalone (Haliotis spp.) populations. Using histology and nuclear magnetic resonance (NMR) spectroscopy based metabolomics, the effects of OTC treatments (10, 20, or 30 days) on WS-RLP infected abalone in seawater temperatures of 13.4 +/- 1.2 and 17.3 +/- 1.3 degrees C were investigated over 160 days. The highly efficacious nature of OTC in combating WS-RLP at both temperatures was demonstrated by histology. Metabolomics revealed, however, that the most significant metabolic changes in foot muscle depended upon posttreatment duration, irrespective of treatment and temperature. This was quite unexpected and would have been overlooked using histology alone. Metabolic changes in all animals at both temperatures included decreased levels of amino acids and carbohydrates and elevated taurine, glycine-betaine, and homarine. Subtle metabolic differences between OTC-treated and untreated abalone were observed at 17.3 degrees C only. These findings provide clear evidence that OTC eradicates WS-RLP which in turn reduces the metabolic decay associated with WS at elevated seawater temperature. Furthermore, this study documents the sequential metabolic changes that occur during pre-clinical WS, and demonstrates the application of metabolic phenotyping for understanding environmental effects on host-pathogen-drug interactions.
Asunto(s)
Interacciones Huésped-Parásitos/efectos de los fármacos , Espectroscopía de Resonancia Magnética/métodos , Metabolismo , Moluscos/microbiología , Temperatura , Animales , Infecciones Bacterianas/tratamiento farmacológico , Oxitetraciclina/farmacologíaRESUMEN
It is important to assess the chronic effects of chemical, physical, and biological stressors on organisms in the environment. Appropriate methods must enable rapid, inexpensive, and multibiomarker analyses of organism health. Here we investigate withering syndrome in red abalone (Haliotis rufescens), an important wild and farmed shellfish species along the Pacific coast, using a metabolomic approach that combines the metabolic profiling capabilities of nuclear magnetic resonance spectroscopy (NMR) with pattern recognition methods. Foot muscle, digestive gland, and hemolymph samples were collected from healthy, stunted, and diseased abalone, and the extracts were analyzed by NMR. Following spectral preprocessing, principal components analyses of the metabolite profiles were conducted. Our results confirm that NMR-based metabolomics can successfully distinguish the biochemical profiles of the three groups of animals, in every type of tissue or biofluid studied. Furthermore, this discovery-based approach successfully identified novel metabolic biomarker profiles associated with withering syndrome. The application of these methods for investigating other environmental stressors is discussed, as are the advantages of NMR-based metabolomics for biomonitoring, particularly in conjunction with gene and protein expression profiling.