Intracellular RNA Levels Research Articles

Identification of Host Factors for Rift Valley Fever Phlebovirus.

Rift Valley fever phlebovirus (RVFV) is a zoonotic pathogen that causes Rift Valley fever (RVF) in livestock and humans. Currently, there is no licensed human vaccine or antiviral drug to control RVF. Although multiple species of animals and humans are vulnerable to RVFV infection, host factors affecting susceptibility are not well understood. To identify the host factors or genes essential for RVFV replication, we conducted CRISPR-Cas9 knockout screening in human A549 cells. We then validated the putative genes using siRNA-mediated knock-downs and CRISPR-Cas9-mediated knock-out studies. The role of a candidate gene in the virus replication cycle was assessed by measuring intracellular viral RNA accumulation, and the virus titers were analyzed using plaque assay or TCID50 assay. We identified approximately 900 genes with potential involvement in RVFV infection and replication. Further evaluation of the effect of six genes on viral replication using siRNA-mediated knock-downs revealed that silencing two genes (WDR7 and LRP1) significantly impaired RVFV replication. For further analysis, we focused on the WDR7 gene since the role of the LRP1 gene in RVFV replication was previously described in detail. WDR7 knockout A549 cell lines were generated and used to dissect the effect of WRD7 on a bunyavirus, RVFV, and an orthobunyavirus, La Crosse encephalitis virus (LACV). We observed significant effects of WDR7 knockout cells on both intracellular RVFV RNA levels and viral titers. At the intracellular RNA level, WRD7 affected RVFV replication at a later phase of its replication cycle (24 h) when compared with the LACV replication, which was affected in an earlier replication phase (12 h). In summary, we identified WDR7 as an essential host factor for the replication of two different viruses, RVFV and LACV, both of which belong to the Bunyavirales order. Future studies will investigate the mechanistic role through which WDR7 facilitates phlebovirus replication.

Extracellular vesicles could be a putative posttranscriptional regulatory mechanism that shapes intracellular RNA levels in Plasmodium falciparum

Plasmodium falciparum secretes extracellular vesicles (PfEVs) that contain parasite-derived RNA. However, the significance of the secreted RNA remains unexplored. Here, we compare secreted and intracellular RNA from asexual cultures of six P. falciparum lines. We find that secretion of RNA via extracellular vesicles is not only periodic throughout the asexual intraerythrocytic developmental cycle but is also highly conserved across P. falciparum isolates. We further demonstrate that the phases of RNA secreted via extracellular vesicles are discernibly shifted compared to those of the intracellular RNA within the secreting whole parasite. Finally, transcripts of genes with no known function during the asexual intraerythrocytic developmental cycle are enriched in PfEVs compared to the whole parasite. We conclude that the secretion of extracellular vesicles could be a putative posttranscriptional RNA regulation mechanism that is part of or synergise the classic RNA decay processes to maintain intracellular RNA levels in P. falciparum.

Characterization of RNase J.

RNase J is involved in RNA maturation as well as degradation of RNA to the level of mononucleotides. This enzyme plays a vital role in maintaining intracellular RNA levels and governs different steps of the cellular metabolism in bacteria. RNase J is the first ribonuclease that was shown to have both endonuclease and5'-3' exonuclease activity. RNase J enzymes can be identified by their characteristic sequence features and domain architecture. The quaternary structure of RNase J plays a role in regulating enzyme activity. The structure of RNase J has been characterized from several homologs. These reveal extensive overall structural similarity alongside a distinct active site topology that coordinates a metal cofactor. The metal cofactor is essential for catalytic activity. The catalytic activity of RNase J is influenced by oligomerization, the choice and stoichiometry of metal cofactors, and the 5' phosphorylation state of the RNA substrate. Here we describe the sequence and structural features of RNase J alongside phylogenetic analysis and reported functional roles in diverse organisms. We also provide a detailed purification strategy to obtain an RNase J enzyme sample with or without a metal cofactor. Different methods to identify the nature of the bound metal cofactor, the binding affinity and stoichiometry are presented. Finally, we describe enzyme assays to characterize RNase J using radioactive and fluorescence-based strategies with diverse RNA substrates.

Enhanced fitness of hepatitis C virus increases resistance to direct-acting antivirals

Drug resistance mutations of hepatitis C virus (HCV) negatively impact viral replicative fitness. RNA viruses are known to change their replication behaviour when subjected to suboptimal selection pressure. Here, we assess whether mutation supply in HCV is sufficiently large to allow the selection of its variants during dual or triple direct-acting antiviral (DAA) treatment associated with augmented virus fitness or impairment. We engineered randomly mutagenized full-genome libraries to create a highly diverse population of replication-competent HCV variants in cell culture. These variants exhibited escape when treated with NS5A/NS5B inhibitors (daclatasvir/sofosbuvir), and relapse on treatment with a combination of NS3/NS5A/NS5B inhibitors (simeprevir or paritaprevir/daclatasvir/sofosbuvir). Analysis of the relationship between virus fitness and drug resistance of JFH1-derived NS5A-5B variants showed a significant positive correlation (P=0.003). At the earliest time points, intracellular RNA levels remain unchanged in both the subgenomic replicon and infection assays, whereas extracellular RNA levels increased upto ten-fold compared to wild-type JFH1. Beneficial substitutions hyperstimulated phosphatidylinositol 4-phosphate during DAA treatment, and showed decreased dependence on cyclophilins during cyclosporine A treatment, indicating an interplay of virus-host molecular mechanisms in beneficial substitution selection that may necessitate infectious virus production. This comprehensive study demonstrates a possible role for HCV fitness of overcoming drug-mediated selection pressure.

Binase Do Not Change Level of Intracellular RNA in Breast Cancer Cells

We analyzed changes of intracellular RNA level in breast cancer cells using BT-20, BT-474, and MCF-7 cell lines. It is the continuation of our former studying the effect of bacterial ribonucleases on physiological and biochemical parameters of breast cancer cells. For the first time, we tested ability of Bacillus pumilus RNase (binase) to effect the level of intracellular RNA in these cells. Differences in total RNA level under of the binase action on the cells of triple negative, triple positive, and double positive breast cancer during 48 h of treatment were not detected. The sensitivity of cells to binase was not associated with a reduction of the intracellular RNA level.

The sirtuin inhibitor sirtinol inhibits hepatitis A virus (HAV) replication by inhibiting HAV internal ribosomal entry site activity

Epigenetics plays a role in the regulation of gene expression. Epigenetic changes control gene expression at the transcriptional level. Our previous study suggested that the La protein, which is mainly localized in the nucleus, was associated with hepatitis A virus (HAV) internal ribosomal entry site (IRES)-mediated translation and HAV replication. The aim of this study was to investigate whether epigenetic compounds have effects on HAV IRES-mediated translation and HAV replication. Sirtinol, a sirtuin inhibitor, inhibited HAV IRES-mediated translation in COS7-HAV-IRES cells. Treatment with 10 μM sirtinol resulted in a significant reduction in the intracellular RNA levels of HAV HA11-1299 genotype IIIA in Huh7 cells. Epigenetic treatment with a sirtuin inhibitor may represent a new treatment option for HAV infection. In conclusion, epigenetic control was involved in HAV IRES-dependent translation and HAV replication. Special attention should also be paid to underlying viral diseases in the clinical use of epigenetic treatments for malignancies.

Procyanidin B1 Purified from Cinnamomi Cortex Suppresses Hepatitis C Virus Replication

A combination of pegylated interferon and ribavirin is the current standard therapy for hepatitis C virus (HCV) infection, but this combination provides relatively low efficacy, especially in some patients with HCV genotype 1 infection; therefore, the development of novel therapeutic agents is required for further improvement in the treatment of chronic HCV infection. HCV pseudotype and subgenomic replicon assays were used in this study. The interaction of compounds with HCV receptors was examined using flow cytometry. Intracellular RNA levels were determined by semi-quantitative reverse transcriptase PCR. Procyanidin B1 (PB1), a dimer of (-)-epicatechin and (+)-catechin, purified from Cinnamomi cortex, inhibits infection by vesicular stomatitis virus and HCV pseudotype virus in Huh-7 cells, with 50% effective concentrations of 29 and 15 microM, respectively. No inhibitory effects were observed in each component of PB1. We found that PB1 does not interfere with viral entry or receptor expression, but inhibits HCV RNA synthesis in a dose-dependent manner. These results indicate that PB1 suppresses HCV RNA synthesis, possibly as a HCV RNA polymerase inhibitor. Our results might contribute towards the development of more effective inhibitors for HCV infection from natural plants.

Immobilized cell biocatalyst activation and pseudo‐steady‐state behavior: Model and experiment

An intrinsic, unstructured model has been utilized to describethe startup dynamics of a continuous Caalginate-immobilized Zymomonas mobilis (ATCC 10988) fermentation. This model predicts, at least qualitatively, transients in the fermenter effluent glucose, ethanol, and biomass concentrations as well as radial gradients in immobilized-cell concentration and activity within the gelbiocatalysts. Predicted intrabiocatalyst gradients in immobilized-cell specific growth rate were used to calculate the corresponding gradients in intracellular RNA level based on a reported linear relationship between the two. Mathematical simulations of immobilized biomass concentration profiles and RNA content were verified using a novel, scanning microfluorimetry technique.

XylE functions as an efficient reporter gene in Streptomyces spp.: use for the study of galP1, a catabolite-controlled promoter.

We describe the development of a convenient and sensitive reporter gene system for Streptomyces spp. based on the use of a promoterless copy of the xylE gene of Pseudomonas putida. The xylE gene product is a catechol dioxygenase, which converts the colorless substrate catechol to an intensely yellow hydroxymuconic semialdehyde. A promoterless copy of xylE was placed under the transcriptional control of galP1, a glucose-repressed and galactose-induced promoter from Streptomyces lividans, and its expression was examined in bacterial colonies on agar plates or in liquid cultures grown in the presence of glucose or galactose as the sole carbon source. On plates, colonies of bacteria grown on galactose turned bright yellow within a few minutes of being sprayed with a solution of catechol, whereas colonies on glucose-containing plates remained white or only slightly colored, even after extensive incubation. Activity of galP1-xylE fusions was conveniently measured in crude cell extracts with a simple colorimetric assay and was shown to faithfully reflect intracellular RNA levels, as determined by quantitative dot blots. Moreover, differences in expression levels of xylE fusions driven by mutant galP1 promoters were readily apparent in color reactions on plates. The properties of xylE as a reporter gene thus make it suitable not only for quantitatively monitoring expression of regulated promoters in Streptomyces spp. but also for recovering mutations that alter the expression levels of promoters of interest.