Bidirectional lipid droplet velocities are controlled by differential binding strengths of HCV Core DII protein

Host cell lipid droplets (LD) are essential in the hepatitis C virus (HCV) life cycle and are targeted by the viral capsid core protein. Core-coated LDs accumulate in the perinuclear region and facilitate viral particle assembly, but it is unclear how mobility of these LDs is directed by core. Herein we used two-photon fluorescence, differential interference contrast imaging, and coherent anti-Stokes Raman scattering microscopies, to reveal novel core-mediated changes to LD dynamics. Expression of core protein’s lipid binding domain II (DII-core) induced slower LD speeds, but did not affect directionality of movement on microtubules. Modulating the LD binding strength of DII-core further impacted LD mobility, revealing the temporal effects of LD-bound DII-core. These results for DII-core coated LDs support a model for core-mediated LD localization that involves core slowing down the rate of movement of LDs until localization at the perinuclear region is accomplished where LD movement ceases. The guided localization of LDs by HCV core protein not only is essential to the viral life cycle but also poses an interesting target for the development of antiviral strategies against HCV

Suppressing RNA silencing with small molecules and the viral suppressor of RNA silencing protein p19

RNA silencing is a gene regulatory and host defense mechanism whereby small RNA molecules are engaged by Argonaute (AGO) proteins, which facilitate gene knockdown of complementary mRNA targets. Small molecule inhibitors of AGO represent a convenient method for reversing this effect and have applications in human therapy and biotechnology. Viral suppressors of RNA silencing, such as p19, can also be used to suppress the pathway. Here we assess the compatibility of these two approaches, by examining whether synthetic inhibitors of AGO would inhibit p19-siRNA interactions. We observe that aurintricarboxylic acid (ATA) is a potent inhibitor of p19's ability to bind siRNA (IC<inf>50</inf>=0.43\u3bcM), oxidopamine does not inhibit p19:siRNA interactions, and suramin is a mild inhibitor of p19:siRNA interactions (IC<inf>50</inf>=430\u3bcM). We observe that p19 and suramin are compatible inhibitors of RNA silencing in human hepatoma cells. Our data suggests that at least some inhibitors of AGO may be used in combination with p19 to inhibit RNA silencing at different points in the pathway.Peer reviewed: YesNRC publication: Ye

Bioinformatic and Physical Characterizations of Genome-Scale Ordered RNA Structure in Mammalian RNA Viruses

By the analysis of thermodynamic RNA secondary structure predictions, we previously obtained evidence for evolutionarily conserved large-scale ordering of RNA virus genomes (P. Simmonds, A. Tuplin, and D.J. Evans, RNA 10: 1337-1351, 2004). Genome-scale ordered RNA structure (GORS) was widely distributed in many animal and plant viruses, much greater in extent than RNA structures required for viral translation or replication, but in mammalian viruses was associated with host persistence. To substantiate the existence of large-scale RNA structure differences between viruses, a large set of alignments of mammalian RNA viruses and rRNA sequences as controls were examined by thermodynamic methods (to calculate minimum free energy differences) and by algorithmically independent RNAz and Pfold methods. These methods produced generally concordant results and identified substantial differences in the degrees of evolutionarily conserved, sequence order-dependent RNA secondary structure between virus genera and groups. A probe hybridization accessibility assay was used to investigate the physical nature of GORS. Transcripts of hepatitis C virus (HCV), hepatitis G virus/GB virus-C (HGV/GBV-C), and murine norovirus, which are predicted to be structured, were largely inaccessible to hybridization in solution, in contrast to the almost universal binding of probes to a range of unstructured virus transcripts irrespective of G + C content. Using atomic force microscopy, HCV and HGV/GBV-C RNA was visualized as tightly compacted prolate spheroids, while under the same experimental conditions the predicted unstructured poliovirus and rubella virus RNA were pleomorphic and had extensively single-stranded RNA on deposition. Bioinformatic and physical characterization methods both identified fundamental differences in the configurations of viral genomic RNA that may modify their interactions with host cell defenses and their ability to persist.</p

Assessing antifreeze activity of AFGP 8 using domain recognition software

Domain recognition software was employed to assess recrystallization-inhibition (RI) activity as an index of antifreeze potential. This represents a key step in the development of a high-throughput analysis for RI activity. Analysis of measurement error indicates an average coefficient of variation for individual crystals of about 8%, which is very small in relation to other sources of variation. Our analysis demonstrates an inverse correlation between AFGP 8 concentration and average crystal size with consistently small, but detectable differences in average crystal size at the edge and centre of the ice wafer. Sensitivity analysis using Monte Carlo re-sampling methods indicate that measuring of 12-15 crystals per field of view are sufficient to obtain accurate estimates of the first two moments (mean and variance) of the crystal size distribution, thereby greatly reducing the time required to assess recrystallization activity. These results suggest that this method has considerable potential for high-throughput analysis of RI activity.NRC publication: Ye

Bioinformatic and physical characterizations of genome-scale ordered RNA structure in mammalian RNA viruses

By the analysis of thermodynamic RNA secondary structure predictions, we previously obtained evidence for evolutionarily conserved large-scale ordering of RNA virus genomes (P. Simmonds, A. Tuplin, and D.J. Evans, RNA 10: 1337-1351, 2004). Genome-scale ordered RNA structure (GORS) was widely distributed in many animal and plant viruses, much greater in extent than RNA structures required for viral translation or replication, but in mammalian viruses was associated with host persistence. To substantiate the existence of large-scale RNA structure differences between viruses, a large set of alignments of mammalian RNA viruses and rRNA sequences as controls were examined by thermodynamic methods (to calculate minimum free energy differences) and by algorithmically independent RNAz and Pfold methods. These methods produced generally concordant results and identified substantial differences in the degrees of evolutionarily conserved, sequence order-dependent RNA secondary structure between virus genera and groups. A probe hybridization accessibility assay was used to investigate the physical nature of GORS. Transcripts of hepatitis C virus (HCV), hepatitis G virus/GB virus-C (HGV/GBV-C), and murine norovirus, which are predicted to be structured, were largely inaccessible to hybridization in solution, in contrast to the almost universal binding of probes to a range of unstructured virus transcripts irrespective of G + C content. Using atomic force microscopy, HCV and HGV/GBV-C RNA was visualized as tightly compacted prolate spheroids, while under the same experimental conditions the predicted unstructured poliovirus and rubella virus RNA were pleomorphic and had extensively single-stranded RNA on deposition. Bioinformatic and physical characterization methods both identified fundamental differences in the configurations of viral genomic RNA that may modify their interactions with host cell defenses and their ability to persist

Multiplex pathogen detection based on spatially addressable microarrays of barcoded resins

Suspension microsphere immunoassays are rapidly gaining recognition in antigen identification and infectious disease biodetection due to their simplicity, versatility and high-throughput multiplex screening. We demonstrate a multiplex assay based on antibody-functionalized barcoded resins (BCRs) to identify pathogen antigens in complex biological fluids. The binding event of a particular antibody on given bead (fluorescence) and the identification of the specific pathogen agent (vibrational fingerprint of the bead) can be achieved in a dispersive Raman system by exciting the sample with two different laser lines. Anthrax protective antigen, Franciscella tularensis lipopolysaccharide and CD14 antigens were accurately identified and quantified in tetraplex assays with a detection limit of 1 ng/mL. The rapid, versatile and simple analysis enabled by the BCRs demonstrates their potential for multiplex antigen detection and identification in a reconfigurable microarray format.Peer reviewed: YesNRC publication: Ye

Spectroscopically encoded microspheres for antigen biosensing

Here we demonstrate the potential of barcoded resins (BCRs) as a reliable platform for immunoassays. Four BCRs were synthesized by dispersion polymerization of 4-methylstyrene, t-butylstyrene, 2,4-dimethylstyrene, and 2,5-dimethylstyrene. Methacrylic acid was included in the polymerization step to provide an anchor point for antibody conjugation. In addition to identifying the BCRs through their unique spectrum in an immunoassay experiment, Raman scattering spectroscopy confirmed the immunoreactivity of the bead-conjugated antibody by detecting 150 ng/mL (~150 pg/bead) of fluorescently labeled rabbit IgG antigen. The simplicity, versatility, and effectiveness of this platform demonstrate its potential for high-throughput multiplexed bioassays.Peer reviewed: YesNRC publication: Ye