Infants look longer at colours that adults like when colours are highly saturated.

The extent to which aesthetic preferences are 'innate' has been highly debated (Reber, Schwarz, & Winkielman, Personality and Social Psychology Review, 8(4), 364-382, 2004). For some types of visual stimuli infants look longer at those that adults prefer. It is unclear whether this is also the case for colour. A lack of relationship in prior studies between how long infants look at different colours and how much adults like those colours might be accounted for by stimulus limitations. For example, stimuli may have been too desaturated for infant vision. In the current study, using saturated colours more suitable for infants, we aim to quantify the relationship between infant looking and adult preference for colour. We take infant looking times at multiple hues from a study of infant colour categorization (Skelton, Catchpole, Abbott, Bosten, & Franklin, Proceedings of the National Academy of Sciences of the United States of America, 114(21), 5545-5550, 2017) and then measure adult preferences and compare these to infant looking. When colours are highly saturated, infants look longer at colours that adults prefer. Both infant looking time and adult preference are greatest for blue hues and are least for green-yellow. Infant looking and adult preference can be partly summarized by activation of the blue-yellow dimension in the early encoding of human colour vision. These findings suggest that colour preference is at least partially rooted in the sensory mechanisms of colour vision, and more broadly that aesthetic judgements may in part be due to underlying sensory biases.

Identification of long non-coding RNAs expressed in knee and hip osteoarthritic cartilage.

OBJECTIVE: Long intergenic non-coding RNAs (lincRNAs) are emerging as key regulators in gene expression; however, little is known about the lincRNA expression changes that occur in osteoarthritis (OA). Here we aimed to define a transcriptome of lncRNAs in OA cartilage, specifically comparing the lincRNA transcriptome of knee and hip cartilage. METHOD: RNA-seq was performed on nucleic acid extracted from hip cartilage from patients undergoing joint replacement surgery because of either OA (n = 10) or because of a neck of femur fracture (NOF; n = 6). After transcript alignment, counts were performed using Salmon and differential expression for ENSEMBL lincRNAs determined using DESeq2. Hip RNA-seq lincRNA expression was compared to a knee dataset (ArrayExpress; E-MTAB-4304). ChIP-seq data from ENCODE was used to determine whether lincRNAs were associated with promoters (plncRNA) or unidirectional enhancer-like regulatory elements (elncRNAs). RESULTS: Our analysis of the hip transcriptome identified 1692 expressed Transcripts Per Million (TPM ≥1) Ensembl lincRNAs, of which 198 were significantly (FDR ≤0.05) differentially expressed in OA vs normal (NOF) cartilage. Similar analysis of knee cartilage transcriptome identified 648 Emsembl lincRNAs with 93 significantly (FDR ≤0.05) differentially expressed in intact vs damaged cartilage. In total, 1834 lincRNAs were expressed in both hip and knee cartilage, with a highly significant correlation in expression between the two cartilages. CONCLUSION: This is the first study to use RNA-seq to map and compare the lincRNA transcriptomes of hip and knee cartilage. We propose that lincRNAs expressed selectively in cartilage, or showing differential expression in OA, will play a role in cartilage homoeostasis.

Density Functional Theory Study on the Complexation of NOTA as a Bifunctional Chelator with Radiometal Ions.

1,4,7-Triazacyclononane-1,4,7-triacetic acid (NOTA) is a key bifunctional chelator utilized for the complexation of metal ions in radiopharmaceutical applications; the ability of these chelators depends on the strength of their binding with ions. The focus of the present work is to evaluate the complexation of Cu2+, Ga3+, Sc3+, and In3+ radiometal ions with NOTA using density functional theory (B3LYP functional) and 6-311+G(2d,2p)/DGDZVP basis sets. The significant role of ion-water interactions in the chelation interaction energies in solution reflects the competition between ion-water and NOTA-ion interaction in the chelation process. There is reasonable agreement between experimental and theoretical binding constants, geometries, and 1H NMR chemical shifts. Chelation interaction energies, Gibbs free energies, and entropies in solution show that the NOTA-Ga3+ and NOTA-Cu2+ are the most and least stable complexes, respectively. The natural atomic charges and second order perturbation analysis reveal charge transfer between NOTA and radiometal ions. The theoretical 1H NMR chemical shifts of NOTA are in good agreement with experiment; these values are influenced by the presence of the ions, which have a deshielding effect on the protons of NOTA. Global scalar properties such as EHOMO/ELUMO, ΔELUMO-HOMO, and chemical hardness/softness confirm that the NOTA-Cu2+ complex, which has a singly occupied molecular orbital, has the lowest ΔELUMO-HOMO value, the least chemical hardness, and the highest chemical softness. The significant variation of the hardness and ΔELUMO-HOMO values of the complexes can be attributed to the different positions of the metal ions on the periodic table. This study affirms that, among the radiometal ions, Ga3+ can be used to effectively radiolabel NOTA chelator for radiopharmaceutical usage as it binds most stably with NOTA.

DFT study of the interaction between DOTA chelator and competitive alkali metal ions.

1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetracetic acid (DOTA) is an important chelator for radiolabeling of pharmaceuticals. The ability of alkali metals found in the body to complex with DOTA and compete with radio metal ions can alter the radiolabeling process. Non-covalent interactions between DOTA complexed with alkali metals Li+, Na+, K+ and Rb+, are investigated with density functional theory using B3LYP and ωB97XD functionals. Conformational possibilities of DOTA were explored with a varying number of carboxylic pendant arms of DOTA in close proximity to the ions. It is found that the case in which four arms of DOTA are interacting with ions is more stable than other conformations. The objective of this study is to explore the electronic structure properties upon complexation of alkali metals Li+ Na+, K+ and Rb+ with a DOTA chelator. Interaction energies, relaxation energies, entropies, Gibbs free energies and enthalpies show that the stability of DOTA, complexed with alkali metals decreases down the group of the periodic table. Implicit water solvation affects the complexation of DOTA-ions leading to decreases in the stability of the complexes. NBO analysis through the natural population charges and the second order perturbation theory, revealed a charge transfer between DOTA and alkali metals. Conceptual DFT-based properties such as HOMO/LUMO energies, ΔEHOMO-LUMO and chemical hardness and softness indicated a decrease in the chemical stability of DOTA-alkali metal complexes down the alkali metal series. This study serves as a guide to researchers in the field of organometallic chelators, particularly, radiopharmaceuticals in finding the efficient optimal match between chelators and various metal ions.

Why on earth did I buy that? A study of regretted appliance purchases.

If targets to reduce greenhouse gas emissions and thereby tackle climate change are to be achieved, it will be necessary to reduce both embodied energy costs (e.g. in terms of producing and manufacturing the products and services that society consumes) and operational energy costs. Reducing the number of purchases that people regret could be a first step in changing the overall dynamic of consumption patterns. This research looks at some potentially adverse effects of consumption on well-being (e.g. negative emotions), applying social practice theory to give insights into why people make purchases that they feel negatively about. This paper draws from: (i) findings of a national survey of over 2000 respondents which found that 53% of adults had reported regretting purchasing an electrical device at some point, and that 23% regretted making such a purchase within the past year; and (ii) a series of walking interviews around people's homes that provide detailed insights into the nature and extent of regretted purchases of electrical goods (e.g. resentment at built-in obsolescence, frustration at the pace of technological change). By combining the qualitative and quantitative data, we develop a typology of regretted consumption and explore the underlying factors that lead to such purchases. The paper concludes with a discussion of the policy implications of this research.This article is part of the themed issue 'Material demand reduction'.

All-atom molecular dynamics simulations of an artificial sodium channel in a lipid bilayer: the effect of water solvation/desolvation of the sodium ion.

All-atom molecular dynamics is used to investigate the transport of Na(+) across a 1,2-dioleoyl-sn-glycero-3-phosphocholine lipid bilayer facilitated by a diazacrown hydraphile. Specifically, the free energy of Na(+) passing through the bilayer is calculated using the adaptive biasing force method to study the free energy associated with the increase in Na(+) transport in the presence of the hydraphile molecule. The results show that water interaction greatly influences Na(+) transport through the lipid bilayer as water is pulled through the bilayer with Na(+) forming a water channel. The hydraphile causes a reduction in the free energy barrier for the transport of Na(+) through the head group part of the lipid bilayer since it complexes the Na(+) reducing the necessity for water to be complexed and, therefore, dragged through with Na(+), an energetically unfavorable process. The free energy associated with Na(+) being desolvated within the bilayer is significantly decreased in the presence of the hydraphile molecule; the hydraphile increases the number of solvation states of Na(+) that can be adopted, and this increase in the number of available configurations provides an entropic explanation for the success of the hydraphile.

Genome sequence of vanilla distortion mosaic virus infecting Coriandrum sativum.

The 9573-nucleotide genome of a potyvirus was sequenced from a Coriandrum sativum plant from India with viral symptoms. On analysis, this virus was shown to have greater than 85 % nucleotide sequence identity to vanilla distortion mosaic virus (VDMV). Analysis of the putative coat protein sequence confirmed that this virus was in fact VDMV, with greater than 91 % amino acid sequence identity. The genome appears to encode a 3083-amino-acid polyprotein potentially cleaved into the 10 mature proteins expected in potyviruses. Phylogenetic analysis confirmed that VDMV is a distinct but ungrouped member of the genus Potyvirus.

The insertion of gas molecules into polyhedral oligomeric silsesquioxane (POSS) cages: understanding the energy of insertion using quantum chemical calculations.

Density functional theory (DFT) calculations using the B3LYP and ωB97XD functionals and Møller-Plesset (MP2) calculations have been used to determine the energy of insertion of seven molecules (CO2, N2, O2, CO, CH4, He and H2O) into two sizes of polyhedral oligomeric silsesquioxane (POSS) cages, T10 and T12. Geometry optimization results of each molecule inserted into the center of the POSS cage and transition-energy searches of the molecules in the largest face of both T10 and T12 (identified as the Si5 face) are discussed. The main factors that affect the energy of a molecule in a particular POSS cage are the size (number of atoms) and shape of the POSS cage and the strength of dispersion and electrostatic interactions. The largest of the molecules, CO2 and CH4, have the largest interaction energies within the center and the face of the T10 and T12 cages. Interaction energies for the diatomic molecules increase in the order H2 < O2 < N2 ∼ CO and differences between interaction energies can be attributed, in part, to differences in electron transfer between the cage and molecule. Electrostatic interactions also play a significant role in determining the interaction energy. For example, H2 and He are only slightly charged when inside the POSS cages, providing little repulsive or attractive interactions, while polar CO provides significant repulsive interaction. In the case of water, the interaction energies are low because of hydrogen bonding between the water hydrogen atoms and the POSS oxygen atoms. The interaction energies of the absorbates at the center of T12 are typically smaller than for T10 due to the larger cavity size of T12. The Si5 face has the same basic structure for both T10 and T12 cages and, consequently, the interaction energies of the absorbate molecules at the face are similar. Interaction energies obtained using MP2 calculations are lower than those obtained from DFT calculations depending on the choice of the functional. For example, use of the ωB97XD functional that treats dispersion interactions more completely than the B3LYP functional, produces values closer to the MP2 values. These results, therefore, suggest that dispersion is important in the interaction between small molecules and POSS cages.

Investigating the quartz (1010)/water interface using classical and ab initio molecular dynamics.

Two different terminations of the (1010) surface of quartz (α and ß) interacting with water are simulated by classical (CMD) (using two different force fields) and ab initio molecular dynamics (AIMD) and compared with previously published X-ray reflectivity (XR) experiments. Radial distribution functions between hydroxyl and water show good agreement between AIMD and CMD using the ClayFF force field for both terminations. The Lopes et al. (Lopes, P. E. M.; Murashov, V.; Tazi, M.; Demchuk, E.; MacKerell, A. D. J. Phys. Chem. B2006, 110, 2782-2792) force field (LFF), however, underestimates the extent of hydroxyl-water hydrogen bonding. The ß termination is found to contain hydroxyl-hydroxyl hydrogen bonds; the quartz surface hydroxyl hydrogens and oxygens that hydrogen bond with each other exhibit greatly reduced hydrogen bonding to water. Conversely, the hydroxyl hydrogen and oxygens that are not hydrogen bonded to other surface hydroxyls but are connected to those that are show a considerable amount of hydrogen bonding to water. The electron density distribution of an annealed surface of quartz (1010) obtained by XR is in qualitative agreement with electron densities calculated by CMD and AIMD. In all simulation methods, the interfacial water peak appears farther from the surface than observed by XR. Agreement among AIMD, LFF, and XR is observed for the relaxation of the near-surface atoms; however, ClayFF shows a larger discrepancy. Overall, results show that for both terminations of (1010), LFF treats the near-surface structure more accurately whereas ClayFF treats the interfacial water structure more accurately. It is shown that the number of hydroxyl and water hydrogen bonds to the bridging Si-O-Si oxygens connecting the surface silica groups to the rest of the crystal is much greater for the α than the ß termination. It is suggested that this may play a role in the greater resistance to dissolution of the ß termination than that of the α termination.

Direct and quantitative comparison of pixelated density profiles with high-resolution X-ray reflectivity data.

A method for comparing pixelated density profiles (e.g. obtained from molecular dynamics or other computational techniques) with experimental X-ray reflectivity data both directly and quantitatively is described. The conditions under which such a comparison can be made quantitatively (e.g. with errors <1%) are determined theoretically by comparing calculated structure factors for an intrinsic continuous density profile with those obtained from density profiles that have been binned into regular spatial increments. The accuracy of the X-ray reflectivity calculations for binned density profiles is defined in terms of the inter-relationships between resolution of the X-ray reflectivity data (i.e. its range in momentum transfer), the chosen bin size and the width of the intrinsic density profile. These factors play a similar role in the application of any structure-factor calculations that involve the use of pixelated density profiles, such as those obtained from iterative phasing algorithms for inverting structures from X-ray reflectivity and coherent diffraction imaging data. Finally, it is shown how simulations of a quartz-water interface can be embedded into an exact description of the `bulk' phases (including the substrate crystal and the fluid water, below and above the actual interface) to quantitatively reproduce the experimental reflectivity data of a solid-liquid interface.

Identification of neryl formate as the airborne aggregation pheromone for the American house dust mite and the European house dust mite (Acari: Epidermoptidae).

The American house dust mite, Dermatophagoidesfarinae Hughes, and European house dust mite, Dermatophagoides pteronyssinus Trouessart, are major pests of medical importance throughout the developed world, causing atopic diseases such as asthma, rhinitis, and atopic dermatitis. Previous studies in our laboratory have shown that the behavioral responses of house dust mites toward volatiles from food sources could be assessed using a Y-tube olfactometer assay. The current study used this Y-tube assay to investigate house dust mite pheromones. A hexane extract of D.farinae, along with fractions of the extract prepared by microscale liquid chromatography over Florisil, were tested for behavioral activity. One of the chromatographic fractions was shown to be significantly attractive (P < 0.05) for D. farinae, compared with a solvent control. Coupled gas chromatography-mass spectrometry analysis of this behaviorally active fraction indicated that neryl or geranyl formate was the major component. Peak enhancement by gas chromatography, using authentic samples of the neryl and geranyl isomers prepared in high purity by chemical synthesis, confirmed the identity of the major peak as neryl formate. In Y-tube assays, male and female D. farinae and D. pteronyssinus both were significantly attracted to synthetic neryl formate at doses of 100 and 10 ng, respectively (P < 0.05). No significant differences were found for D. farinae and D. pteronyssinus when synthetic neryl formate and house dust mite extracts containing natural neryl formate were tested at the same level. Dynamic headspace collection of D. farinae and D. pteronyssinus colonies showed that neryl formate was released as a volatile organic compound by both species. Our study shows that neryl formate is an aggregation pheromone for D. farinae and D. pteronyssinus, and has the potential to be used as part of a novel lure-and-kill system for house dust mite control.

Preclinical characterization of the antiviral activity of SCH 900518 (narlaprevir), a novel mechanism-based inhibitor of hepatitis C virus NS3 protease.

Small-molecule hepatitis C virus (HCV) NS3 protease inhibitors such as boceprevir (SCH 503034) have been shown to have antiviral activity when they are used as monotherapy and in combination with pegylated alpha interferon and ribavirin in clinical trials. Improvements in inhibitor potency and pharmacokinetic properties offer opportunities to increase drug exposure and to further increase the sustained virological response. Exploration of the structure-activity relationships of ketoamide inhibitors related to boceprevir has led to the discovery of SCH 900518, a novel ketoamide protease inhibitor which forms a reversible covalent bond with the active-site serine. It has an overall inhibition constant (K*(i)) of 7 nM and a dissociation half-life of 1 to 2 h. SCH 900518 inhibited replicon RNA at a 90% effective concentration (EC(90)) of 40 nM. In biochemical assays, SCH 900518 was active against proteases of genotypes 1 to 3. A 2-week treatment with 5x EC(90) of the inhibitor reduced the replicon RNA level by 3 log units. Selection of replicon cells with SCH 900518 resulted in the outgrowth of several resistant mutants (with the T54A/S and A156S/T/V mutations). Cross-resistance studies demonstrated that the majority of mutations for resistance to boceprevir and telaprevir caused similar fold losses of activity against all three inhibitors; however, SCH 900518 retained more activity against these mutants due to its higher intrinsic potency. Combination treatment with alpha interferon enhanced the inhibition of replicon RNA and suppressed the emergence of resistant replicon colonies, supporting the use of SCH 900518-pegylated alpha interferon combination therapy in the clinic. In summary, the results of the preclinical characterization of the antiviral activity of SCH 900518 support its evaluation in clinical studies.

Canna yellow streak virus: a new potyvirus associated with severe streaking symptoms in canna.

A new potyvirus has been found in canna. A 1700-nucleotide region at the 3' end of the genomic RNA has been sequenced from two isolates. The sequence reveals the virus to be a distinct member of the genus Potyvirus but most closely related to Johnsongrass mosaic virus. A specific primer pair was designed that enabled canna material to be screened specifically for this virus. The virus was consistently found in cannas showing severe virus symptoms. This virus has been found in different canna varieties from the UK, Belgium, Netherlands, France and Israel. The name Canna yellow streak virus (CaYSV) has been proposed for this new virus.

Olfactory responses of medically and economically important mites (Acari: Epidermoptidae and Acaridae) to volatile chemicals.

Dermatophagoides farinae Hughes (Acari: Epidermoptidae), the American house dust mite, and Tyrophagus putrescentiae (Schrank) (Acari: Acaridae), the mold mite, are medically and economically important but controlling them has proved difficult, and recolonization is commonplace. Their behavioral responses to different sources of volatile chemicals are still not fully elucidated. For the first time, the Y-tube olfactometer, which is an enclosed bioassay to resolve responses to test and control volatiles, has been successfully used with these mites. Mites were tested individually, and both T. putrescentiae and D. farinae responded to food volatiles. Y-tube olfactometers may be used to test for potential semiochemicals, thereby increasing knowledge of our behavior of astigmatic mites.

SCH 503034, a mechanism-based inhibitor of hepatitis C virus NS3 protease, suppresses polyprotein maturation and enhances the antiviral activity of alpha interferon in replicon cells.

Cleavage of the hepatitis C virus (HCV) polyprotein by the viral NS3 protease releases functional viral proteins essential for viral replication. Recent studies by Foy and coworkers strongly suggest that NS3-mediated cleavage of host factors may abrogate cellular response to alpha interferon (IFN-alpha) (E. Foy, K. Li, R. Sumpter, Jr., Y.-M. Loo, C. L. Johnson, C. Wang, P. M. Fish, M. Yoneyama, T. Fujita, S. M. Lemon, and M. Gale, Jr., Proc. Natl. Acad. Sci. USA 102:2986-2991, 2005, and E. Foy, K. Li, C. Wang, R. Sumpter, Jr., M. Ikeda, S. M. Lemon, and M. Gale, Jr., Science 300:1145-1148, 2003). Blockage of NS3 protease activity therefore is expected to inhibit HCV replication by both direct suppression of viral protein production as well as by restoring host responsiveness to IFN. Using structure-assisted design, a ketoamide inhibitor, SCH 503034, was generated which demonstrated potent (overall inhibition constant, 14 nM) time-dependent inhibition of the NS3 protease in cell-free enzyme assays as well as robust in vitro activity in the HCV replicon system, as monitored by immunofluorescence and real-time PCR analysis. Continuous exposure of replicon-bearing cell lines to six times the 90% effective concentration of SCH 503034 for 15 days resulted in a greater than 4-log reduction in replicon RNA. The combination of SCH 503034 with IFN was more effective in suppressing replicon synthesis than either compound alone, supporting the suggestion of Foy and coworkers that combinations of IFN with protease inhibitors would lead to enhanced therapeutic efficacy.

The use of real-time RT-PCR (TaqMan) and post-ELISA virus release for the detection of Beet necrotic yellow vein virus types containing RNA 5 and its comparison with conventional RT-PCR.

Real-time RT-PCR (TaqMan) assays were developed for the specific detection of Beet necrotic yellow vein virus (BNYVV). The two assays designed were a broad-spectrum one that detected RNA 2 from all types and a second designed to detect types containing RNA 5. The assays were validated against a range of different isolates from Europe and the Far East. These real-time assays were compared to a conventional RT-PCR assay for the detection of RNA 5. Sensitivity comparisons showed that for the detection of RNA 5, TaqMan was 10,000 times more sensitive than the conventional RT-PCR assay. Further improvements were made to the test procedure by using post-ELISA virus release (VR), as an alternative to RNA extraction. This significantly increased the speed of processing samples and reduced the staff input required, allowing the TaqMan assay to be used routinely as part of an annual survey of UK field samples.

Tailoring the substrate specificity of the beta-glycosidase from the thermophilic archaeon Sulfolobus solfataricus.

The substrate specificity of the thermophilic beta-glycosidase (lacS) from the archaeon Sulfolobus solfataricus (SSbetaG), a member of the glycohydrolase family 1, has been analysed at a molecular level using predictions from known protein sequences and structures and through site-directed mutagenesis. Three critical residues were identified and mutated to create catalysts with altered and broadened specificities for use in glycoside synthesis. The wild-type (WT) and mutated sequences were expressed as recombinant fusion proteins in Escherichia coli, with an added His(6)-tag to allow one-step chromatographic purification. Consistent with side-chain orientation towards OH-6, the single Met439-->Cys mutation enhances D-xylosidase specificity 4.7-fold and decreases D-fucosidase activity 2-fold without greatly altering its activity towards other D-glycoside substrates. Glu432-->Cys and Trp433-->Cys mutations directed towards OH-4 and -3, respectively, more dramatically impair glucose (Glc), galactose (Gal), fucose specificity than for other glycosides, resulting in two glycosidases with greatly broadened substrate specificities. These include the first examples of stereospecificity tailoring in glycosidases (e.g. WT-->W433C, k(cat)/K(M) (Gal):k(cat)/K(M) (mannose (Man))=29.4:1-->1.2:1). The robustness and high utility of these broad specificity SSbetaG mutants in parallel synthesis were demonstrated by the formation of libraries of beta-glycosides of Glc, Gal, xylose, Man in one-pot preparations at 50 degrees C in the presence of organic solvents, that could not be performed by SSbetaG-WT.

Evolution not revolution? The struggle for the recognition and development of patient education in the UK.

In this paper, a review of patient education in the United Kingdom (UK) is presented. Some of the reasons for the development of patient education are identified and its relationship to other educational activities in health care settings is discussed. The gap between the theory and practice of UK patient education is then examined and emerging new practices are considered. The concluding section of the paper identifies a number of important questions to ask when considering the future development of patient education. These are relevant questions for patients, practitioners, researchers and politicians.

Histopathologic findings in the alopecia associated with Vogt-Koyanagi-Harada disease.

BACKGROUND: Vogt-Koyanagi-Harada (VKH) disease is a rare autoimmune disease primarily involving pigmented structures. The commonly associated alopecia usually precedes leukodermic changes seen in the hair and skin. OBJECTIVE: We present a 46-year-old oriental woman with VKH disease who developed diffuse alopecia followed by the regrowth of nonpigmented hairs. The biopsy specimens showed a peribulbar mononuclear infiltrate with increased telogen/catagen:anagen follicles. The most prominent additional histologic finding was melanin pigment release from the matrix into the dermal papillae, fibrous tracks, and surrounding perifollicular sheaths. The peribulbar mononuclear cells showed diffuse immunohistochemical staining of approximately 80 to 90% of the cells for CD3, CD45RO. More than 50% of the mononuclear cells showed positive staining for CD4, whereas approximately 10 to 20% showed staining for TIA with TIA+ cells within the follicular epithelium. CONCLUSION: Although the histologic features seen in the alopecia associated with VKH are consistent with alopecia areata, the prominent pigment release does suggest that the prime target are the melanocytes and that keratinocytes may be secondarily involved.

RNA-dependent RNA polymerase activity encoded by GB virus-B non-structural protein 5B.

Phylogenetic analysis and polyprotein organization comparison have shown that GB virus-B (GBV-B) is closely related to hepatitis C virus (HCV). In this study, the coding region for GBV-B non-structural protein 5B (NS5B) was isolated by reverse transcription-polymerase chain reaction (RT-PCR) from pooled serum of GBV-B-infected tamarins. Expression of soluble GBV-B NS5B protein in Escherichia coli was achieved by removal of a 19-amino acid hydrophobic domain at the C-terminus of the protein. The truncated GBV-B NS5B (NS5BDeltaCT19) was purified to homogeneity and shown to possess an RNA-dependent RNA polymerase (RdRp) activity in both gel-based and scintillation proximity assays. NS5BDeltaCT19 required the divalent cation Mn2+ for enzymatic activity, at an optimal concentration of 15 mM. Interestingly, Mg2+, at concentrations up to 20 mM, did not support the GBV-B NS5B activity. This differs from HCV NS5B where both Mn2+ and Mg2+ can support RdRp activity. Zn2+ was found to inhibit the activity of GBV-B NS5B, with a 50% inhibitory concentration (IC50) of 5-10 microM. Higher concentrations of monovalent salts (NaCl or KCl > 100 mM) and glycerol (> 3%) were also inhibitory. NS5BDeltaCT19 was able to bind to RNA homopolymers, but utilized most efficiently poly(C), the one with the lowest binding affinity for RNA synthesis. Mutational analysis of GBV-B NS5B demonstrated the importance of several conserved sequence motifs for enzymatic activity. Based on sequence homology ( approximately 37% identity and 52% similarity) between GBV-B and HCV NS5B proteins, the active GBV-B RdRp provides a good surrogate assay system for HCV polymerase studies.