Identification of a key determinant of hepatitis C virus cell culture adaptation in domain II of NS3 helicase

Efficient replication of hepatitis C virus (HCV) replicons in cell culture is associated with specific sequences not generally observed in vivo. These cell culture adaptive mutations dramatically increase the frequency with which replication is established in vitro. However, replicons derived from HCV isolates that have been shown to replicate in chimpanzees do not replicate in cell culture even when these adaptive mutations are introduced. To better understand this apparent paradox, we performed a gain-of-function screen to identify sequences that could confer cell culture replication competence to replicons derived from chimpanzee infectious HCV isolates. We found that residue 470 in domain II of the NS3 helicase is a critical determinant in cell culture adaptation. Substitutions in residue 470 when combined with the NS5A-S232I adaptive mutation are both necessary and sufficient to confer cell culture replication to otherwise inactive replicons, including those derived from genotype 1b HCV-BK and genotype 1a HCV-H77 isolates. The specific substitution at residue 470 required for replication is context-dependent, with R470M and P470L being optimal for the activity of HCV-BK and HCV-H77 replicons, respectively. Together these data indicate that mutations in the NS3 helicase domain II act in concert with previously identified adaptive mutations and predict that introduction of compatible residues at these positions can confer cell culture replication activity to diverse HCV isolates.     

Expression screen by enzyme-linked immunofiltration assay designed for high-throughput purification of affinity-tagged proteins

High-throughput purification of affinity-tagged fusion proteins is currently one of the fastest developing areas of molecular proteomics. A prerequisite for success in protein purification is sufficient soluble protein expression of the target protein in a heterologous host. Hence, a fast and quantitative evaluation of the soluble-protein levels in an expression system is one of the key steps in the entire process. Here we describe a high-throughput expression screen for affinity-tagged fusion proteins based on an enzyme linked immunofiltration assay (ELIFA). An aliquot of a crude Escherichia coli extract containing the analyte, an affinity-tagged protein, is adsorbed onto the membrane. Subsequent binding of specific antibodies followed by binding of a secondary antibody horseradish peroxidase (HRP) complex then allows quantitative evaluation of the analyte using tetramethylbenzidine as the substrate for HRP. The method is accurate and quantitative, as shown by comparison with results from western blotting and an enzymatic glutathione S-transferase (GST) assay. Furthermore, it is a far more rapid assay and less cumbersome than western blotting, lending itself more readily to high-throughput analysis. It can be used at the expression level (cell lysates) or during the subsequent purification steps to monitor yield of specific protein.     

Fluorescent in situ sequencing on polymerase colonies

Integration of DNA isolation, amplification, and sequencing can be achieved by the use of polymerase colonies (polonies) and cycles of fluorescent dNTP incorporation. In this paper, we present four advances that bring us closer to sequencing genomes cost-effectively using the polony technology. First, a polymerase trapping technique enables efficient nucleotide extension by DNA polymerase in a polyacrylamide matrix and eliminates loss of enzyme during sequencing cycles. Next, we present two novel types of reversibly dye-labeled nucleotide analogues, show that DNA polymerase can incorporate these analogues, and demonstrate that the dyes can be removed by thiol reduction or light exposure. Using these nucleotides, we have sequenced multiple polonies in parallel. In addition, we have found that a high density of polonies can be achieved with minimal overlap between adjacent polonies by limiting the concentration of free primer in the polony amplification reactions. Finally, we have developed software for automated image alignment and sequence calling.     

An immobilized metal ion affinity adsorption and scintillation proximity assay for receptor-stimulated phosphoinositide hydrolysis

A novel approach to measuring receptor-stimulated phosphoinositide hydrolysis was developed based on the principles of immobilized metal ion affinity chromatography (IMAC) and scintillation proximity assay (SPA). Hard Lewis metal ions, such as Zr(4+), Ga(3+), Al(3+), Fe(3+), Lu(3+), and Sc(3+), were immobilized on SPA beads via metal chelate and utilized as affinity ligands to entrap inositol phosphates. [3H]Inositol phosphates bound to IMAC-SPA beads through the strong interaction of their phosphate group with the immobilized metal ions. The binding brought [3H]inositol phosphates in close proximity to the scintillant embedded in the SPA beads, thereby allowing the radioactivity to be quantified. Quantification of [3H]inositol phosphate production in cells preincubated with [3H]inositol provided a highly sensitive measurement of phosphoinositide hydrolysis. The utility of this approach was demonstrated in measuring the response mediated by the G-protein-coupled neurokinin NK1 receptor and the tyrosine kinase-linked platelet-derived growth factor (PDGF) receptor. Substance P stimulated phosphoinositide hydrolysis concentration-dependently in CHO cells expressing NK1 receptors with a maximal 12-fold increase in inositol phosphate production. Similarly, PDGF-BB stimulated a 5-fold increase in phosphoinositide hydrolysis in quiescent Swiss 3T3 cells. This new approach is highly sensitive, fast, simple, easily performed on 96-well plates, and amenable for high-throughput screening.     

Cloning,heterologous expression,and characterization of recombinant class II cytochromes c from Rhodopseudomonas palustris

The cytochrome (cyt) c', cyt c(556), and cyt c(2) genes from Rhodopseudomonas palustris have been cloned; recombinant cyt c' and cyt c(556) have been expressed, purified, and characterized. Unlike mitochondrial cyt c, these two proteins are structurally similar to cyt b(562), in which the heme is embedded in a four-helix bundle. The hemes in both recombinant proteins form covalent thioether links to two Cys residues. UV/vis spectra of the Fe(II) and Fe(III) states of the recombinant cyts are identical with those of the corresponding native proteins. Equilibrium unfolding measurements in guanidine hydrochloride solutions confirm that native Fe(II)-cyt c(556) is more stable than the corresponding state of Fe(III)-cyt c(556) (DeltaDeltaG(f)(o) =22 kJ/mol).     

Identification of sites in the cysteine-rich domain of the class P-III snake venom metalloproteinases responsible for inhibition of platelet function

Atrolysin A and jararhagin are class P-III snake venom metalloproteinases (SVMPs) with three distinct domains: a metalloproteinase, a disintegrin-like and a cysteine-rich. The metalloproteinase and the disintegrin-like domains of atrolysin A and jararhagin contain peptide sequences that interact with alpha2beta1 integrin and inhibit the platelet responses to collagen. Recently, the recombinant cysteine-rich domain of atrolysin A was shown to have similar effects, but the sequence(s) responsible for this is unknown. In this report, we demonstrate two complete peptide sequences from the homologous cysteine-rich domains of atrolysin A and jararhagin that inhibit both platelet aggregation by collagen and adhesion of alpha2-expressing K562 cells to this protein. In addition, the peptide effects on platelets do not seem to involve an inhibition of GPVI. These results identify, for the first time, sites in the cysteine-rich domain of SVMPs that inhibit cell responses to collagen and reveal the complexity of the potential biological effects of these enzymes with multifunctional domains.     

Testing for genetic evidence of population expansion and contraction: an empirical analysis of microsatellite DNA variation using a hierarchical Bayesian model

The role of past climatic change in shaping the distributions of tropical rain forest vertebrates is central to long-standing hypotheses about the legacy of the Quaternary ice ages. One approach to testing such hypotheses is to use genetic data to infer the demographic history of codistributed species. Population genetic theory that relates the structure of allelic genealogies to historical changes in effective population size can be used to detect a past history of demographic expansion or contraction. The fruit bats Cynopterus sphinx and C. brachyotis (Chiroptera: Pteropodidae) exhibit markedly different distribution patterns across the Indomalayan region and therefore represent an exemplary species pair to use for such tests. The purpose of this study was to test alternative hypotheses about historical patterns of demographic expansion and contraction in C. sphinx and C. brachyotis using a coalescent-based analysis of microsatellite variation. Specifically, we used a hierarchical Bayesian model based on Markov chain Monte Carlo simulations to estimate the posterior distribution of genealogical and demographic parameters. The results revealed strong evidence for population contraction in both species. Evidence for a population contraction in C. brachyotis was expected on the basis of biogeographic considerations. However, similar evidence for population contraction in C. sphinx does not support the hypothesis that this species underwent a pronounced range expansion during the late Quaternary. Genetic evidence for population decline may reflect the consequences of habitat destruction on a more recent time scale.     

Antipredator benefits of group living in colonial web-building spiders: the ‘early warning’ effect

The Mexican colonial web-building spider Metepeira incrassata is frequently attacked by predatory wasps from a number of families. Previous studies have shown that wasps often attack more than one spider during a ‘run’ on a colony, but capture success declines as colony size increases, and that spiders in the central core of the colony have lower risk (Rayor & Uetz 1990, Behavioral Ecology and Sociobiology, 27, 77-85; Uetz & Hieber 1994, Behavioral Ecology, 5, 326-333). We used data from direct observation of attacks and field experiments to test the hypothesis that colonial web-building spiders benefit from ‘early warning’ of predator approach through vibrations in the colony web. Analysis of 135 naturally occurring wasp attack ‘runs’ (attacks on 454 spiders) showed that the per-attack run and per-spider capture success of wasps decreased significantly with increased spider colony size. Spider defensive and evasive behaviours observed in a subset of these attacks varied with the attack sequence. Evasive responses by spiders were more frequent later in the attack, suggesting advance warning of predator approach. Experiments using a predator-simulating vibration source demonstrated that mean reaction distance of spiders increased with increasing colony size. Adult female spiders in core positions reacted at greater distances than those on the periphery, but immature spiders, whose capture risk is lower, showed no difference. Behaviour of spiders during simulated attacks was similar to observed encounters with wasps: evasive responses were more frequent and response latency was shorter in spiders attacked later in the sequence, and in many cases, spiders took evasive action prior to any contact with the stimulus. Additional experiments testing isolated cues (web contact, airborne vibration, web-borne vibration) suggest spiders respond to web-borne vibrations generated by predators and evasive behaviours of other spiders. Together, these results support the ‘early warning’ hypothesis of antipredator benefits for colonial web-building spiders.