Abstract. In this study we tested the hypothesis that fusion mediated by the influenza virus hemagglutinin (HA) is a cooperative event. To do this we characterized 3T3 cell lines that express HA at nine different defined surface densities. HA densities ranged from 1.0 to 12.6 × 103 HA trimers/~m 2 as determined by quantitative fluorescent antibody binding. The lateral mobility and percent mobile fraction of HA did not vary significantly among these cells, nor did the contact area between HA-expressing cells and target RBCs. The fusion reaction of each HA-expressing cell line was analyzed using a fluorescence dequenching assay that uses octadecylrhodamine (R18)-labeled RBCs. For each cell line we measured the lag time preceding the onset of fusion, the initial rate of fusion, and the final extent of fusion. The final extent of fusion was similar for all cell lines, and the initial rate of fusion as a function of HA surface density displayed a MichaelisMenten-type dependence. However, the dependence of the lag time preceding the onset of fusion on HA surface density was clearly sigmoidal. Kinetic analysis of the data for the reciprocal lag time vs HA surface density, by both a log/log plot and a Hill plot, suggested that the observed sigmoidicity does not reflect cooperativity at the level of formation of HA aggregates as a prerequisite to fusion. Rather, the cooperativity of the process(es) that occur(s) during the lag time arises at a later step and involves a minimum of three, and most likely four, HA trimers. A model is proposed to explain HA cooperativity during fusion.
Kinesin-5 motors fulfil essential roles in mitotic spindle morphogenesis and dynamics as slow, processive microtubule (MT) plus-end directed motors. The Saccharomyces cerevisiae kinesin-5 Cin8 was found, surprisingly, to switch directionality. Here, we have examined directionality using single-molecule fluorescence motility assays and live-cell microscopy. On spindles, Cin8 motors mostly moved slowly (∼25 nm/s) towards the midzone, but occasionally also faster (∼55 nm/s) towards the spindle poles. In vitro, individual Cin8 motors could be switched by ionic conditions from rapid (380 nm/s) and processive minus-end to slow plus-end motion on single MTs. At high ionic strength, Cin8 motors rapidly alternated directionalities between antiparallel MTs, while driving steady plus-end relative sliding. Between parallel MTs, plus-end motion was only occasionally observed. Deletion of the uniquely large insert in loop 8 of Cin8 induced bias towards minus-end motility and affected the ionic strength-dependent directional switching of Cin8 in vitro. The deletion mutant cells exhibited reduced midzone-directed motility and efficiency to support spindle elongation, indicating the importance of directionality control for the anaphase function of Cin8.
Hepatitis C virus (HCV) is a major cause of viral hepatitis. There is no effective therapy for most patients. We have identified a nucleotide binding motif (NBM) in one of the virus's nonstructural proteins, NS4B. This structural motif binds and hydrolyzes GTP and is conserved across HCV isolates. Genetically disrupting the NBM impairs GTP binding and hydrolysis and dramatically inhibits HCV RNA replication. These results have exciting implications for the HCV life cycle and novel antiviral strategies.Over 150 million people are infected with hepatitis C virus (HCV) worldwide (1). Current therapies are inadequate for most of these individuals (18). HCV is a positive singlestranded RNA virus. Its 9.6-kb genome encodes a single ϳ3,000-amino-acid polyprotein, which is proteolytically processed into structural proteins, which are components of the mature virus, and nonstructural proteins, which are involved in replicating the viral genome (26). A characteristic feature of positive-strand RNA viruses is their use of cytoplasmic membranes as platforms for replication (27). These membranes can either be preexisting host cell compartments or novel structures induced by the virus (2,4,8,17,27). HCV is also believed to replicate in association with intracellular membranes, although how the RNA replication complex is assembled and maintained remains unknown. Recently the HCV NS4B protein has been shown to induce the formation of a distinct membranous structure designated the membranous web (5), which represents the candidate site for HCV RNA replication (12). The mechanism whereby NS4B mediates its function(s) in membrane-associated RNA replication, however, remains to be elucidated and may offer insights for the development of novel antiviral strategies. Here we report the identification of a nucleotide binding motif (NBM) within NS4B and show that this motif mediates both binding and hydrolysis of GTP and HCV RNA replication. MATERIALS AND METHODSCell cultures. Cell monolayers of the human hepatoma cell line Huh-7 were routinely grown in complete medium consisting of equal volumes of Dulbecco's modified minimal essential medium (Gibco) and RPMI 1640 (Gibco), supplemented with 1% L-glutamine (Gibco), 1% penicillin, 1% streptomycin, and 10% fetal bovine serum. Cell lines were passaged twice weekly after treatment with 0.05% trypsin-0.02% EDTA and seeding at a dilution of 1:10. Antibodies.A rabbit polyclonal antibody against green fluorescent protein (GFP) and an anti-rabbit secondary antibody were purchased from Molecular Probes. A monoclonal antibody against glutathione S-transferase (GST) was purchased from Cell Signaling Technology.Plasmids. Standard recombinant DNA technology was used to construct and purify all plasmids. All regions that were amplified by PCR were analyzed by automated DNA sequencing. Plasmid DNAs were prepared from large-scale bacterial cultures and purified by a Maxiprep kit (Marligen Biosciences). Restriction enzymes were purchased from New England Biolabs. The Bart79I plasmid was described previou...
a b s t r a c tExpression of recombinant proteins in Escherichia coli (E. coli) remains the most popular and costeffective method for producing proteins in basic research and for pharmaceutical applications. Despite accumulating experience and methodologies developed over the years, production of recombinant proteins prone to aggregate in E. coli-based systems poses a major challenge in most research applications. The challenge of manufacturing these proteins for pharmaceutical applications is even greater. This review will discuss effective methods to reduce and even prevent the formation of aggregates in the course of recombinant protein production. We will focus on important steps along the production path, which include cloning, expression, purification, concentration, and storage.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.