Labeling of surface proteins of herpes simplex virus type 1 using a modified biotin-streptavidin system

Skulstad, Siri; Rødahl, Eyvind; Langeland, Nina; Haarr, Lars

doi:10.1016/0168-1702(95)00036-p

Cited by 8 publications

(6 citation statements)

References 53 publications

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“…Although biotin is small molecule (224 Da) and can bind to proteins without affecting their biological function, the physicochemical interactions involved with viral adherence to produce are complex, and a demonstration that the biotinylation process did not otherwise influence virus attachment is critical for the practical use of this approach to study virus-matrix interactions. In addition, the biotinylation process did not appear to impact HAV or MNV-1 infectivity, which is somewhat different from the results of Skulstad et al (19), who reported that the infectivity of herpes simplex virus type 1 was reduced by 40% when the virus is biotinylated. However, herpes simplex virus is an enveloped virus, whereas both HAV and MNV-1 are nonenveloped, which may explain why their infectivity was not affected by the biotinylation process.…”

Section: Discussioncontrasting

confidence: 56%

Development of a Fluorescent In Situ Method for Visualization of Enteric Viruses

Rawsthorne

Phister

Jaykus

2009

Appl Environ Microbiol

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Studying the interactions between enteric pathogens and their environment is important to improving our understanding of their persistence and transmission. However, this remains challenging in large part because of difficulties associated with tracking pathogens in their natural environment(s). In this study, we report a fluorescent labeling strategy which was applied to murine norovirus (MNV-1), a human norovirus surrogate, and hepatitis A virus (HAV). Specifically, streptavidin-labeled Quantum dots (Q-Dots) were bound to biotinylated capsids of MNV-1 and HAV (bio-MNV-1 and bio-HAV); the process was confirmed by using a sandwichtype approach in which streptavidin-bound plates were reacted with biotinylated virus followed by a secondary binding to Q-Dots with an emission range of 635 to 675 nm (Q-Dots 655). The assay demonstrated a relative fluorescence of 528 ؎ 48.1 and 112 ؎ 8.6 for bio-MNV-1 and control MNV-1, respectively. The biotinylation process did not impact virus infectivity, nor did it interfere with the interactions between the virus and host cells or model produce items. Using fluorescent microscopy, it was possible to visualize both bio-HAV and bio-MNV-1 attached to the surfaces of permissive mammalian cells and green onion tissue. The method provides a powerful tool for the labeling and detection of enteric viruses (and their surrogates) which can be used to track virus behavior in situ.Over the last 2 decades, production and consumption of fresh fruits and vegetables have increased dramatically, with an accompanying rise in the number of produce-associated foodborne disease outbreaks (12). It is clear from review of the CDC Food-borne Disease Outbreak database (1996 to 2006; available at: http://www.cdc.gov/foodborneoutbreaks/outbreak _data.htm) that the human noroviruses (HuNoV) are the leading cause of produce-associated food-borne disease. Although most HuNoV outbreaks are small and limited in scope, vegetable salads and berries are notable vehicles (8,11,17,18). Hepatitis A virus (HAV) is the other human enteric virus of concern in fresh produce, and several high-profile outbreaks have occurred in association with HAV-contaminated strawberries and green onions in the last decade (9, 21).Little is known about the behavior of the enteric viruses in fresh produce items although virus persistence in such products is well documented (reviewed by Papafragkou et al. [14]). It is likely that a complex set of physicochemical interactions governs such persistence, which may be associated with attachment and/or internalization of the virus in the produce item(s). Unfortunately, there are no known means by which to visualize the HuNoV and HAV in situ in order to study such purported virus-surface interactions or other mechanisms by which these viruses persist in fresh produce. In the only study of its kind, Chancellor et al. (5) used fluorescent microspheres as a surrogate to examine the uptake of HAV into green onions. The fluorophores were observed microscopically on the surface and within the green o...

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Section: Discussioncontrasting

confidence: 56%

Development of a Fluorescent In Situ Method for Visualization of Enteric Viruses

Rawsthorne

Phister

Jaykus

2009

Appl Environ Microbiol

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“…Cell surface receptors, after binding with the biotinylated ligand, could be visualized by fluorescein isothiocyanate (FITC)-conjugated streptavidin [Skulstad et al, 1995]. A total of 200,000 CHO-HSR cells and vector transfected control CHO cells were seeded separately into P35G-01 capsules (MatTet, Ashland, MA) 2 days before the experiment.…”

Section: Confocal Microscopy Of Cho-hsr Cellsmentioning

confidence: 99%

Real-time evaluation of human secretin receptor activity using cytosensor microphysiometry

Pang

Chow

et al. 1999

J. Cell. Biochem.

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Human secretin receptor is a G protein-coupled receptor that is functionally linked to the cAMP second messenger system by stimulation of adenylate cyclase. To functionally characterize the receptor and evaluate its signal transduction pathway, the full-length human secretin receptor cDNA was subcloned into the mammalian expression vector pRc/CMV and expressed in cultured CHO cells. Intracellular cAMP accumulation of the stably transfected cells was measured by a radioimmunoassay (RIA), while the extracellular acidification rate was measured by the Cytosensor microphysiometer. Human secretin and biotinylated human secretin were equipotent in both assays in a dose-dependent manner. The EC50 values of stimulating the intracellular cAMP accumulation and the extracellular acidification rate were 0.2-0.5 nM and 0.1 nM, respectively, indicating that microphysiometry is more sensitive than the cAMP assay in monitoring ligand stimulation of the human secretin receptor. The secretin-stimulated response could be mimicked by forskolin and augmented by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, indicating that the extracellular acidification response is positively correlated with intracellular cAMP level. The response could be abolished by the protein kinase A inhibitor H-89, suggesting that protein kinase A plays an essential role in the intracellular signaling of the receptor. Upon repeated stimulation by the ligand, the peak acidification responses did not change significantly at both physiological (0.03 nM and 3 nM) and pharmacological (0.3 microM) concentrations of human secretin, suggesting that the human secretin receptor did not exhibit robust homologous desensitization.

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“…1 Three approaches have been developed for virus labeling, such as fusion of viral target proteins with fluorescent protein (FP) by genetic engineering, 2,3 directly labeling the virus envelope with fluorophors by chemical coupling or physicochemical association. [4][5][6] Viruses were chemically bonded with fluorophors through the functional groups of the envelope proteins in chemical coupling methods, while physicochemical association made use of electrostatic or hydrophobic interactions to incorporate the organic dye into the membrane of the envelope virus.…”

Section: Introductionmentioning

confidence: 99%

Site-specific labeling of baculovirus in an integrated microfluidic device

et al. 2013

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Labeling of viruses can be used to reveal viral infection pathways and screen potential anti-viral drugs. Complex procedures, including virus cultivation, purification and labeling are involved in traditional virus labeling methods. And the manipulation of living virus brings risk to researcher health. In this work, we report a general method for site-specific labeling of the envelope virus in an integrated microfluidic device with simple procedures and high security. Site-specific labeling of virus was achieved by fusing the biotin acceptor peptide (AP-tag) and the biotin ligase enzyme (BirA enzyme) with the envelope protein GP64 of baculovirus. The AP-tag could be modified by BirA enzyme to introduce the biotin moiety onto the viral envelope. Western blots and fluorescence colocalization analysis proved that the baculoviruses were biotinylated and labeled with high efficiency. The integrated device incorporated several operation steps including cell seeding, cell culture, cell transfection, virus culture and virus labeling. Since virus biotinylation was achieved during the process of virus cultivation, the complex procedures of virus labeling were simplified in our device. Furthermore the whole process could be completed in the integrated microfluidic device, and direct contact between viruses and researchers could be eliminated in our method, which could greatly reduce the risk to researcher health during living virus labeling.

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Labeling of surface proteins of herpes simplex virus type 1 using a modified biotin-streptavidin system

Cited by 8 publications

References 53 publications

Development of a Fluorescent In Situ Method for Visualization of Enteric Viruses

Development of a Fluorescent In Situ Method for Visualization of Enteric Viruses

Real-time evaluation of human secretin receptor activity using cytosensor microphysiometry

Site-specific labeling of baculovirus in an integrated microfluidic device

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