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1 (199-210)病毒14-4期3475换.Indd VIROLOGICA SINICA 2014, 29 (4): 199-210 DOI 10.1007/s12250-014-3475-1 RESEARCH ARTICLE Labeling of infl uenza viruses with synthetic fl uorescent and biotin-labeled lipids Natalia A Ilyushina1#, Evgeny S Chernyy2#, Elena Y Korchagina2, Aleksandra S Gambaryan3, Stephen M Henry4, Nicolai V Bovin2 1. Food and Drug Administration, 29 Lincoln Drive, Bethesda 20892, MD, USA 2. Shemyakin Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho Maklaya 16/10, Moscow 117997, Russia 3. Chumakov Institute of Poliomyelitis and Viral Encephalitides, Russian Academy of Medical Sciences, Moscow Region 142782, Russia 4. Biotechnology Research Institute, AUT University, Private Bag 92006, Auckland 1142, New Zealand Direct labeling of virus particles is a powerful tool for the visualization of virus–cell interaction events. However, this technique involves the chemical modification of viral proteins that affects viral biological properties. Here we describe an alternative approach of influenza virus labeling that utilizes Function-Spacer-Lipid (FSL) constructs that can be gently inserted into the virus membrane. We assessed whether labeling with fl uorescent (fl uo-Ad-DOPE) or biotin-labeled (biot- CMG2-DOPE) probes has any deleterious effect on influenza virus hemagglutinin (HA) receptor specificity, neuraminidase (NA) activity, or replicative ability in vitro. Our data clearly show that neither construct signifi cantly affected infl uenza virus infectivity or viral affi nity to sialyl receptors. Neither construct infl uenced the NA activities of the infl uenza viruses tested, except the A/Puerto Rico/8/34 (H1N1) strain. Our data indicate that lipid labeling provides a powerful tool to analyze infl uenza virus infection in vitro. KEYWORDS bioimaging; Function-Spacer-Lipid (FSL) constructs; labeling of influenza viruses; synthetic lipids antibodies or serum has a number of methodological INTRODUCTION limitations, including higher risk of artifacts due to a multistep protocol. Unfortunately, direct labeling with Interaction of influenza virus with the surface of the fl uorescent molecules such as fl uorescein isothiocyanate host target cells and its subsequent internalization are (FITC) (Yoshimura A, et al., 1984) has several disadvan- the subject of numerous studies (Lakadamyali M, et al., tages due to reduced viral infectivity and the chemical 2003; Nichols J E, et al., 1992; Rust M J, et al., 2004; modification of external influenza virus proteins that Yoshimura A, et al., 1984). Influenza virion labeling is are responsible for virus interactions with target cell re- an experimental necessity for such studies because the ceptors (Freistadt M S, et al., 2006; Nichols J E, et al., alternative approach that utilizes specific anti-influenza 1992). Modern protein labeling techniques with different Received: 18 May 2014, Accepted: 17 July 2014 lipid traces (e.g., lipophilic dialkylcarbocyanines) utilize Published online: 31 July 2014 milder labeling conditions compared to FITC modifi- #These authors contributed equally to this work. cation and demonstrate the improved reproducibility of Correspondence: Natalia A Ilyushina, Phone: +1-301-8271730, Fax: +1-301-4803256, labeling and/or fl uorescence intensity signals (Ewers H., Email: [email protected] et al., 2012; Fecek R J, et al., 2006; Lakadamyali M, et Nicolai V Bovin, Phone: +7-495-3307138, Fax: +7-495-3305592; al., 2003; 1992; Rust M J, et al., 2004; Zhang S L, et al., Email: [email protected] 2010). However, such tracers do not exclude the possible © WIV, CAS and Springer-Verlag Berlin Heidelberg 2014 AUGUST 2014 VOLUME 29 ISSUE 4 199 Labeling of infl uenza viruses with FSL-constructs disturbance of functional properties of the viral surface DOPE (Figure 1B) (Abbreviations: Ad, the adipoyl proteins due to covalent attachment of the label to the vi- linker -OC(CH2)4CO-; CMG2, the carboxymethylglycyl rions, and they require highly purifi ed initial material and linker), into the membrane of human and avian infl uenza have low solubility in aqueous solutions. In addition, a A and B viruses. We assessed whether FSL labeling had recently developed membrane association-based strategy any deleterious effect on the biological properties of in- of fl uorescence molecular painting that labels the viruses fl uenza viruses, such as viral attachment, hemagglutinin with a green fl uorescent protein (GFP) carrying a specifi c (HA) receptor specificity, neuraminidase (NA) activity, glycosylphosphatidylinositol (GPI) moiety is not suitable and virus growth in vitro. for molecules that form di- or multimers and is absolute- ly dependent on the presence of the lipophilic parts of the MATERIALS AND METHODS GPI-anchored GFP for association with enveloped virus particles (Metzner C, et al., 2013). Cells and viruses KODETM technology, which utilizes a series of Function- Madin-Darby canine kidney (MDCK) cells were ob- Spacer-Lipid (FSL) constructs, is optimized for effi cient, tained from the Institute of Cytology RAS (Moscow, reliable, and gentle cell integration of different dyes Russia) or from the American Type Culture Collection into cellular and viral lipid membranes (Blake D, et al., (Manassas, VA, USA) and were maintained as previously 2011; Frame T, et al., 2007; Hadac E, et al., 2011; Henry described (Ilyushina N A, et al., 2008). S, 2009). In particular, unsaturated oleic acid chains of Avian influenza viruses A/FPV/Rostock/34 (H7N1), dioleoylphosphatidylethanolamine (DOPE) in FSL con- A/mallard/Pennsylvania/10218/84 (H5N2), A/duck/ structs provide a rapid and higher degree of construct in- France/46/82 (H1N1), and human A/Puerto Rico/8/34 sertion into lipid cellular/viral membranes, and the spac- (H1N1) strain were obtained from the Institute of er makes the whole structure dispersible in physiological Poliomyelitis and Viral Encephalitides RAMS (Moscow, buffers. KODETM technology is a membrane-targeting Russia). Human A/California/07/09 (H1N1), A/ labeling method that is dependent on the presence of the Perth/16/09 (H3N2), and B/Brisbane/60/08 were ob- lipophilic parts in cellular/viral membranes. However, tained from the Influenza Division of the Centers for in contrast to covalent chemical modifi cation with direct Disease Control and Prevention (Atlanta, GA, USA). FITC labeling (Yoshimura A, et al., 1984), attachment Stock viruses were prepared by one passage in the allan- of fl uorescein and biotin to the cells or virions in a lipid toic cavities of 10-day-old embryonated chicken eggs for form is carried out under physiological conditions, which 48 h at 37°C (or at 33°C for B virus), then purifi ed and allows maximum preservation of cellular/viral proteins concentrated by ultracentrifugation through 30% sucrose and ensures the integrity of supramolecular ensembles on as previously described (Mochalova L, et al., 2003). the membrane surface. Virus samples were stored at 4°C in phosphate-buffered In this study, we describe the method of synthesis and saline (PBS) with 0.01% NaN3. The protein concentra- insertion of highly stable fl uorescein- and biotin-labeled tion of the concentrated virus was determined by the constructs, fl uo-Ad-DOPE (Figure 1A) and biot-CMG2- Bradford assay, as described previously (Bradford M, Figure 1. Chemical structures of (A) the synthetic fl uorescent lipid, fl uo-Ad-DOPE, where a fl uorescein residue is conjugated to dioleoylphosphatidylethanolamine (DOPE) residue by a 15-atom linker, and (B) the synthetic biotin- conjugated lipid, biot-CMG2-DOPE, where biotin is conjugated to DOPE by hydrophilic N-carboxymethylglycyl (CMG2) linker. 200 AUGUST 2014 VOLUME 29 ISSUE 4 VIROLOGICA SINICA Natalia A Ilyushina et al 1976). All experimental work was performed in biosafety and human A/Puerto Rico/8/34 (H1N1) influenza vi- level 2 laboratories approved for use with these strains ruses were labeled with all three concentrations of fl uo- by the Russian Ministry of Health or the U.S. Department Ad-DOPE. Avian A/mallard/Pennsylvania/10218/84 of Agriculture and the U.S. Centers for Disease Control (H5N2) and A/duck/France/46/82 (H1N1) strains were and Prevention. labeled with fl uo-Ad-DOPE at a concentration of 50 μg/ mL. All mixtures were incubated at room temperature Synthesis of fl uorescent lipid fl uo-Ad-DOPE in the dark for 2 h with periodic stirring. Any excess We first synthesized an activated 1,2-O-dioleoyl- of fluo-Ad-DOPE was removed using chromatography sn-glycero-3-phosphatidylethanolamine (DOPE)-Ad- with a Sephadex G-50. In addition, sucrose density ONSu) substrate. For this purpose, DOPE dissolved in gradient experiments demonstrated that all influen- chloroform together with triethylamine was added to a za viruses studied co-localized with the fluorescent solution of bis (N-hydroxysuccinimidyl) adipate in dry fluo-Ad-DOPE label. Labeled viruses were stored at N,N-dimethylformamide. The mixture was stirred for 4°C in the dark in PBS containing 0.01% NaN3 for 2 h at room temperature, neutralized with acetic acid, 4–6 weeks. Labeling of human A/California/07/09 and then partially concentrated. After purifi cation using (H1N1), A/Perth/16/09 (H3N2), and B/Brisbane/60/08 column chromatography (Sephadex LH-20, 1:1 chlo- infl uenza viruses with biot-CMG2-DOPE was performed roform-methanol, 0.2% acetic acid), condensation of by the same procedure described above. activated DOPE with 1,5-diaminopentane (DOPE-Ad- Labeling of influenza viruses with FITC (Sigma- cadaverine) was performed. DOPE-Ad-ONSu dissolved Aldrich, St. Louis, MO, USA) was performed as de- in dichloromethane was added to a solution of cadaver- scribed previously (Yoshimura A, et al., 1984) with some ine in dichloromethane. The mixture was kept for 30 min modifications. Briefly, concentrated viruses (1.5 mg/ at room temperature, and then acetic acid was added mmol/L in PBS), A/FPV/Rostock/34 (H7N1), A/mallard/ slowly during 5 min. Then the solution was concentrated Pennsylvania/10218/84 (H5N2), A/duck/France/46/82 and purifi ed using a Sephadex LH-20 column (1:2 chlo- (H1N1), and A/Puerto Rico/8/34 (H1N1) were mixed roform-methanol).
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