Enveloped Virus Labeling via Both Intrinsic Biosynthesis and

Publication Date (Web): April 22, 2013. Copyright ... For the first time, it is by natural propagation of the virus in its host cells in the presence ...
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Enveloped Virus Labeling via Both Intrinsic Biosynthesis and Metabolic Incorporation of Phospholipids in Host Cells Li-Li Huang,†,∥ Gui-Hong Lu,†,‡,∥ Jian Hao,† Hanzhong Wang,§ Du-Lin Yin,‡ and Hai-Yan Xie*,† †

School of Life Science, Beijing Institute of Technology, Beijing 100081, China Chemistry and Chemical Engineering, Hunan Normal University, Hunan, P. R. China § Wuhan Institute of Virology, Chinese Academy of Sciences, Hubei, P. R. China ‡

S Supporting Information *

ABSTRACT: An alternative method for labeling fully replicative enveloped viruses was developed, in which both the biosynthesis and metabolic incorporation of phospholipids in host cells were simultaneously utilized to introduce an azide group to the envelope of the vaccinia virus by taking advantage of the host-derived lipid membrane formation mechanism. Such an azide group could be subsequently used to fluorescently label the envelope of the virus via a bioorthogonal reaction. Furthermore, simultaneous dual-labeling of the virus through the virus replication was realized skillfully by coupling this envelope labeling strategy with “replication-intercalation labeling” of viral nucleic acid. For the first time, it is by natural propagation of the virus in its host cells in the presence of fluorophores that simultaneous dual-labeling of living viruses can be mildly realized with high efficiency in facile and mild conditions. first metabolically incorporating biotin-functionalized phosphatidylethanolamine (Biotin-Cap-PE) into Vero cells and then naturally incorporating the Biotin-Cap-PE into enveloped viruses during the virus in situ assembling process.20 This strategy is simple in technique and reliable and does not decrease the virulence of the viruses. However, the intact Biotin-Cap-PE was used instead of taking advantage of the intracellular biosynthesis. Moreover, as well-known, PE is less abundant in eukaryote membranes. Therefore, it is inefficient and inconvenient to label the harvested virions with the biotin on the incorporated Biotin-Cap-PE. Furthermore, Biotin-CapPE may disturb the normal function of PE to some extent since their structures are evidently different from each other. In 2009, Jao and co-workers biosynthesized and incorporated choline (Cho) analogues, such as azidoethyl-choline (AECho), azidopropyl-choline (APCho), or propargyl-choline (propargylCho), into the phospholipids of cells and tissues.21,22 The Choanalogues-containing phospholipids bear a terminal azide or alkyne moiety, which can be labeled by the effective cycloaddition reactions. Since the structure of Cho-analoguescontaining phospholipids is very similar to that of native Chocontaining phospholipids, the function of the membranes will be native to the largest extent. Moreover, Cho-containing phospholipids are the most common and important structural

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eciphering the viral invasion process is of great importance for augmenting our understanding of the pathogenic mechanism of a virus and improving viral disease diagnosis and therapeutics. For this purpose it is necessary to develop both a highly sensitive labeling technique and highresolution microscopic imaging technique. Despite several strategies for labeling viruses reported,1−5 some key problems still need to be solved. In most cases, viruses were fluorescently labeled via their surface proteins or glycoproteins, which commonly act as recognition ligands to their host cells. Additionally, almost all strategies were for extracellular labeling, which is time-consuming and destructive, especially for the enveloped viruses with a fragile lipid membrane, which is formed when the nucleocapsid core protrudes through the cell membranes. By intercepting the intracellular biosynthesis of molecules, a small chemical moiety can be metabolically exchanged and derived to the cellular biomolecules, with the functionalized biomolecules obtained, whose innate character was not remarkably changed. This is emerging as an appealing approach for studying macromolecules in cells and remodeling the surface structure of cells.6−19 Can the metabolic incorporation be coupled with the intrinsic biosynthetic machinery of cellular utilization of molecules to naturally label the viruses in host cells? In theory, it is practicable since the viruses are completely parasitic organisms. For enveloped viruses, their envelope is derived from the host cells during the duplicate and assembling process. Recently, Pang’s group demonstrated this concept by © XXXX American Chemical Society

Received: March 18, 2013 Accepted: April 22, 2013

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Figure 1. Schematic illustration. (A) The chemical formulas of choline (Cho) and azide-Cho (AECho and APCho). (B) Biosynthesis of azide-Chocontaining phospholipids and its labeling by strain-promoted azide−alkyne cycloaddition using DBCO-Fluor 525. (C) The vaccinia virus (VACV) propagation in the presence of both azide-Cho and [Ru(phen)2(dppz)]2+ in the host cell. The biosynthesis and incorporation of azide-Chocontaining phospholipids in host cells were carried out at first (①). Then the cells were infected with VACV, and the [Ru(phen)2 (dppz)]2+ was added to the medium at 2 h postinfection, which could enter the cells through the permeable cytomembrane resulting from cytopathogenic effects of the virus to label the nucleic acid of the virions (②). At 24 h postinfection, DBCO-Fluor 525 was added to the medium to label the envelope of the VACV (③). After another 24 h infection, the dual-labeled virions were finally assembled and released.

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EXPERIMENTAL SECTION Materials and Reagents. The 1,2-dibromoethane and 1,3dibromopropane were purchased from J&K Scientific Ltd. (Beijing, China), and dimethyl-ethanolamine was purchased from Alfa Aesar (Massachusetts). Vaccinia virus (VACV) tiantan strain, H9N2 influenza virus (H9N2), and MDCK cells (Medin-Darby canine kidney cells) were obtained from Wuhan Institute of Virology, Chinese Academy of Sciences. Vero cells (African green monkey kidney cells) were purchased from Peking Union Medical College Hospital, Phospholipase C (PLC type XIV from C. perf ringens) was purchased from SigmaAldrich, and dibenzocyclooctynes-derived Fluor 525 (DBCOFluor 525) was purchased from Click Chemistry Tools (Scottsdale, AZ). [Ru(phen)2(dppz)]2+ was supplied by Prof. Zhi-Ke He of Wuhan University, China. Dylight 488 conjugated Goat Anti-Mouse IgG antibody was from Earthox LLC (San Francisco, CA), anti-H3 protein (vaccinia virus) and anti-HA protein (H9N2 influenza viruses) mouse monoclonal antibody (mAb) were from Immune Technology Corp. All other chemical reagents were supplied by Beijing Chemical Reagent Company. Synthesis of Azide-Cho. A colorless solution of 1,2dibromoethane (8.5 mL, 98.52 mmol) and sodium azide (3.20 g, 49.22 mmol) was added to 25 mL of N,N-dimethylformamide (DMF). The reaction mixture was heated to 80 °C for 20 h, then cooled to room temperature, put over a mixture of satd. NaCl and ice, and extracted with cold pentane three times.

components of membranes in eukaryotes. Accordingly, it will be of great significance for virus labeling if the Cho-analoguescontaining phospholipids on the membranes of host cells can be further transferred to enveloped viruses. In this paper, both the intrinsic biosynthesis and metabolic incorporation of phospholipids have been coupled to label the living enveloped virus in its host cells. The Cho analogue azidecholine (azide-Cho) was biosynthetically bonded to phospholipids and incorporated into the host cells, and subsequently the model enveloped virus, vaccinia virus (VACV) or H9N2 influenza virus (H9N2), was propagated, with progeny virions with the azide-modified envelope formed from the membranes of the azide-modified host cells being harvested. The virions could be labeled with fluorophores conveniently through strainpromoted azide−alkyne cycloaddition (SPAAC)23 with a high efficiency and good biocompatibility. Furthermore, the living virus was also simultaneously dual-labeled in host cells by integrating this viral envelope labeling with the “virus replication-intercalated labeling” of the viral nucleic acid (Figure 1). The dual labeling efficiency was about (81 ± 3.6)% (mean ± SD). The dual labeled viruses also could recognize their host cells and still retain their infectivity. This method is facile, fast, highly efficient, and universal for all dsDNA enveloped viruses. B

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each well was measured with a Microplate System (354 model). Cell viability was determined by comparing the ratio of absorbance of the cells incubated with AECho or APCho to that of the cells incubated with culture medium only. Flow Cytometry Assay. Vero cells were seeded in 6-well culture dish (2.6 × 105 cells per well) and incubated with AECho at different concentrations in the culture medium for 24 h. Subsequently, the azide-modified cells were infected by VACV at a multiplicity of infection (MOI, ratio of infectious virus particles to cells) of 10 for 12 h before the flow cytometry (BD Calibur) assay of GFP+ cells. All samples were counted over 10 000 cells, and all data were processed with WinMDI and Origin 8.0. The percentage of GFP+ cells was calculated from the flow cytometry plots. Data from three independent experiments were normalized as a percentage of the control. Virus Propagation. The vaccinia virus (VACV) was propagated in monolayer cultures of Vero cells or azidemodified Vero cells at 37 °C in the presence of 2% FBS. The propagation lasted for 48 h. After three rounds of freeze−thaw, the cell debris was removed by centrifugation at 2 000g for 15 min at 4 °C. The control virus or azide-modified virus was purified by differential centrifugation and density gradient centrifugation as previously described.25 H9N2 influenza virus (H9N2) was propagated in monolayer cultures of MDCK cells or azide-modified MDCK cells at 37 °C in viral growth medium (VGM, DMEM with 0.3% BSA) supplemented with 0.5 μg/mL trypin as previously described.26 The control virus or azide-modified virus was purified by differential centrifugation and density gradient centrifugation as previously described.27 Virus Titer Assays. The titer of VACV was quantified by 50% Tissue Culture Infective Dose (TCID50). Vero cells were cultured in 96-well plates in culture medium until the cells reached 80−90% confluence. The virus samples were 10-fold diluted ranging from 10−1 to 10−8 in DMEM. They were subsequently added to the cells and infected for 1 h. TCID50 cultures were washed and fed with DMEM containing 2% FBS. Then the infected cells were cultured in an incubator at 37 °C with 5% CO2 for about 5 days. Count the number of wells which have CPE on Vero cells. The TCID50 was calculated based on the Reed and Muench method.28 The titer of VACV can also be quantified by flow cytometry assay. The azide-virus (MOI = 10) harvested from Vero cells preincubated with AECho at different concentrations were used to infect Vero cells for 12 h. The GFP+ cells were analyzed by flow cytometry. One-Step Growth Assays. Control virus or AEChomodified virus was inoculated in Vero cells (MOI = 5), respectively. After 1 h of adsorption at 4 °C, the inoculum was replaced by prewarmed DMEM medium containing 2% FBS and incubated for 4, 8, 12, 24, 36, 48, 60, or 72 h, respectively. The infected cells were scraped into the medium, after three rounds of freeze−thaw; the cell debris was removed by centrifugation at 2000g for 15 min. The infectivities of collected virus were determined by TCID50. Fluorescence Co-Localization Assay of the Virus on the Glass Slides. The purified control virus or azide-modified virus solution was dropped onto slides (22 mm × 22 mm, Citoglas) coated by anti-H3 protein (VACV) mouse mAb or anti-HA protein (H9N2) mouse mAb for 1 h. The excessive viruses were washed out by 0.01 M phosphate buffer solution (1× PBS, pH 7.2). Then the virus was fixed with heat. After being blocked with 2% (w/v) bovine serum albumin (BSA), the

Then the organic layer was washed with cold brine followed by drying over Na2SO4 and concentrated under reduced pressure to give 1-azido-2-bromoethane as yellow oil used in the subsequent reaction without further purification. Azide-cho was synthesized according to reported procedures22,24 with a little modification. For the preparation of azidoethyl-choline (AECho), the above azido-2-bromoethane was slowly added to dimethyl-ethanolamine (49.22 mmol) in 15 mL of dry tetrahydrofuran (THF) while stirring under argon gas. The reaction mixture was allowed to react for 6 h at 0 °C. Then the white precipitate was washed with diethylether and dried under vacuum with pure azidoethyl-choline with the white solid obtained. 1H NMR (600 MHz, CD3OD) (Varian VNMR): δH 4.81 (H2O), 4.01(4H, m), 3.68 (2H, t), 3.58 (2H, m), 3.25 (6H, s). 13C NMR (600 MHz, CD3OD): δC 66.33, 63.40, 55.51, 51.65, 47.53, 44.80. ESI-LC/MS (Agilent Technologies 1200 series, 6460 Triple Quad LC/MS): [M]+ = 159.1. The azidopropyl-choline (APCho) was prepared similar with that of azidoethyl-choline by using 1,3- dibromopropane as the material. 1H NMR (600 MHz, CD3OD): δH 4.82 (H2O), 3.98 (2H, m), 3.50 (6H, m), 3.18 (6H, s), 2.37 (2H, m). 13C NMR (600 MHz, CD3OD): δC 65.37, 63.93, 55.30, 51.09, 44.78, 28.03, 22.17. ESI-LC/MS: [M]+ = 173.1. Cell Culture. Vero cells or MDCK cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 IU/mL penicillin and 100 μg of streptomycin (Lonza). Cell cultures were incubated in a 5% CO2 incubator at 37 °C. Biosynthesis and Metabolic Incorporation of Phospholipids in Vero Cells. Vero cells were placed in glassbottom dishes (NEST Corp), cultured with azide-Cho (AECho or APCho) at different concentrations in complete medium (DMEM with 10% FBS) for 24 h, with the azide-modified cells obtained. For the fluorescence imaging, the azide-modified cells were washed with phosphate buffer solution (PBS), after which they were fixed with 4% (w/v) paraformaldehyde for 15 min at room temperature. The samples were reacted with 10 μM DBCO-Fluor 525 for 1 h, washed with Tris buffer solution (TBS), 0.5 M NaCl, and again TBS. The nucleic acid of the cells was stained with Hoechst 33342 by incubating with 10 μg/ mL Hoechst 33342 solution for 10 min. After washing, the cells were imaged by Leica TCS SP5 laser scanning confocal microscope equipped with argon and HeNe lasers. The DBCOFluor 525 was excited using a 514 nm laser; emission was collected by photomultiplier tubes in the range of 550−570 nm. Hoechst 33342 was excited with UV, emitting 450−500 nm fluorescence. To test the phospholipase sensitivity of the azideCho biosynthetical conjugation and metabolic incorporation, three dishes of Vero cells, having incubated with 400 μM AECho for 24 h, were fixed with 4% (w/v) PFA and rinsed with TBS. Then they were incubated in TBS with 0.02 U/mL phospholipase C, both 0.02 U/mL phospholipase C and 10 mM CaCl2 or both 0.02 U/mL phospholipase C and 10 mM EDTA (negative control) for 1 h at 37 °C, respectively. Finally, the cells were washed with TBS and then stained with DBCOFluor 525 as described above.21 Cells Viability Assay. Cell viability assays were performed using Cell Counting Kit-8 (CCK-8) (Dojindo, Kumamoto, Japan). Vero cells were incubated with AECho or APCho at different concentrations in the culture medium for 24 h. After having been washed with PBS, they were incubated with the CCK-8 solution for 4 h. Finally, the absorbance (450 nm) for C

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Figure 2. Fluorescence labeling and viability detection of azide-modified Vero cells. (A, C) The biosynthetical bonding and metabolic incorporation of azide-Cho (A, APCho; C, AECho) into Vero cells. All Vero cells were grown in complete media together with or without azide-Cho at certain concentrations, thus azide-Cho could compete with native Cho in the culture media for bonding to phospholipids during the biosynthesis of Chocontaining phospholipids, which would subsequently incorporate into the membranes of the cells. After having incubated for 24 h, the cells were fixed with 4% (w/v) paraformaldehyde and incubated with DBCO-Fluor 525. The nucleus of the cells was stained with Hoechst 33342 (Blue, Hoechst 33342; red, DBCO-Fluor 525). (B, D) CCK-8 detection of the viability of Vero cells incubated with azide-Cho (B, APCho; D, AECho) at different concentrations for 24 h. Error bars represented the standard deviation from three replicated experiments.

fluorescence. Hoechst 33342 was excited with UV, emitting 450−500 nm fluorescence. Effect of DBCO-Fluor 525 on the Cell Viability. Vero cells were allowed to incubate with DBCO-Fluor 525 at different concentrations for 24 h. Then, the cell viability was determined by CCK-8 as described above. Preparation and Imaging of the Dual-Labeled VACV. Vero cells were cultured in DMEM added with 400 μM azideCho for 24 h and then inoculated with VACV for 2 h. Subsequently, the medium was changed into DMEM supplemented with [Ru(phen)2(dppz)]2+ at a final concentration of 15 μM.28 At 24 h postinfection, 5 μM DBCO-Fluor 525 was added into the medium. After another 24 h infection, the infected cells were centrifuged at 2000g for 15 min and the cell pellets were suspended in 20 mL of PBS. After three rounds of freeze−thaw, the cell debris was removed by centrifugation at 2000g for 15 min. The dual-labeled virus was purified by differential centrifugation and density gradient centrifugation. The fluorescence imaging and the phospholipase sensitivity assay were carried out as mentioned above. Transmission Electron Microscope (TEM) Imaging. The size and the morphology of the virions were characterized by TEM (Hitachi, H-7650B), for which, control virus, azidevirus or dual-labeled virus (10 μL) was dropped onto a carboncoated copper grid. After 2 min, unabsorbed virus was removed with filter paper. To negative staining, phosphotungstate (1%, 10 μL) was applied to the sample-loaded grid and blotted off after 1.5 min. The samples were observed with the TEM. Fluorescence Microscopic Imaging of Cells Interacted with Dual-Labeled Virus. Cells, cultured in glass-bottom dishes, were grown to 70% confluence. Then the cells were incubated with as-prepared virus for 30 min at 4 °C to allow virus binding, the unbound virions were removed by washing

virus was incubated with 10 μM DBCO-Fluor 525 for 1 h. The nucleic acid of the virus was stained with Hoechst 33342 or SYTO 13 by incubating with 2 mg/mL Hoechst 33342 or 10 mM SYTO 13 solutions for 30 min. The excessive fluorophores were washed out by 1× PBS. The fluorescence images were acquired using a Leica laser confocal fluorescence microscope (TCS SP5). In VACV labeling, DBCO-Fluor 525 was excited using a 514 nm laser, emitting 550−570 nm fluorescence. Hoechst 33342 was excited with UV, emitting 450−500 nm fluorescence. In H9N2 labeling, DBCO-Fluor 525 was excited using a 543 nm laser, emitting 560−600 nm fluorescence. SYTO 13 was excited with a 488 nm laser, emitting 500−520 nm fluorescence.29 Immunofluorescence Assay of the Virus. Azide-virus was incubated with 10 μM DBCO-Fluor 525 for 1 h at room temperature. Unreacted DBCO-Fluor 525 was removed with a desalting NAP-5 column (GE Healthcare). Then Vero or MDCK cells were coincubated with the as-prepared virions at 4 °C for 0.5 h, after which the nonspecific adsorbed virions were washed out. The cells were fixed with 4% PFA for 15 min at room temperature. After washing with 1× PBS, they were blocked in PBST containing 1% BSA for 1 h at room temperature, then the cells were incubated with anti-H3 protein (VACV) mouse mAb or anti-HA protein (H9N2) mouse mAb for 1 h at room temperature, followed by incubating with Dylight 488 conjugated goat antimouse IgG antibody for 1 h and then with 10 μg/mL Hoechst 33342 for 10 min at room temperature in dark. The samples were rinsed three to four times with PBS between two successive steps. The fluorescence images were acquired using a Leica laser confocal fluorescence microscope (TCS SP5). Dylight 488 was excited with 488 nm laser, emitting 500−520 nm fluorescence. DBCO-Fluor 525 was excited with 543 nm laser, emitting 560−600 nm D

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form phosphatidylcholine (PC) in the endoplasmic reticulum (ER). The PC was next transported to other cellular membranes (such as the mitochondrial and plasma membranes) by the vesicular trafficking pathway.30 The azide-Cho was selected to functionalize the phospholipids in cells since the azide was most widely used because of its small size, metabolic stability, and no interference with biosystems. Moreover, the PC can be detected by the dibenzylcyclooctyne-Fluor 525 (DBCO-Fluor 525) fluorescence probe by covalent attachment of the azide group to the cyclooctyne via a SPAAC,31,32 which is nontoxic and biocompatible.29 We synthesized two different Cho analogues, AECho and APCho (Figure 1A and see Figure S1 in the Supporting Information), and tested their biosynthetical conjugation to phospholipids, followed by metabolic incorporation into Vero cells. All Vero cells were grown in complete medium together with or without azide-Cho at certain concentrations, thus azide-Cho could compete with native Cho in the culture media for bonding to phospholipids during the biosynthesis of Cho-containing phospholipids, which would subsequently incorporate into the membranes of the cells. After incubating for 24 h, the cells were fixed with 4% paraformaldehyde and labeled with DBCO-Fluor 525. The cells showed strong fluorescence proportional, in intensity, to the concentration of added azide-Cho (Figure 2A,C). When they were incubated with phospholipase C (PLC type XIV from C. perf ringens) under different conditions, the fluorescence disappeared if both PLC and calcium ions (Ca2+) were present. However, this is not the case when there was only PLC or both PLC and ethylene diamine tetraacetic acid (EDTA) were present (Figure 3), suggesting that azide-Cho indeed was bonded to phospholipids since PLC could specifically hydrolyze Cho head groups into Cho phosphate in the presence of Ca2+.7 For the azide-Cho at the same concentration, the fluorescence of the AECho-installed cells was much stronger than that of the APCho-installed cells (see Figure S2 in the Supporting Information), demonstrating that AECho was bonded much more efficiently than APCho. The cell viability could be kept more than 90% even when they incubated with AECho at a concentration of 1.6 mM for 24 h, indicating that it was biocompatible. For AECho and APCho at the same concentration, the viability of the cells treated with AECho

with PBS containing 1% glucose, after which the cells were imaged by a Leica laser confocal fluorescence microscope (TCS SP5). DBCO-Fluor 525 was excited with a 514 nm laser, emitting 550−570 nm fluorescence. [Ru(phen)2(dppz)]2+ was excited with UV, and the fluorescence was collected in the wavelength range of 600−650 nm.

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RESULTS AND DISCUSSION Biosynthesis and Metabolic Incorporation of Azide Modified Phosphatidylcholine in Vero Cells. Phospholi-

Figure 3. The fluorescence imaging of azide-modified Vero cells treated with phospholipase C at different conditions. The phospholipase C (PLC) specifically hydrolyzes Cho head groups to Cho phosphate in the presence of Ca2+. The fluorescence disappeared if both PLC and calcium ions were present. But this is not the case when there was only PLC or both PLC and EDTA present.

pids are the major components of all biological membranes, and phosphatidylcholine (PC) is the most abundant phospholipid in most eukaryotic cells, comprising almost 50% of the phospholipid pool. Choline (Cho) itself cannot be synthesized de novo in animal cells; it must be imported from culture media via specific transporters.7 Upon entry, Cho was phosphorylated and converted to CDP-Cho, which then transferred to diacylglycerol by the CDP-choline pathway to

Figure 4. Fluorescence colocalization imaging of azide-installed VACV. (A) Fluorescence colocalization imaging of azide-virions (a−c) and control virions (a′−c′). The virions were captured onto the glass slides by the antibody of VACV. After having fixed with heat, DBCO-Fluor 525 was dropped and incubated for 1 h. The nucleic acid of the virions was stained with Hoechst 33342. (B) Fluorescence colocalization imaging of VACV on cells. The azide-virions (d−g) or control virus (d′−g′) was incubated with 10 μM DBCO-Fluor 525 for 0.5 h. Unreacted DBCO-Fluor 525 was removed with a desalting NAP-5 column. Then, Vero cells were coincubated with as-prepared virions at 4 °C for 0.5 h, and then the unbound virions were washed out. The nucleic acid of the cells was stained with Hoechst 33342. Subsequently, one of the envelope proteins of VACV, H3 protein was labeled with Dylight 488. Hoechst 33342 was excited with UV, emitting 450−500 nm fluorescence. Dylight 488 was excited with 488 nm laser, emitting 500−520 nm fluorescence. DBCO-Fluor 525 was excited with a 543 nm laser, emitting 560−600 nm fluorescence. E

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Figure 5. Detection of infectivity of modified VACV: (A) Flow cytometry assay of Vero cells infected by virus. (a) The azide-virus (MOI = 10) harvested from Vero cells modified with AECho at different concentrations were used to infect Vero cells for 12 h before the flow cytometry assay of GFP+ cells. All samples were counted over 10 000 cells, and all data were processed with WinMDI and Origin 8.0. The first line, Vero cells without being infected with virus; the second line, Vero cells infected with control virus; the third to eighth lines, Vero cells infected with azide-virus which was harvested from cells modified with AECho at different concentrations (0.025 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, 0.8 mM). (b) Percentage of GFP+ cells calculated from the flow cytometry plots of part a. Data from three independent experiments were normalized as a percentage of the control. (c) Microscopic imaging of Vero cells infected by control virus (a′−d1′) or azide-virus (e′−h1′) (The concentration of the azide was 400 μM) for various durations (a′−h′, bright-field; a1′−h1′, fluorescence of GFP). (B) Titers of virus. Vero cells were cultured until they reached 80%−90% of confluence. Serially diluted virus samples (control virus, azide-virus, or dual-labeled virus) of the same batch were added to the cells, followed by infecting for 1 h. TCID50 cultures were washed and fed with DMEM containing 2% FBS. Then the cells were cultured for about 5 days. TCID50 in Vero cells was calculated based on the Reed-Muench formula. (C) Growth curves of virus. Vero cells were infected with virus samples at MOI = 5 for the time indicated. Titers were determined by TCID50. Error bars represent the standard deviation from three repeated experiments. (D) Transmission electron microscope (TEM) images of virus. Virus samples (control VACV, azide-VACV, or dual-labeled VACV) were dropped onto a carbon-coated copper grid. After 2 min, unabsorbed virus was removed with filter paper. To negative staining, phosphotungstate (1%, 10 μL) was applied to the sample-loaded grid and blotted off after 1.5 min. The samples were observed with the TEM.

normally expressed with increasing propagation time. Moreover, there was no significant difference between the control cells and the azide-modified cells (see Figure S3 in the Supporting Information), demonstrating that the azide modification did not obviously alter the capability of the Vero cells to propagate the virus. VACV is one of the enveloped viruses containing dsDNA. It is composed of a core surrounded by one or more membranes, derived from the membranes of the host cells.34,35 To determine that the azide-

was higher than that of the cells treated with APCho (Figure 2B,D). Therefore, AECho was used in the following study. Production and Labeling of the Azide-Installed Virus. The azide-modified Vero cells were used to propagate the recombinant VACV with a green fluorescence protein (GFP) reporter gene,33 where the GFP expressed in the cytoplasm of the host cells could accordingly be used to evaluate the infectivity of the virus. Obviously, the VACV could be propagated in the azide-modified cells since GFP could be F

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protein was labeled with a Dylight 488 labeled antibody against H3. As shown, both viruses were located on the surface of the Vero cells since the fluorescence of Dylight 488 was circled around all the Vero cells, whereas the signal of Fluor 525 could only be seen on the Vero cells incubated with the azide-virus (Figure 4B and Figure S4 in the Supporting Information). Moreover, most of the signal of the Fluor 525 was colocalized with that of Dylight 488, and the colocalization efficiency was (83 ± 2.5)% (mean ± SD) (Figure 4B), suggesting that the azide-virus could still specifically recognize its host cells. In the infectivity assays, as seen, the azide-virus could induce the cytopathogenic effects (CPE) and the expression of GFP in cytoplasm was remarkable at 12 h postinfection. Moreover, the CPE in the cells infected by azide-virus was always similar to that in the cells infected by control virus (Figure 5A), suggesting that the azide-virus is infectious. The titers of control virus and azide-virus were 2.20 × 107 TCID50 mL−1 and 1.47 × 107 TCID50 mL−1, respectively (Figure 5B), which had no significant difference. In general, the “one-step growth curves” for the azide-virus resembled closely those for the control virus of the same batch. Both the azide-virus and control virus could reach 108 TCID50 mL−1 about 30 h postinfection (MOI = 5) (Figure 5C). It could be concluded that the azide-virus well preserved its infectivity. Unsurprisingly, since the azide was extremely small, the azide-Cho containing phospholipids were almost the same as native Cho-containing phospholipids in structure (Figure 1A). Accordingly, they behave like native Cho-containing phospholipids. As well, the incorporation of azide-Cho containing phospholipids into the envelope of virus occurs spontaneously when progeny virions bud through the azide-modified cells and the SPAAC is highly biocompatible, thus the infectivity of the virus can be kept to the greatest extent. Preparation and Imaging of the Dual-Labeled VACV. Simultaneously labeling the viral nucleic acid and the envelope is vital for tracking its invasion process because they will separate generally from each other during the process, undergoing completely different entry pathways. We developed a “replication-intercalated labeling strategy” of viral nucleic acid to label the viral nucleic acid during the replication process in host cells, when the membrane would become permeable for labels.28 Since the azide-Cho could be readily labeled with DBCO-derived fluorophores under physiological conditions, simultaneous dual labeling of living viruses in host cells should be feasible by integrating our nucleic acid labeling with this envelope labeling. For this purpose, the biosynthesis and incorporation of azide-Cho containing phospholipids in host cells were carried out. Then the VACV was inoculated into the cells. The [Ru(phen)2(dppz)]2+, where phen = 1,10-phenanthroline and dppz = dipyrido[3,2-α:2′,3′-c]phenazine was added to the medium at 2 h postinfection. The DBCO-Fluor 525, which was confirmed to be no effect on the viability of Vero cells (see Figure S6 in the Supporting Information), was added to the medium at 24 h postinfection. The harvested virions were of the same morphology and size as the control virions (Figure 5D). The fluorescence imaging results showed that (81 ± 3.6)% (mean ± SD) of virions were both [Ru(phen)2(dppz)]2+ and Fluor 525 signal positive (Figure 6). The signal of Fluor 525 could hardly be seen when the duallabeled virus were incubated with both of PLC and Ca2+ (Figure 6), suggesting that azide indeed was modified into the envelope of the virions. They also could recognize their host cells (see Figure S7 in the Supporting Information). The

Figure 6. Fluorescence imaging of the dual-labeled VACV. The virions captured onto the glass slides were treated with Tris buffer solution (TBS) (a−c), TBS added with PLC and Ca2+ (d−f) or TBS added with PLC and EDTA (g−i). The DBCO-Fluor 525 was excited using a 514 nm laser, emitting 550−570 nm fluorescence. [Ru(phen)2 (dppz)]2+ was excited with UV and the fluorescence was collected in the wavelength range of 600−650 nm.

Cho containing phospholipids in host cells could really incorporate into the virus envelope, the harvested virions were labeled and fluorescently imaged. The virions harvested from the azide-modified Vero cells were fixed with heat and incubated with DBCO-Fluor 525. The nucleic acid of the virions was stained with Hoechst 33342. We supposed that the virions would be dual-labeled if the azide was present on the envelope of the virions. Consistent with this hypothesis, nearly (85 ± 4.4)% (mean ± SD) of virions were both Hoechst 33342 and DBCO-Fluor 525 signal positive. On the contrary, there was no signal of Fluor 525 on the control virions (Figure 4A). Because the azide group does not naturally exist in host cells, it can be concluded that the azide-Cho had been successfully biosynthetically bonded and metabolically incorporated into the virions with a high efficiency, thereby the azide-installed virus (azide-virus) is being produced. They were of an intact structure like that of the control virus (Figure 5D). Different viruses have different budding sites including the endoplasmic reticulum, nucleus membrane, golgi, and other membrane organelles.36 Because PC is abundant in most membrane structure, this method is universal for all kinds of enveloped viruses. One of the other important model enveloped viruses, H9N2 influenza viruses, was also successfully installed with azide by this method (see Figure S5 in the Supporting Information). Infectivity Detection of the Azide-Installed Virus. For any functionalization and labeling method for viruses, it is indispensable to ensure the infectivity of viruses. To confirm the infectivity of azide-virus, the specific recognition ability of the azide-virus was detected at first. The azide-virus was labeled with DBCO-Fluor 525. Then it was allowed to incubate with Vero cells at 4 °C for 0.5 h, afterward the unbound virions were washed out. Then, one of the envelope proteins of VACV, H3 G

dx.doi.org/10.1021/ac4008144 | Anal. Chem. XXXX, XXX, XXX−XXX

Analytical Chemistry

Article

titers of control virus and dual-labeled virus were 2.20 × 107 TCID50 mL−1 and 9.02 × 106 TCID50 mL−1, respectively (Figure 5B), which had no significant difference, showing that the dual-labeled virus still retained their infectivity.

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CONCLUSIONS We developed an original method to introduce an azide group to the envelope of the viruses by taking advantage of the hostderived lipid membrane formation mechanism. Such an azide group could be subsequently used to fluorescently label through strain-promoted azide−alkyne cycloaddition. Coupling this strategy with the “replication-intercalated labeling strategy” of viral nucleic acid, we realized simultaneous dual-labeling of living virus in the host cells in the presence of DBCO-Fluor 525 and [Ru(phen)2(dppz)]2+. The whole process of viral labeling performed spontaneously in the viral replication cycle process and additional precise purification of the virus before and/or after the labeling can be avoided. Thus, viral bioactivity and the structure can be kept to the greatest extent. This method is facile, fast, highly efficient, and universal for all dsDNA enveloped viruses, and it will be helpful for dynamic visualization of the virus infection process.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: (+86)-10-68915940. Author Contributions ∥

Li-Li Huang and Gui-Hong Lu contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (973 Program, Grant No. 2011CB933600) and the National Natural Science Foundation of China (Grant No. 20975013). We thank Prof. Zhi-Ke He of Wuhan University for providing the [Ru(phen)2(dppz)]2+ and Prof. Steven R. Kirk from Hunan Normal University for polishing the manuscript.

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REFERENCES

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dx.doi.org/10.1021/ac4008144 | Anal. Chem. XXXX, XXX, XXX−XXX