Integrating Two Efficient and Specific Bioorthogonal Ligation

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Integrating two efficient and specific bioorthogonal ligation reactions with natural metabolic incorporation in one cell for virus dual labeling Li-Li Huang, Kejiang Liu, Qianmei Zhang, Jin Xu, Dongxu Zhao, Houshun Zhu, and Hai-yan Xie Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03043 • Publication Date (Web): 03 Oct 2017 Downloaded from http://pubs.acs.org on October 3, 2017

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Analytical Chemistry

Integrating two efficient and specific bioorthogonal ligation reacreactions with natural metabolic incorporation in one cell for virus dual labeling Li-Li Huang,1a Kejiang Liu,1a Qianmei Zhang,a Jin Xu,a Dongxu Zhao,a Houshun Zhua and Hai-Yan Xie*a a

School of Life Science, Beijing Institute of Technology, Beijing 100081, China

1

Contributed Equally, *Corresponding Author

Author Contributions Li-Li Huang and Kejiang Liu contributed equally to this work.

Corresponding author Hai-Yan Xie School of Life Science, Beijing Institute of Technology, Beijing 100081, China Tel: (+86)-10-68915940; E-mail: [email protected]

ABSTRACT: Though techniques in bioorthogonal chemistry and metabolic incorporation have been developed over the past decade, it remains difficult to integrate different bioorthogonal reactions or metabolic incorporations into one system. In this report, the protein and DNA metabolic incorporations were combined with two bioorthogonal reactions in one cell to develop a facile and universal method for virus dual labeling. Azide group and vinyl group were introduced into the proteins or genomes of viruses, respectively, through the intrinsic biosynthesis of biomolecules, which were subsequently fluorescently labeled via copper-free click chemistry or alkene-tetrazine ligation reactions during natural propagation process in host cells. Both the envelope viruses and capsid viruses could be labeled, and the dual labeling efficiency was more than 80%. The labeled progeny virions were structurally intact and fully infectious, and their fluorescence was strong enough to track single virions.

Metabolic incorporation is a novel strategy to modify and label biomolecules in vitro and in vivo.1-3 To perform metabolic incorporation, cells are cultivated in the presence of nonnatural derivatives carrying a chemical reporter group, which are usually small bioorthogonal and chemical reactive groups and accepted by the intrinsic biosynthetic machinery of cells. Once presented on or in cell, the reporter group-containing biomolecules can be labeled and visualized through bioorthogonal reactions under physiological conditions with high efficiency.4-15 This strategy is highly efficient; meanwhile, the innate function of biomolecules is not remarkably changed.

Among all the bioorthogonal reactions, click chemistry ligations, especially copper-free click chemistry reactions, are most attractive due to their high efficiency in mild conditions. Because of the azide-alkyne "click" cycloaddition reaction capability, azide group has been widely used in identifying de novo-synthesized proteins, glycans and other metabolites.4,5,6,8, 10,11 For example, proteins can be modified by incorporating azidohomoalanine (AHA) instead of natural methionine, and then labeled by fluorescent alkyne.11,16 5-ethynyl-2'-deoxy uridine (EdU) can be incorporated into nucleic acids and detected by fluorescent azide.6,17 Though “click” cycloaddition reaction is commonly used, it is still a challenge to couple two

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or more orthogonal ligations in one cell. Recently, inverse electron demand Diels-Alder (iEDDA) reactions between 1,2,4,5-tetrazines and strained dienophiles or terminal alkenes Scheme 1. Schematic illustration of labeling strategy during virus propagation in the presence of both AHA and VdU. (A) (a) The chemical formulas of L-methionine (Met) and L-azidohomoalaine (AHA). (b) AHA-containing protein was labeled by copper-free click chemistry reaction. (B) (a) The chemical formulas of 2’-deoxythymidine (T) and 5-Viny-2’-deoxyuridine (VdU). (b) VdU-containing nucleic acid was labeled by inverse electron demand Diels-Alder reactions. (C) The cells were first infected with VACV. AHA and VdU were added at 24 h postinfection. After another 12 h infection, Cy5-Tz was added to label the nucleic acids. When the virions were finally assembled and released, DBCO-Fluor 525 was added to label the envelope proteins of VACV for 1 h.

emerged as attractive alternative bioorthogonal reactions. 5vinyl-2'-deoxyuridine (VdU) is the first reported metabolic probe for cellular DNA synthesis that can be labeled through iEDDA with a comparable reaction rate to that of copper-free click chemistry.7 More significantly, the two reactions show hardly any cross-reactivity.18 Therefore, the alkene-tetrazine ligation reaction provides a possible alternative to azidealkyne click chemistry reaction for bioorthogonal chemical labeling. It is uncertain, however, whether these two reactions can be integrated to label different intracellular components with sufficient selectivity and efficiency. Virus labeling, especially multi-labeling, remains a big challenge due to complex viral structures and invasion process. It is well known that viruses are obligate parasites whose vital activities take place exclusively in host cells. Moreover, self-assembly of an intact virion is dependent on viral components being replicated or synthesized. Therefore, we aimed to develop efficient multi-labeling methods to label virions during natural replication and assembling process. By making use of the host cell deriving formation mechanism of viral envelope and the intercalating capability for nucleic acids of [Ru(phen)2(dppz)]2+, we succeeded in labeling fully duplicative envelope viruses by incorporating [Ru(phen)2(dppz)]2+ into DNA and azide group into the envelope of vaccinia virus.12,19 The azide group was subsequently labeled via copper-

free click chemistry.20 Simultaneously virus dual labeling was realized in natural propagation process through this approach. However, only one of the metabolic incorporations was used, and this method could not be applied in capsid viruses. Developing a universal method that combines different metabolic incorporation strategies to label both envelope viruses and capsid viruses is greatly anticipated. In this paper, a universal and efficient dual labeling strategy for viruses was developed, in which two metabolic incorporations were integrated with two bioorthogonal ligation reactions in cells. Taking advantage of intrinsic biosynthetic machinery, the proteins were incorporated with AHA during translation, and nucleic acids were incorporated with VdU during replication under extensively optimized conditions and were then labeled via copper-free click chemistry or iEDDA during the natural viral assembly process (Scheme 1). Fluorescence imaging and dot blotting results clearly showed the success of dual labeling. Both envelope virus and capsid virus could be labeled with a high efficiency. The labeled viruses were structurally intact and fully infectious. They could recognize host cells and induce cytopathogenic effects. Single particle tracking was easily performed.

EXPERIMENTAL SECTION


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Materials and Reagents. The IdU, bis(triphenylphosphine)palladium(II)-dichloride and tributylvinylstannane were purchased from Sigma Aldrich. Vaccinia virus (VACV) Tiantan strain and poliovirus were obtained from Wuhan Institute of Virology, Chinese Academy of Sciences. Vero cells were purchased from Peking Union Medical College Hospital. Dibenzocyclooctynes-derived Fluor 525 (DBCO-Fluor 525, catalog#: A109-5), DBCO-Fluor 568 (catalog#: A107-5) and Cy5 Tetrazine (Cy5-Tz, catalog#: A1019-5) were purchased from Click Chemistry Tools (Scottsdale, AZ). Anti-H3 protein (vaccinia virus) mouse monoclonal antibody (mAb) was from Immune Technology Corp. Alexa 488 conjugated Goat AntiMouse IgG antibody was from Earthox LLC (San Francisco, CA). FITC-phalloidin, Hoechst 33342 were purchased from Beyotime Biotechnology (Shanghai, China). Total protein extraction kit and viral genomic DNA extraction kit were purchased from Solarbio (Beijing Solarbio Science & Technology Co., Ltd). Celllight® actin-GFP/tubulin-GFP, bacmam 2.0 were purchased from Thermo Fisher Scientific Inc. All other chemical reagents were supplied by Beijing Chemical Reagent Company. Synthesis of 5-ivinyl-2'-deoxyuridine (VdU) and Lazidohomoalanine (L-AHA). VdU was synthesized according to reported procedure7 with a little modification. L-AHA was synthesized according to reported procedures.21 Metabolic Labeling of Newly Synthesized DNA with VdU in Vero Cells.7 Vero cells were seeded in glassbottom dishes (NEST Corp), cultured with medium containing variable concentrations of VdU for 48 h. To evaluate background labeling, control cells were treated with 40 µM VdU in the presence of DNA synthesis inhibitor (e.g., 10 µM aphidicolin or 1 µM doxorubicin). Then the VdU-modified cells were washed, treated with 4% PFA, quenched with PBS containing 50 mM glycine and 50 mM NH4Cl, washed with Triton X-100 and denatured using 2 M HCl. After which, they were neutralized with 0.1 M Borax (Na2B4O7 • 10 H2O) and incubated with 5 µM Cy5 Tetrazine (Cy5-Tz). Cellular DNA was stained with Hoechst 33342 and imaged by Leica TCS SP5 laser scanning confocal microscope, Cy5-Tz was excited with a 633 nm laser; emission was collected by photomultiplier tubes in the range of 650-700 nm. Hoechst 33342 was excited with UV, emitting 431-496 nm fluorescence. Metabolic Labeling of Newly Synthesized Proteins with AHA in Vero Cells.11 Vero cells were seeded in glassbottom dishes (NEST Corp), cultured with methionine-free medium for 24 h, and then transferred into methionine-free medium containing 10% FBS (basic medium), supplemented with 1 mM or 10 mM AHA, and incubated for another 24 h, with AHA-modified Vero cells obtained. To evaluate background labeling, the control cells were treated with 10 mM AHA in the presence of protein synthesis inhibitor (e.g., 10 µM anisomycin or puromycin). To detect incorporated AHA, the AHAmodified cells were fixed with 4% (w/v) paraformaldehyde (PFA), and then reacted with 10 µM DBCO-Fluor 525 for 1 h, washed with Tris buffer solution (TBS), 0.5 M NaCl, and again TBS. In order to label the microfilament cytoskeleton of cells, 5 µg/mL FITC-phalloidin was added to the cells, followed by incubation for 45 min, washed twice with PBS, The nucleic acids of the cells was stained with Hoechst 33342. After washing, the cells were imaged by laser scanning confocal microscope (Leica TCS SP5). The DBCO-Fluor 525 was

excited with a 543 nm laser; emission was collected by photomultiplier tubes in the range of 550-570 nm. FITCphalloidin was excited using a 488 nm laser; emitting 500-520 nm fluorescence. Hoechst 33342 was excited with UV, emitting 431-496 nm fluorescence. Flow Cytometry Analysis of Viral Infectivity. Vero cells were seeded in 6-well culture dishes (2.6 × 105 cells/well) and incubated for 24 h. Then they were infected by VACV at MOI (multiplicity of infection, ratio of infectious virus particles to cells) of 2, with AHA or VdU at different concentrations in the culture medium 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. Dual labeling Vaccinia Virus with AHA and VdU. Vero cells were maintained in DMEM containing 10% FBS at 37 °C until they reached 70% confluency, then the medium was changed into methionine-free medium supplemented with 10% FBS and incubated for 24 h. Remove the medium and rinse with PBS. Then they were infected by VACV at MOI of 1 in methionine-free medium for 24 h. After which, 400 µM AHA and 40 µM VdU were added and incubated for 12 h. Subsequently, viral nucleic acid was labeled by 5 µM Cy5-Tz at 37 °C for another 12 h. After three rounds of freezeingthawing, the cell debris was removed. The viral envelope proteins were labeled by 5 µM DBCO-Fluor 525 at 37 °C for 1 h. The virus was finally layered on to a 36% sucrose cushion and centrifuged at 80,000 × g with a Beckman rotor SW40. The resulting virus pellet was suspended in PBS and layered onto a continuous 40% to 60% sucrose gradient and centrifuged at 58, 000 × g. The clearly visible virus band was collected and washed with PBS. The final virus pellet was suspended in PBS and stored at -80 °C. Fluorescence Colocalization Assay of the Virus on the Glass Slides. The purified control virus or modified virus was dropped onto slides (22 mm × 22 mm, Citoglas) coated with anti-H3 protein (VACV) mouse mAb for 1 h. The excessive viruses were washed out with 0.01 M PBS (pH 7.2). Then the virus was treated with heat. After being blocked with 2% (w/v) bovine serum albumin, the AHA was labeled with 10 µM DBCO-Fluor 525 for 1 h; the VdU was labeled with 5 µM Cy5-Tz. The viral nucleic acids were stained with Hoechst 33342. DBCO-Fluor 525 was excited using a 543 nm laser, emitting 550-570 nm fluorescence. Hoechst 33342 was excited with UV, emitting 431-496 nm fluorescence. Cy5-Tz was excited using a 633 nm laser, emitting 650-700 nm fluorescence. Transmission Electron Microscope (TEM) Imaging. Control virus or dual labeled virus was dropped onto a carbon coated copper grid. After 2 min, unabsorbed viruses were 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. Immunofluorescence Analysis and Stochastic Optical Reconstruction Microscopy (STORM) Imaging of Dual labeled Vaccinia Virus. Control virus or dual labeled virus was dropped onto chamber slides (Lab-Tek® Chamber Slide™ System) coated by poly-L-lysine. The excessive viruses were washed out. After being blocked with 2% (w/v) BSA, the



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samples were incubated with anti-H3 protein (vaccinia virus) mouse monoclonal antibody, followed by goat anti-mouse Alexa 488 conjugate. The samples were rinsed with PBS between two successive steps. Alexa 488 was excited with 488 nm laser, emitting 500-530 nm fluorescence. DBCO-Fluor 568 was excited using a 543 nm laser, emitting 560-590 nm fluorescence. Cy5-Tz was excited using a 633 nm laser, emitting 650-700 nm fluorescence. For STORM imaging, DBCO-Fluor 568 was detected with 561 nm laser and the signal was collected through a 607/73 nm band-pass filter. Cy5-Tz was detected with 640 nm laser and the signal was collected through a 680/40 nm band-pass filter. Dot Blotting. The proteins and nucleic acids of vaccinia virus were purified by total protein extraction kit and viral genomic DNA Extraction Kit according to the manufacturer’s protocols. The same concentrations of AHA-modified or control viral proteins were individually mixed with 1 µL DBCO-Fluor 568 for 1 h, then the mixtures were dropped on PVDF membrane (0.22 µm, Millipore) after being treated with methanol. Subsequently, the membrane was dried at 57 °C for 10 min, rinsed with PBS. Similarly, the same concentrations of VdUmodified or control viral nucleic acids were respectively mixed with 1 µL Cy5-Tz for 1 h, then the mixtures were absorbed on nitrocellulose membrane (0.45 µm, Millipore), dried at 57 °C and rinsed with PBS. Fluorescence of DBCO-Fluor 568 and Cy5-Tz were visualized using a DNR Bio-Imaging Systems (MF-ChemiBIS 3.2). Dynamic Tracking of Virions. Vero cells were plated on chamber slides and grew to 70% confluence. Then the cells were transfected with 2 µL of actin-GFP and tubulin-GFP. After 18 h transfection, the cells were incubated with dual labeled VACV. The dynamic behaviors of virions were fluorescently tracked with a spinning-disk confocal microscope (Andor Revolution WD), equipped with an Olympus IX 81 microscope, a Nipkow disk-type confocal unit (CSU 22, Yokogawa), a CO2 online culture system (INUBG2-PI), and an electron microscope charge-coupled device (EMCCD) (Andor iXon DV885K single photon detector). The cytoskeleton was labeled by CellLightTM Actin-GFP and CellLightTM Tubulin-GFP, viral nucleic acids and envelope proteins were individually labeled by Cy5-Tz and DBCO-Fluor 568.

RESULTS AND DISCUSSION DISCUSSION Modification and labeling the nucleic acids of cells. The analogue deoxythymine, 5-vinyl-2’-deoxyuridine (VdU) (Fig. S1), can be incorporated into the genomes of replicating cells by endogenous enzymes.7 When variable doses of VdU were individually left to incubate with Vero cells, which were used as host cells for vaccinia virus (VACV) Tiantan strain, for 48 h, the cells were stained with tetrazine derived Cy5 (Cy5-Tz) and imaged. Concentration-dependent nuclear staining was observed, and the nucleus was clearly visible when 40 µM of VdU was used. Whereas the nuclear staining was greatly decreased if a DNA polymerase inhibitor (Aphidicolin) or DNA topoisomerase inhibitor (doxorubicin hydrochloride, hereafter referred to as Dox) was added in conjunction with the VdU, confirming that newly synthesized DNA was specifically labeled (Fig. 1A, 1B). CCK-8 detection results showed that for cells treated with VdU for 48 h, which was the time needed for

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virus propagation, more than 90% of cells remained viable if the concentration of VdU was lower than 40 µM (Fig. 1C).

Figure 1. Fluorescence labeling (A), flow cytometry assay (B) and viability detection (C) of VdU-modified Vero cells. Cells were treated with different concentrations of VdU or VdU accompanied with DNA synthesis inhibitor for 48 h, followed by fixation, DNA denaturation, and incubation with 5 µM Cy5-Tz, total cellular DNA was stained with Hoechst 33342. Scale bars: 25 µm. Error bars represented the standard deviation from three replicated experiments. * p＜0.05.

Incorporating VdU into the nucleic acids of virus. Though the metabolic incorporation of VdU in cells was feasible, it was not sure if it could occur in propagating viruses, since the enzymes for viral genome replication is not the same as those for cells. To verify the feasibility, varying doses of VdU were added to Vero cells during VACV propagation and incubated for 48 h. A recombinant VACV carrying a green fluorescent protein (GFP) reporter gene was used in our experiments. By quantifying Vero cells expressing GFP after VACV infection, infectivity can be calculated.22-23 Flow cytometry analysis results showed that the replication ability of VACV was maintained at about 80% if the VdU concentration was lower than 40 µM (Fig. S2A). To further reduce the effects of VdU on virus infectivity, the VACV was first propagated in normal media for 24 h and another 24 h after 40 µM VdU addition. It was found that the mean fluorescence intensity of GFP+ cells was similar to that of control VACV that was propagated in normal media for 48 h (Fig. 2A and 2B), suggested that the infectivity could be well kept in this condition. The virions were harvested and labeled by Cy5-Tz.24 The microscopy imaging showed that more than 85% of the virions could be labeled (the labeling efficiency was calculated by colocalization analysis of Image-Pro Plus software), while the wild virions hardly can, indicated that reactive VdU could be recognized by endogenous enzymes and incorporate into DNA of newly replicated virions (VdU-VACV), and vinyl modified DNA could be assembled into virions as normal (Fig. 2C).


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changes happened in the host cells, such as the expansion of cells and damage of membrane integrity, which is due to the budding of initial progeny virus particles, resulting in the dramatically increased cellular uptake level of small molecules,25 at the same time, the alkene-tetrazine ligation has a rapid reaction rate.7 Third, that labeling should not impede the assembling and compromise viral infectivity. For these reasons, we optimized the concentration of Cy5-Tz, which was determined to be 5 µM and confirmed to be of no obvious influence on the viability of host cells (Fig. S4, S5). Then 5 µM Cy5-Tz was added to the media and incubated for 12 h after the propagation had lasted for 36 h, so that enough nucleic acids were available for labeling and there was sufficient time for labeling. The fluorescence imaging results showed that 81.7 ± 1.9% (mean ± SD) of the harvested virions were labeled with Cy5Tz (Fig. S2 B). When they were left to incubate with host cells, they could induce cytopathogenic effects (CPE) and express GFP report gene as control virions (Fig. S6), indicating the infectivity was well maintained. These results together verified the success of in situ labeling of viral nucleic acids.

Figure 2. Fluorescence imaging and flow cytometry assay of VACV-infected Vero cells and VdU-modified virions. (A) Vero cells were infected by VACV for 48 h (a’-c’), or 24 h and then 40 µM VdU was added in the following 24 h (a-c). Scale bars: 200 µm. (B) (a) Flow cytometry plots of Vero cells infected by VACV for 48 h or 24 h and then 40 µM VdU was added in the following 24 h before flow cytometry assay of GFP+ cells. (b) Mean fluorescence intensity of GFP+ cells calculated from panel a. Data from three independent experiments were normalized as a percentage of control. *p＜0.05; **p＜0.01. (C) VdU-modified virions (a-c) and control virions (a’-c’) were captured onto the glass slides. After being treated with heat, Cy5-Tz was dropped and incubated for 1 h. The nucleic acids of the virions were stained with Hoechst 33342. Scale bars: 25 µm.

Labeling the nucleic acids of virus through alkene– tetrazine ligation reaction in situ. We then tried to in situ label alkene-modified viral nucleic acids before assembly. This was of special importance because it is difficult for labeling reagents penetrating assembled viral particles and thus accessing viral DNA (Fig. S3). The in situ labeling feasibility of our research depended on three factors: first, the addition of Cy5-Tz should not affect both the viability of host cells and replication of viruses. Second, Cy5-Tz should can enter host cells and interact with the incorporated VdU before virus assembling.25-26 This was possible since many pathological

Figure 3. Viability detection and fluorescence labeling of AHAmodified Vero cells. (A) CCK-8 detection of Vero cells incubated with AHA. (B) Metabolic labeling of newly synthesized proteins in Vero cells using 1 mM (a-e) or 10 mM (a’-e’) AHA. The newly synthesized proteins were labeled with DBCO-Fluor 525; the DNA and microfilament of cells was stained with Hoechst 33342 and FITC-phalloidin, separately. The control cells received identical treatment except being incubated with 10 mM Met instead of AHA (a1-e1). Scale bars: 75 µm.

Modification and labeling the proteins of cells. Azidohomoalanine (AHA) can be taken up by cells and incorporates into nascent proteins. The small bioorthogonal chemical reactive



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azide group can subsequently be labeled via copper-free click chemistry.11,27 To assess the influence of AHA on the viability of cells, Vero cells were incubated with AHA added or normal media, and then detected through CCK-8. No significant viability difference between AHA treated and control cells was detected, even at a high (25.6 mM) AHA concentration (Fig. 3A), indicating the robust toleration of AHA by the cell. To confirm whether AHA was incorporated into newly synthesized cellular proteins, DBCO-Fluor 525 was added to cells and incubated for 1 h. The microscopy imaging results showed that strong fluorescent signal was detected for AHA treated cells while no signal was detectable for the control (Fig. 3B). Furthermore, the signal greatly increased with the increasing of AHA concentration, even the nucleus could also be stained when the concentration was 10 mM. And the signal clearly decreased when living Vero cells were treated with 10 mM of AHA in the presence of protein synthesis inhibitor puromycin or anisomycin for 24 h (Fig. S7), suggesting that this procedure labeled newly synthesized proteins with high specificity. Modification and labeling proteins of both enveloped viruses and capsid viruses. Next, we explored the possibility of incorporating AHA into virions and then labeling virus during propagation. The key for this was the conflict between incorporation efficiency and infectivity maintaining, which was attempted to mitigate by optimizing reaction conditions. To assess the effects of AHA on the virus infectivity, Vero cells individually incubated in media supplemented with different concentrations of AHA were infected by the same amount of VACV, and then GFP expression was measured by flow cytometry assay. As could be seen, the infectivity could be maintained at more than 80%, as long as the concentration of AHA was no higher than 0.4 mM (Fig. S8 A). As for the incubation time, it was found that if VACV propagated in normal media for 24 h, and then 0.4 mM AHA was added and incubated for another 24 h, the replication capability was similar to the control, which had propagated in normal media for 48 h (Fig. S8 B and S8 C). After having propagated for 48 h, 5 µM DBCOFluor 525, which had been tested to have no evident toxicity (Fig. S4) and the highest labeling efficiency to AHA-virus (Fig. S9), was added. The specific and on site labeling efficiency of the virions (Fig. 4A) was analyzed by Mander’s Colocalization coefficients for channel 1 (M1) and channel 2 (M2).28-30 The tMr, tMg and intensity correlation quotient (ICQ) values were 0.89 ± 0.05, 0.91 ± 0.07 and 0.39 ± 0.04 (mean ± standard deviation), respectively (Figure 4C a). These results suggested that almost 90% signals of DBCO-Fluor 525 strongly colocalized with Hoechst 33342. The labeled virions could induce CPE and GFP expression comparably to the control virus in various durations (Fig. S10), which means that the infectivity of AHA labeled virions was well preserved. This did not surprise us, since the virions replicated as normal in the optimized conditions and DBCO-Fluor 525 was added after 48 h of viral propagation, when most of the virions were fully assembled and released. Moreover, it has been shown that infectivity is not affected if the virus surface is modified through copper-free click chemistry.12,20 Because this labeling method is based on the modification of proteins, which all viruses have, it should be universal to both envelope viruses and capsid viruses. Actually, when one of the capsid viruses, poliovirus (PV), was incubated with AHA during propagation at optimized concentration for 48 h, followed with 5 µM

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DBCO-Fluor 525, the harvested virions were also labeled with a high efficiency (Fig. 4B), the tMr, tMg and ICQ values were 0.89 ± 0.06, 0.96 ± 0.11 and 0.31 ± 0.03 (mean ± standard deviation), respectively (Figure 4C b).

Figure 4. Fluorescence colocalization imaging of AHAincorporated VACV (A) and PV (B). Azide-virions (a-c) and control virions (a’-c’) were separately captured onto the glass slides. After being treated with heat, the proteins were labeled with DBCO-Fluor 525 or DBCO-Cy5. The nucleic acids of virions were stained with Hoechst 33342 or SYTO 82. The green signal was pseudo color of nucleic acids which made merge image clear. Scale bars: 25 µm. (C) Histograms of tMr, tMg and ICQ values obtained from random fields of c in A and B. tMr (tMg) value indicated the percentage of the red (green) signals colocalized with green (red) signals in the corresponding thresholded image.

Simultaneously dual labeling of virions via two bioorthogonal ligation reactions. The above results indicated that envelope/capsid and DNA could be individually labeled during propagation. We next considered the possibility of selectively labeling both viral DNA and proteins by coupling VdUtetrazine ligation and AHA-DBCO cycloaddition. To this end, we first confirmed the chemical orthogonality of the two reac-


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tions. Vero cells were successively treated with VdU and DBCO-Fluor 525 or AHA and Cy5-Tz, and then detected by fluorescence imaging. The results did not show any crossreactivity, since DBCO-Fluor 525 did not label VdU-treated cells and AHA-treated cells were not stained by Cy5-Tz (Fig. S11). Then, optimized concentrations of VdU (40 µM) and AHA (0.4 mM) were added to the media in sequence during virions propagation, followed by the addition of Cy5-Tz and DBCO-Fluor 525. The proteins and DNA of the harvested virions were purified and detected by dot blotting. It was found that they could be successfully labeled with DBCOFluor 525 or Cy5-Tz, while those of the control virions hardly could (Fig. 5A b). The super-resolution stochastic optical reconstruction microscopy (STORM) imaging result showed that the core of the virion was yellow and the peripheral was green (Fig. 5B). Then, immunofluorescence experiments were performed and the H3 protein of VACV was further labeled with Alexa 488 tagged antibodies. As shown in Figure S12, DBCO-Fluor 525 and Cy5-Tz signals well overlaid with Alexa 488, suggesting that DBCO-Fluor 525 and Cy5-Tz were indeed coupled with VACV. These results confirmed the success of dual labeling. The labeling efficiency was assessed according to the fluorescence colocalization results (Fig. 5B). Then, the colocalization of DBCO-Fluor 525 and Cy5-Tz was performed by intensity correlation analysis (ICA).28-30 Both the dot clouds distributed on the right side (Figure 5d, e) and most particles appeared yellow in the product of the differences from the mean (PDM) image (Figure 5f), indicated that most signals of DBCO-Fluor 525 and Cy5-Tz colocalized with each other. The tMr, tMg and ICQ values were 0.88 ± 0.05, 0.83 ± 0.01 and 0.32 ± 0.05 respectively, which indicated that more than 83% viruses were dual labeled.

Figure 5. Characterization of dual labeled VACV. (A) TEM imaging (a) and Dot blotting assay (b). Scale bars: 100 nm; (B) Fluo-

rescence colocalization imaging of dual labeled virions (a-c) and control virions (a’-c’), STORM image of c in the upper right corner; (d, e) Intensity correlation plots (ICP) of green and red signals in (a) and (b). (f) PDM image for visualizing the extent of colocalization. (g) Histograms of tMr, tMg and ICQ values obtained from random fields.

The dual labeled virions were of the same morphology and size as the control (Fig. 5A a). If they were left to incubate with Vero cells, they could bind to cell surface and then induce CPE. Cytoplasmic GFP was detectable at 12 h postinfection. Moreover, the CPE was similar to that of cells infected by control virus (Fig. S13 and Fig. 6), indicating that the dual labeled virus was still infectious. This should be attributed to the following points: on one hand, AHA and VdU were almost the same as natural methionine or thymidine in structure since the azide and vinyl groups were very small. Accordingly, they almost behaved as native biomolecules. On the other hand, the incorporation of AHA and VdU spontaneously occurred during intrinsic biosynthesis and natural propagation. Moreover, copper-free click chemistry and alkene-tetrazine ligation are highly biocompatible and bioorthogonal. Thus, the infectivity should be maintained. Tracking the dynamic behavior of dual labeled virions in Vero cells transfected with actin-GFP and tubulin-GFP, the movement of individual virions could clearly be discerned. Many virions initially stayed on the cell surface. Over time, they gradually moved into the cytoplasm along the cytoskeleton (Movie S1), showing that this technique could be used to decipher the dynamic infection process of viruses. This was of special significance since the infection mechanism of the VACV Tiantan strain we studied here is still unclear.

Figure 6. Fluorescence colocalization imaging of VACV on cells. (A) Vero cells were co-incubated with dual labeled virus (a-e) or control virus (a’-e’) at 4 °C for 0.5 h, and then unbound virions



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were washed out. The nucleic acids of cells were stained with Hoechst 33342. (f, g) ICP of green and red signals in (c) and (d), (h) PDM image for visualizing the extent of colocalization. Scale bars: 25 µm. (B) flow cytometry assay of Vero cells infected by VACV, AHA or/and VdU modified VACV, DBCO-Fluor 525 or/and Cy5-Tz labeled VACV for 12 h.

CONCLUSIONS In summary, we report a dual labeling method universal for both envelope and capsid viruses, which integrates two natural metabolic incorporations with two bioorthogonal and rate comparable ligation reactions. Taking advantage of the intrinsic biosynthesis of biomolecules, AHA and VdU were spontaneously incorporated into the proteins or nucleic acids of duplicating viruses, and then were labeled through copperfree click chemistry or alkene-tetrazine ligation during natural propagation process. The dual labeling efficiency was high, and the viral structure and infectivity was well maintained. The labeled virions could recognize their host cells, and then, the dynamic invasion behavior could be tracked, which will prove to be useful for deciphering viral entry mechanism.

ASSOCIATED CONTENT Supporting Informatio The Supporting Information is available free of charge on the ACS Publications website. Detection of cells viability and viral infectivity over time; fluorescence colocalization imaging of AHA- and/or VdU incorporated virions (PDF)

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Author Contributions All authors have given approval to the final version of the manuscript. 1

Li-Li Huang and Kejiang Liu contributed equally to this work. Notes

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The authors declared that no competing interest exists.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 21422502, No. 21372028).

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Integrating Two Efficient and Specific Bioorthogonal Ligation

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