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Simultaneous Visualization of Parental and Progeny Viruses by a Capsid-Specific HaloTag Labeling Strategy An-An Liu, Zhenfeng Zhang, En-Ze Sun, Zhenhua Zheng, ZhiLing Zhang, Qinxue Hu, Hanzhong Wang, and Dai-Wen Pang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b06438 • Publication Date (Web): 31 Dec 2015 Downloaded from http://pubs.acs.org on January 3, 2016

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Simultaneous Visualization of Parental and Progeny Viruses by a Capsid-Specific HaloTag Labeling Strategy An-An Liu1, Zhenfeng Zhang2, En-Ze Sun1, Zhenhua Zheng2, Zhi-Ling Zhang1, Qinxue Hu2, Hanzhong Wang3,*, Dai-Wen Pang1,*

1

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education),

College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, The Institute for Advanced Studies, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, P.R. China 2

State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences,

Wuhan 430071, P.R. China 3

Key Laboratory of Special Pathogens and Biosafety, Center for Emerging Infectious Diseases,

Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, P.R. China *

Corresponding authors: D.-W.P. (E-mail: [email protected]) and H.W. (E-mail:

[email protected]).

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Abstract Real-time, long-term single particle tracking (SPT) provides us an opportunity to explore the fate of individual viruses toward understanding the mechanisms underlying virus infection, which in turn could lead to the development of therapeutics against viral diseases. However, the research focusing on the virus assembly and egress by SPT remains a challenge, because established labeling strategies could neither specifically label progeny viruses nor make them distinguishable from the parental viruses. Herein, we have established a temporally controllable capsid-specific HaloTag labeling strategy based on reverse genetic technology. VP26, the smallest pseudorabies virus (PrV) capsid protein, was fused with HaloTag protein and labeled with HaloTag ligand during virus replication. The labeled replication-competent recombinant PrV harvested from medium can be applied directly in SPT experiments without further modification. Thus, virus infectivity, which is critical for the visualization and analysis of viral motion, is retained to the largest extent. Moreover, progeny viruses can be distinguished from parental viruses using diverse HaloTag ligands. Consequently, the entire course of virus infection and replication can be visualized continuously, including: virus attachment and capsid entry; transportation of capsids to the nucleus along microtubules; docking of capsids on the nucleus; endonuclear assembly of progeny capsids; and the egress of progeny viruses. In combination with SPT, the established strategy represents a versatile means to reveal the mechanisms and dynamic global picture of the life cycle of a virus.

Keywords Virus, HaloTag, Capsid, Labeling, Tracking

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Understanding viral pathogeneses is crucial for diagnosis and treatment of viral diseases. In recent years, single particle tracking (SPT) technique has become a powerful means capable of tracing the journey of a virus in living cells, defining the virus-host interactions as well as revealing previously unobservable infection processes.1-3 To exploit the superiority of SPT, fluorophores should be efficiently labeled to viruses without impairing virus infectivity. In general, viruses could be labeled by nonspecifically conjugating fluorophores via chemical reactions.1, 4-6 By contrast, genetic engineering is more effective in labeling a virus at specific sites with hereditability.1 Although fluorescent proteins (FPs) are commonly employed as the tag in this strategy, their relatively poor photostability and long maturation period limit their application in real-time, long-term tracking. HaloTag protein, an engineered derivative of bacterial dehalogenase, is a promising alternative fluorescent tag. Based on a rapid, specific, and irreversible covalent reaction between HaloTag and its fluorescent ligands at a 1:1 molar ratio, the protein of interest fused with HaloTag can be illuminated.7 Unlike FPs, HaloTag protein is not fluorescent until conjugating with a fluorescent ligand, making the labeling temporally controllable. Pseudorabies virus (PrV), a pathogen of swine resulting in devastating disease and huge economic losses worldwide, has been studied for several years as a model of herpesvirus infection and neurotropism.8 PrV is composed of an envelope, tegument proteins, an icosahedral capsid and a large linear dsDNA genome.8 Compared with other components, capsid is an ideal target for labeling and tracking to reveal the life cycle of PrV because it is continuously associated with the genome during intracellular transport, progeny virus assembly and egress. Herein, we constructed a capsid-specific HaloTag labeled chimeric recombinant PrV through reverse genetic technology. The recombinant PrV harvested from medium can be applied directly in SPT experiments without further modification. Thus, virus infectivity, which is critical for the visualization and analysis of viral motion, is retained to the largest extent. Moreover, progeny viruses can be distinguished from parental viruses using diverse HaloTag ligands, which is not achievable for previously established strategies. Consequently, the entire course of virus infection and replication can be visualized continuously, including: virus attachment and capsid entry; transportation of capsids to the nucleus along microtubules; docking of capsids on the nucleus; 3


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endonuclear assembly of progeny capsids; and egress of progeny viruses. In combination with SPT, the established strategy represents a promising tool to reveal the dynamic global picture of the life cycle of a virus.

Results and Discussion Characterization of VP26-Specific HaloTag Labeled Recombinant PrV. Capsid protein VP26, encoded by UL35 gene, was determined as the target protein for labeling because approximately 900 copies of VP26 per PrV virion would ensure the labeling efficiency and image quality.1, 8 Moreover, previous studies have already demonstrated that fusing a relatively large GFP to VP26 would not affect the assembly and infectivity of PrV.9-10 As shown in Scheme 1, the UL35-HaloTag recombinant gene was inserted into viral genome through transposition, generating the recombinant viral genome.11-12 Thereafter, VP26-specific HaloTag labeled recombinant PrV (rPrV_HT) was produced through transfecting Vero cells with the recombinant viral genome. Fluorescent HaloTag ligands-labeled rPrV_HT was generated by adding the HaloTag ligands to the medium during the virus propagation. To examine whether HaloTag was integrated into the recombinant virus, virus cultivation was successively passaged for four times and the purified rPrV_HT from the fifth passage was analyzed using Tricine-SDS-PAGE.13 As for why rPrV_HT from the fifth passage was used, there were two major reasons. On one hand, the titer and amount of the recombinant virus rescued by reverse genetic technology could be increased to meet the requirements of western blot following successive passages. On the other hand, the rPrV_HT from the fifth passage could facilitate the examination of stable expression and incorporation of VP26-HaloTag into recombinant virus for successive passages, which is critical for the simultaneous labeling and visualization of parental and progeny viruses. In the presence of antibody against VP26, both VP26 (11.5 kD) and VP26-HaloTag fusion protein (44.5 kD) were detected in rPrV_HT, whereas only VP26 was detected in wtPrV (Figure 1A, left). The same samples were also probed with anti-HaloTag antibody. Only VP26-HaloTag fusion protein was detected in rPrV_HT, whereas no band in wtPrV (Figure 1A, right). According to the intensity of the bands of VP26-HaloTag and

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Scheme 1. Schematic diagram for the generation of rPrV_HT. HaloTag gene was inserted into the C-terminal of the UL35 gene amplified from pBecker3 to comprise one open reading frame, and

this

recombinant

gene

was

cloned

into

pFB-CMV

vector.

The

resultant

pFB-CMV_UL35-HaloTag was transformed into modified competent E. Coli cells containing a shuttle vector that encodes the entire genome of PrV and a helper plasmid for transposition. The CMV_UL35-HaloTag expression cassette from pFB-CMV_UL35-HaloTag was specifically inserted into pBeZF1 through transposition, producing a recombinant bacmid named pBeZF1_UL35-HaloTag. Afterwards, Vero cells were transfected with the recombinant bacmid to generate the capsid-specific HaloTag labeled recombinant PrV. During the propagation, HaloTag ligands were added into the medium to label VP26-HaloTag fusion proteins, generating the fluorescent-ligand labeled recombinant PrV.

VP26, the ratio of VP26 to VP26-HaloTag that was incorporated into viral capsid was 2:1. Since there are 900 copies of VP26 in one PrV virion, approximately one third (around 300 copies on average) of VP26 protein were labeled with HaloTag in a chimeric rPrV_HT. These results were also evidenced by immunoelectron micrographs of the purified viral capsids. The immune-gold particles only attached to the capsid of rPrV_HT but not to that of wtPrV (Figure 1B). Moreover, the capsid morphology of rPrV_HT did not show any distinction in comparison with that of wtPrV, indicating that HaloTag was site-specifically and effectively incorporated into rPrV_HT for several (at least five) passages, and it exerted negligible effects on the structure and assembly of the viral capsid. Virus infectivity was determined using one-step growth curve assay. The growth kinetics of rPrV_HT was similar to that of wtPrV (Figure 1C), indicating that the infectivity of rPrV_HT is as robust as that of wtPrV. The labeling efficiency was estimated by fluorescence colocalization assay of viral capsid with nucleic acid. Because of the generation of empty enveloped capsids (without genome), light particles (without viral nucleocapsids) and immature viruses, the labeling efficiency was approximately 80% (Figure 1D, top panel).14 Moreover, the non-specific labeling of HaloTag ligands was negligible (Figure 1D, bottom panel, 1E and S1). Above results demonstrated that the viral capsids were specifically and efficiently labeled with HaloTag without 6


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impairing viral structure and infectivity, and the resultant rPrV_HT was an appropriate candidate for visualizing the life cycle of PrV by SPT.

Figure 1. Characterization of rPrV_HT. A) Western blot analysis of rPrV_HT and wtPrV. B) Immunoelectron micrographs of purified viral capsids of rPrV_HT and wtPrV. HaloTag proteins were detected using anti-HaloTag pAb and gold-conjugated secondary antibody. C) One-step growth curves of wtPrV and rPrV_HT in Vero cells. Each data point represents mean ± standard deviation (N=3). D) Fluorescence colocalization assay of rPrV_HT with wtPrV. Viral genomes and capsids were labeled with SYTO 13 and TMR, respectively. E) Histogram of the statistical analysis of colocalization of rPrV_HT shown in (D).

Virus Attachment and Entry. The life cycle of PrV initiates by attaching itself to the cell surface followed by diffusing on the cell membrane before entering the host cell (Stage I, Figure 2A, Movie S1). We tracked and analyzed the trajectories of the viruses that internalized. The initial motion of virus attachment was slow, and restricted (Figures 2B and 2C, blue).15 Upon 7


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entering the cell (membrane fusion),8 the viral capsid experienced a relatively slow and directed movement in the cytoplasm (Figures 2B and 2C, red).15 These results indicate that viruses experience a slow diffusion before the internalization, and do not directly enter the cell after they bind to their receptor on the plasma membrane.

Figure 2. Visualization of virus entry and transport in the cytoplasm by parental rPrV_HT. A, D) Confocal images of rPrV_HT entered Vero cells (A) and transported in the cytoplasm (D). Yellow lines indicate the typical trajectories of viruses. Dashed lines represent the plasma membrane. N indicates the position of nuclei. B, E) The instantaneous velocity of the virus shown in (A) and (D). Blue and red parts represent the slow diffusion of viruses in the cell periphery region and the rapid movement of viruses in the cytoplasm, respectively. C, F) MSD-time plots of viral movement. The colors are in accordance with those in (B) and (E). Scale bar: 2 µm. G-J) Orthogonal slice images of the fate of viral capsids (red) at 9 hours post infection (hpi). Nuclei of living Vero cells (G) or ultrathin cryosection of infected Vero cells (H) were stained with Hoechst33342 (blue). Nuclei of fixed Vero cells were immunostained with anti-Lamin A/C mAb (I) or anti-NPC mAb (J) (green). Arrow heads indicate the viral capsids. Scale bar: 10 µm.

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Directed Motion of PrV Along Microtubules in the Cytoplasm. Most viral capsids that transported toward the nucleus experienced a rapid and directed movement in the cytoplasm (Stage II, Figures 2D-2F, Movie S2), and we further demonstrated that this kind of movement depended on microtubules through dual-color observation of rPrV_HT and microtubules (Figure S2).16-19 Moreover, in some cases, viral capsids experienced a rapid and bidirectional directed movement in the cytoplasm (Figure S3), implying that both minus/plus-end-directed motor dynein and kinesin participate in the transportation of the viral capsids along microtubules.20 Capsid Docking on the Nucleus to Release the Viral Genome. Viral capsids were eventually transported to the cell nucleus where the viral genome was released (Stage III).21 To investigate the fate of viral capsids, cell nuclei were labeled (Figures 2G-2J), and our results indicate that viral capsids stayed on the surface of the nuclear for several hours, and did not enter the nuclear throughout the observation. These results are consistent with the fact that viral capsid (diameter is approximately 100 nm, Figure 2B) is too large to cross the nuclear pore (the maximum of particle diameter is 39 nm).22 Assembly of Progeny Viral Capsids in the Nucleus. Assembly of progeny viral capsids occurred in the cell nucleus (Stage IV, Figure 3A, Movie S3). To label the progeny capsids and make them distinguishable from their parents, HaloTag ligand R110 was added to the medium during the observation. Excitation and emission wavelengths of R110 were different from TMR, therefore they were used in pairs for simultaneous dual-color visualization. Both orthogonal confocal imaging (Figure 3C) and ultrastructural analysis (Figure 3B) of the infected Vero cells demonstrated that viral capsids became observable in nuclei, indicating that the nucleus was the place where capsid proteins assembled into capsid. Namely, newly synthesized VP26-HaloTag fusion proteins were labeled in the cytoplasm, and then transported to the nucleus for the assembly. These assembled capsids performed a slow and restricted diffusion in the nucleus (Figures 3D, 3E), and the mean value of diffusion coefficient was 2.11×10-3 µm2/s (Figure 3F). This is the first time that the restricted motion of assembled progeny capsids in the nucleus was visualized and dissected.

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Figure 3. Assembly of progeny capsids in the nucleus. A) A typical confocal image of assembled progeny viral capsids (green) in the nucleus (white dashed line). Parental rPrV_HT could also be observed (red). B) TEM images of ultrathin section of infected Vero cells at 9 hpi. Higher magnification image indicates that the assembled capsids in the nucleus. C) Orthogonal image of progeny capsids in the nucleus. D) The instantaneous velocity of the virus shown in (A, arrow head). Inset is the trajectory of this virus. E) MSD-time plot of the progeny viral capsids in the nucleus. F) Statistical analysis of diffusion coefficient of the progeny capsids in the nucleus. The black triangle indicates the mean value of diffusion coefficient. Scale bar: 10 µm.

Nuclear Egress and Transportation of Progeny Viral Capsids in the Cytoplasm. Mature capsids were exported to the cytoplasm for further assembly (Stage V, Figure 4A, Movie S4). Depending on the motion mode of the progeny viral capsids in the cytoplasm, their behaviors were divided into two typical types: directed motion (Figures 4B, 4D, blue) which suggested that the transportation of progeny viruses was likely assisted by the cytoskeleton23 and restricted motion (Figures 4B, 4D, red) which implied that progeny capsids were trapped by some cellular organisms, such as trans Golgi network, for further assembly.8 The relatively low velocity (approximately 0.2 µm/s, Figure 4C) of the movement was likely caused by the interaction 10


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between viral capsid and the cellular organisms.8 In the final stage of replication, R110 puncta were recruited to the plasma membrane (Figure 4A), indicating that the released progeny viruses remained cell associated until very late stages in replication.24

Figure 4. Transport of progeny viral capsids in the cytoplasm. A) A confocal image of an infected Vero cell at 20 hpi. Assembled progeny viral capsids (green) egressed to the cytoplasm. Scale bar: 5 µm. B) Two typical trajectories shown in yellow rectangles in (A). C) The instantaneous velocity of the trajectories shown in (B), with corresponding colors. D) MSD-time plot of the progeny capsids in (B).

All of the observed processes are summarized in Scheme 2. On the basis of reverse genetic technology and HaloTag technology, we have developed a strategy for temporally controllable capsid-specific labeling of parental and progeny viruses using different fluorophores. The TMR labeled parental PrV experienced virus attachment, entry, capsid transportation toward the nucleus and docking on the nucleus, while the temporally-controllable R110 labeled progeny virus underwent the assembly of capsids in the nucleus and egress processes. The life cycle of PrV was globally depicted by tracking different fluorescent ligands labeled capsid-specific HaloTag labeled 11


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PrV through SPT.

Scheme 2. Schematic diagram for the life cycle of PrV by visualization of capsid-specifically HaloTag labeled rPrV_HT. Virus attachment, entry (Stage I), capsid transportation toward the nucleus along microtubules (Stage II) and capsid docking on the nucleus (Stage III) could be investigated via TMR labeled parental viruses; while the endonuclear assembly (Stage IV) and egress (Stage V) of progeny capsids could be visualized via the R110 labeled progeny viral capsids.

The established labeling strategy is universal for labeling of various viruses. In combination with existing labeling materials and methods,5, 25-28 we could further obtain dual or triple labeled viruses, which would provide us a versatile means to reveal the fate of different viral components in virus life cycle and facilitate the development of novel prophylactic and therapeutic strategies against viral diseases.

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Conclusions On the basis of reverse genetic technology and HaloTag technology, we have developed an effective strategy for temporally controllable capsid-specific labeling of parental and progeny viruses using different HaloTag ligands. Consequently, the entire course of virus infection and replication can be consecutively visualized. The infection processes including virus attachment, capsid entry, transportation along microtubules in the cytoplasm, and docking on the nucleus can be visualized via TMR labeled parental recombinant viruses. Meanwhile, the assembly and egress of progeny capsid can be also visualized via the R110 labeled progeny viruses. The virus-cell interactions in the five stages of PrV life cycle have been dissected. In particular, assembly and transportation of progeny viral capsids have been monitored, which is difficult to be accomplished by conventional labeling approaches.

Methods Cell Culture Vero cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco). PK15 and BHK cells were cultured in minimum essential medium (MEM, Gibco), both media were supplemented with 10% fetal bovine serum (FBS, Gibco). Cells were cultured at 37°C in an atmosphere of humidified 5% CO2. The Vero cell line (CCL-81), PK15 cell line (CCL-31) and BHK-21 cell line (CCL-10) were from American Type Culture Collection (ATCC, Manassas, VA).

Production of Recombinant Viruses A transfer plasmid named pFB-CMV_UL35-HaloTag for transposition was constructed by inserting the HaloTag open reading frame into the C-terminal of the UL35 gene (PrV strain Becker1 genomic DNA, pBecker3,11 as the template) linked by 3×SGGGG. This linker was designed to diminish the potential influence of HaloTag on the structure and function of VP26. The pFB-CMV vector was derived from pFastBac Dual (Invitrogen) by replacing the p10 promoter and the polyhedrin promoter with the cytomegalovirus promoter. The generated pFB-CMV_UL35-HaloTag plasmid was transformed into modified competent DH10B 13


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Escherichia coli cells containing a shuttle vector that encodes the entire genome of PrV (a bacmid named pBeZF1) and a helper plasmid.12 A recombinant bacmid named pBeZF1_UL35-HaloTag was produced after the transposition of the CMV_UL35-HaloTag expression cassette from pFB-CMV_UL35-HaloTag to the genome of PrV, pBeZF1. Wild-type virus (wtPrV) and recombinant virus (rPrV_HT) were generated by transfecting Vero cells with pBeZF1 and pBeZF1_UL35-HaloTag, respectively. The supernatants were harvested approximately 48 h after transfection, and cell debris was removed through centrifugation at 6000 g for 30 min at 4°C. The supernatants were aliquoted and stored at -80°C as the first passage of wtPrV and rPrV_HT.

Purification of Viruses The supernatants of cell culture were clarified first and then concentrated by ultracentrifugation at 95,400 g for 3 h in a Beckman SW28 rotor on 6 mL 30% (W/V) sucrose cushion in PBS buffer. The pellets were suspended in PBS buffer and further centrifuged at 5,700 g for 5 min to remove the aggregation. Subsequently, the supernatants were centrifuged at 95,400 g for 1.5 h using a Beckman SW55 rotor on 1 mL 30% (W/V) sucrose cushion in PBS buffer for purification. The pellets were suspended in PBS buffer and further centrifuged at 110,000 g for 1h to remove sucrose. The pellets (purified viruses) were then suspended in PBS buffer and stored at -80°C in aliquots. All centrifugations were performed at 4°C.

Labeling of Viruses For the fourth propagation of recombinant viruses (the fifth passage), 200 nM HaloTag TMR ligand (TMR) was supplemented to the virus growth medium (MEM medium containing 2% FBS). The generated TMR ligand labeled recombinant PrV (rPrV_HT-TMR) was observed in the living cell after clarification without further purification. To examine the specificity of the interaction between HaloTag protein and TMR, 200 nM TMR was supplemented to the virus growth medium during the propagation of wtPrV. The generated wtPrV (TMR) was served as a negative control in the following experiments. All operations and storage after the addition of TMR were protected from light. 14


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Virus Titration and One-Step Growth Assay The titer of viruses was determined by 50% tissue culture infective dose (TCID50). In brief, Vero cells cultured in 96-well plates were infected with 10-fold serially diluted viruses (range from 10-2 to 10-8) at 37°C in 5% CO2 incubator for 4-5 days. The number of wells with CPE was counted, and TCID50 was calculated using Reed-Muench method. One-step growth assay was performed to determine the growth kinetics of the recombinant virus. Vero cells were infected with wtPrV and rPrV_HT at an MOI of 10 for 1 h at 4°C, and then the inoculums were replaced with pre-warmed DMEM. Subsequently, to inactivate extracellular viruses, cells were treated with citrate solution (40 mM sodium citrate, 10 mM KCl, and 135 mM NaCl, pH3.0) for 1 min after 1 h of incubation at 37°C. Cells were then cultured in fresh medium supplemented with 2% FBS at 37°C for 4, 8, 12, 24, and 36 h. To release infectious intracellular virus, cells were exposed to two freeze-thaw cycles prior to harvest. Titers of all samples were determined on Vero cells. The average titer of three measurements was calculated to plot the one-step growth curves.

Western Blot Analysis of Viruses Purified viruses were treated with gel-loading buffer for Tricine-SDS-Polyacrylamide gel electrophoresis (Tricine-SDS-PAGE). 40 µg samples of viruses were electrophoresed in a 16% polyacrylamide gel at 10 mA for 6.5 h and then transferred electrically onto polyvinylidene difluoride (PVDF) membranes for immunoblotting.13 The membranes were blocked with 3% BSA and 0.5% casein in TBS-T buffer (TBS buffer+0.1% TweenTM20) overnight at 4°C. Thereafter, primary antibody (anti-VP26 pAb (1:250, Abmart) or anti-HaloTag pAb (1:1000, Promega)) was incubated with the membrane overnight at 4°C. After washing with TBS-T buffer extensively, the blots were then incubated with Alkaline-Phosphatase conjugated secondary antibody (1:2500, Thermo) at 37°C for 1 h and washed with TBS-T buffer as above. The bands of the detected proteins were developed by the color reaction (NBT/BCIP, Biosharp).

Immunoelectron Microscopy Imaging of Purified Viral Capsids 15


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Purified viral capsids were obtained by following procedures. BHK cells were infected with viruses at MOI of 5 in virus growth medium. At 9 h post infection (hpi), the medium was removed and cells were lysed in cold lysis buffer (20 mM Tris·HCl (pH7.5), 0.5 M NaCl, 1% Triton X-100, 1 mM EDTA, and 1 mM DTT) by intermittent sonication for 10 min in total on ice, and the lysates were clarified by centrifugation for 15 min at 11,400 g. Subsequently, the supernatants were subjected to ultracentrifugation at 104,000 g for 1.5 h in a Beckman SW55 rotor. The pellets were suspended in PBS buffer and then centrifuged at 110,000 g for 1.5 h in an SW55 rotor on 2 mL 30% (W/V) sucrose cushion in PBS buffer. The pellets containing capsids were suspended in 200 µL PBS buffer, and further centrifuged at 110,000 g for 1 h in a Beckman SW40 rotor to remove sucrose. All centrifugations were performed at 4°C. Purified capsids were dispended into aliquots and stored at -80°C. 10 µL of 1:2 diluted purified viral capsids (diluted in dilution buffer, PBS containing 0.2% BSA) was dropped onto a sheet of Parafilm, and a carbon-coated copper grid was covered on the top of the droplet using a forceps (Drop-to-Drop Method) for 5 min at room temperature (RT). After wicking away excess fluid with filter paper, the copper grid was blocked with 1% BSA at RT for 2 h, and then incubated with 10 µL Anti-HaloTag pAb (1:40, Promega) in a humidity chamber at 37°C for 2 h. Subsequently, the grid was washed with 100 µL dilution buffer on the sheet of Parafilm on a shaker for 5 min at RT, and the procedure was repeated for 5 times. The grid was incubated with 10 µL gold-labeled secondary antibody (1:500, Abcam) at 37°C for 1 h followed by washing as described above. Finally, the grid was placed on the top of 2% phosphotungstic acid (PTA) solution for 5 min at RT. All operations followed Drop-to-Drop Method. The capsids on the copper grids were examined under transmission electron microscope (H-7000 FA, Hitachi).

Ultrastructural Assay of Progeny Virus Vero cells were infected with wtPrV and rPrV_HT at MOI of 5 in virus growth medium. At 9 hpi, the medium was removed and cells were washed thrice with PBS, and then fixed with 2% glutaraldehyde in PBS for 15 min on ice. The fixed cells were collected and washed with PBS thrice, and then fixed with prechilled 1% osmic acid at 4°C for 2 h. The cells were dehydrated using serial concentrations of alcohol (50%, 70%, 80%, 90%, 95%, 100%), 15 min for each 16


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concentration, and then the cells were completely dehydrated twice absolute of alcohol. Subsequently, the fixed cells were permeated and embedded in epoxy resin. The samples were sectioned to yield 60-100 nm ultrathin sections with a cryo-ultramicrotome (EM UC7, Leica). The sections were transferred to carbon-coated copper grids for further negative staining. The preparation of ultrathin sections was accomplished by the core facility center in Wuhan Institute of Virology, Chinese Academy of Sciences. Ultrathin sections were examined using a transmission electron microscope (Tecnai G20 TWIN, FEI).

Immunofluorescence Vero cells were infected with rPrV_HT-TMR, fixed with 4% paraformaldehyde at 10 hpi for 20 min, and then treated with 0.1% Triton-100 for 10 min to increase the permeability of the cell membrane. After blocking with 5% BSA in PBS at RT for 30 min, the cells were incubated with anti-Beta Tubulin mAb (1:400, Earthox), anti-Lamin A/C mAb (1:100, Cell Signaling Technology), and anti-nuclear pore complexes (NPC) mAb (1:100, Abcam) at 37°C for 1.5 h. After extensive washes with PBS, cells were incubated with DyLight488-conjugated secondary antibody at 37°C for 45 min. Immunostained cells were observed after extensive washes with PBS. All operations were protected from light.

Ultrathin Cryosection Vero cells were infected with rPrV_HT at MOI of 5 in virus growth medium. At 9 hpi, cells were scraped from the flask, and collected by centrifugation. The pellets were resuspended in optical cutting temperature (OCT) freezing medium, and mounted in a cryo-ultramicrotome (Leica). The sample was sectioned to yield a 5 µm ultrathin section. The cryosections were transferred to a glass slide, and stained with Hoechst33342 at 37°C for 10 min (5 µg/mL, Invitrogen). The prepared ultrathin cryosections were observed with the confocal microscope.

Cell Transfection To visualize microtubules, Vero cells were transiently transfected with plasmid encoding GFP-microtubule-associated protein 4 microtubule binding domain (GFP-MAP4BD)29 by using 17


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LipofectamineTM LTX and PLUS reagents (Invitrogen) in accordance with the manufacturer’s instructions. 36 h after transfection, the transfected cells were infected with rPrV_HT-TMR and observed under a confocal microscope.

Colocalization Assay of Viral Genomes/Envelopes and Viral Capsids A 10 µL aliquot of wtPrV or rPrV_HT-TMR was dropped and spread on plasma-cleaned coverslips (22 mm × 22 mm, Citoglas) and then fixed with heat. After washing thrice with PBS, the genome and envelope of viruses were stained with the nucleic acid stain (SYTO 13, 5 µM, 30 min, Invitrogen) and the lipophilic tracer (DiO, 5 µM, 20 min, Invitrogen), respectively. The excessive dyes were removed with PBS, and the prepared coverslips were observed under a confocal microscope. Intensity correlation analysis was performed by Image J to quantify the colocalization efficiency of ﬂuorescent signals from two channels.30 The extent of colocalization was demonstrated by the following parameters: tMr and tMg and intensity correlation quotient (ICQ). tMr and tMg indicate the percentage of red/green signals colocalized with green/red signals in the image, and ICQ value was used for statistical analysis (+0.1~+0.5 implies a strong covariance).31 The product of the difference from the mean (PDM) image was used to exhibit the colocalization extent for each pair of images. Line proﬁle, which indicates the intensity of green and red signals distributed on a line, was obtained using Image Pro Plus (IPP).

Microscope Setup SPT was performed on a spinning-disk confocal microscope equipped with an Olympus IX81 microscope, an online culture system, and an EMCCD.32 All observations were protected from light, and all images were acquired with an Olympus 100× oil immersion objective with a numerical aperture of 1.4 and Andor iQ software. We selected a 405 nm laser with a BP447/60 nm filter for Hoechst33342, a 488 nm laser with a BP 525/50 nm filter for SYTO 13/DiO/GFP/DyLight488, a 561 nm laser with a BP605/20 nm filter for TMR, and a 640 nm laser with a BP685/40 nm filter for CellMaskTM Deep Red plasma membrane stain (CellMask, Invitrogen). For simultaneous dual-color observation, the signals from two channels were alternately captured onto the EMCCD. 18


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SPT in Living Cells Vero cells were seeded onto a 35-mm glass-based dish 24 h prior to observation and incubated with rPrV_HT-TMR at 4°C for 30 min for attachment. Excessive viruses were removed by washing with DMEM. For cell nuclear and plasma membrane staining, Vero cells were incubated with Hoechst33342 (5 µg/mL, at 37°C for 10 min, Invitrogen) and CellMask (1:1500, at RT for 3 min, Invitrogen) prior to the virus infection, respectively. For progeny capsid labeling, 250 nM HaloTagR110Direct ligand (R110 ligand) was added to the medium at 4 hpi. To maintain cells in good morphology during long-term imaging, cells were cultured in DMEM containing 2% FBS throughout the observation. All observations were performed at 37°C. Puncta that moved within the focal plane throughout the capture were selected for further analysis. The movements of labeled viruses were tracked and analyzed using IPP.33 The trajectories of labeled viruses were generated by tracking the representative single viruses. Mean square displacement (MSD) was calculated using a home-compiled program based on Matlab (MathWorks).34 The time trajectories of the velocity indicate the instantaneous velocity of virus in the living cell. MSD against time was plotted to reveal the motion mode of virus. If the plot could be fitted with an equation of MSD = 4 Dt α , the trajectory performed a restricted motion, which implies the virus interacted with either its receptor or other organelles. By contrast, if the plot was fitted with 2

an equation of MSD = 4 Dτ + (Vτ ) , the trajectory experienced a directed movement, which indicates the virus likely transported along the cytoskeleton.15

Acknowledgements We thank Wan-Po Zhang for his help in preparing the samples of ultrathin cryosections. We also would like to thank An-Na Du and Pei Zhang from The Core Facility and Technical Support, Wuhan Institute of Virology, for their help with producing EM micrographs. This work was supported by the National Basic Research Program of China (973 Program, No. 2011CB933600), 19


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the National Natural Science Foundation of China (21535005; 31170874), the 111 Project (111-2-10), and Collaborative Innovation Center for Chemistry and Molecular Medicine.

Supporting Information Available: The Supporting Information is available free of charge via the ACS Publications website at http://pubs.acs.org.

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Additional information

Competing financial interests: The authors declare no competing financial interests.

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