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Labeling and Single Particle Tracking Based Entry Mechanism Study of Vaccinia Virus from the Tiantan Strain Li-Li Huang, Li-Li Wu, Xue Li, Kejiang Liu, Dongxu Zhao, and Hai-yan Xie Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 02 Feb 2018 Downloaded from http://pubs.acs.org on February 2, 2018

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Analytical Chemistry

Labeling and single particle tracking based entry mechanism study of Vaccinia Virus from the Tiantan Strain Li-Li Huang, Li-Li Wu, Xue Li, Kejiang Liu, Dongxu Zhao, Hai-Yan Xie* School of Life Science, Beijing Institute of Technology, Beijing 100081, China *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: Entry is the first and critical step of viral infection, while the entry mechanisms of many viruses are still unclear due to the lack of efficient technology. In this report, by taking advantage of the single-virion fluorescence tracking technique and simultaneous dual-labeling methods for viruses, we developed; the entry pathway of vaccinia virus from tiantan strain (VACV-TT) was studied in real-time. By combining with the technologies of virology and cell biology, we found that VACV-TT moved toward the Vero cell body along the filopodia induced by the virions interaction, and then, they were internalized through macropinocytosis, which was an actin-, ATP-dependent but clathrin-, caveolin-independent endocytosis. These results are of significant importance for VACV-TT based vaccine vectors and oncolytic virus study.

Viruses are obligatory parasites that hijack intrinsic cellular pathways to duplicate themselves. Virus infection is a multistep and complicated process, and entry is the first and most important step. Deciphering the pathway of virus entry is critical for unraveling the viral infection mechanisms and promoting the diagnoses and therapeutics of viral diseases. In the past 10 years, the development of new fluorescence labeling methods combined with the emerging dynamic imaging technologies greatly promoted the study on entry mechanisms of viruses.1-8 Zhuang’s group labeled the viruses with lipophilic dyes, amino reactive dyes or nucleic acid intercalating dyes and studied their entry pathways.1-3 Pang’s group labeled viruses with quantum dots and dissected the endocytic routes of influenza A virus in detail.4 Our group developed several unique and universal methods to label viruses during natural replication and assembling process.6-8 For example, the Ru(II) complex was successfully used to label the nucleic acid of fully replicative vaccinia virus by a ‘virus replication-intercalated labeling’ strategy.6 By making use of the host cell deriving formation mechanism of viral envelope, we succeeded in labeling fully duplicative viruses by incorporating azide choline (azide-Cho) into the envelope, which was subsequently labeled via copper-free click chemistry in natural propagation process.7 By taking advantage of the intrinsic biosynthetic machinery of biomacromolecules, the viral proteins were incorporated with azidohomoalanine (AHA) during translation and the nucleic acids of viruses were incorporated with 5vinyl-2'-deoxyuridine (VdU) during replication, and then they were individually labeled via copper-free click chemistry and inverse electron demand Diels-Alder (iEDDA) reactions dur-

ing the natural viral assembly process.8 The dual-labeled virions were of intact structure and full infectivity. They could recognize host cells and induce cytopathogenic effects; meanwhile the single particle tracking could easily be carried out due to the strong fluorescence. Viruses are generally divided into enveloped viruses and capsid viruses. Enveloped viruses are composed of nucleic acids, proteins and one or more envelope. Partly sourced from their host cell structure, there are two primary possible entry strategies: membrane fusion and endocytosis. The former is realized through the fusion of viral envelope and host cell membrane, resulting in the formation of fusion pores, through which the viral genome is released. As for the endocytosis pathway, the whole virions penetrate into the cell, and then encapsulate into endocytic vesicles.9-10 It can be subdivided into clathrin-mediated endocytosis, macropinocytosis, caveolin-mediated endocytosis and other pathways still poorly characterized.9-11 Vaccinia viruses (VACV) are typical enveloped viruses that replicate in cytoplasm. They include several variants, such as Western Reserve (WR), International Health Department-J (IHD-J), Tiantan strain (TT), and so on. Despite having been studied for several decades, the entry mechanisms of VACV are still far from being conclusively identified. Early studies by means of electron microscopy suggested that intracellular mature VACV (IMV) from WR strain entered cells through membrane fusion,12 while the other report claimed that IMV could induce transient macropinocytosis.13-14 More recent studies demonstrated that the entry pathways of IMV were strain

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type and cell type dependent. The IMV from WR strain entered cells by activating transient plasma membrane blebbing, while IMV from the IHD-J strain induced the rapid formation (and lengthening) of filopodia during entry.15 VACV from TT strain (VACV-TT) is the most widely used VACV in China. Recently, it is also developed as vaccine vectors to fight against infectious diseases and its oncolytic potential has led to increasing interest in developing various recombinants for cancer therapy.16 All these studies rely on elucidating its entry mechanism in detail. In this study, we systematically studied the entry mechanisms of VACV-TT by using of the developed labeling methods and single particle tracking technologies. Combined with the methods of virology and cell biology, it was found that instead of membrane fusion, VACV-TT entered Vero cells through macropinocytosis, one of the subways of endocytosis that was actin- and ATP- dependent but clathrin-, caveolinindependent. During the entry process, cellular protrusions were actively formed, and then folded back to the plasma membrane together with the virions, leading to the formation of endocytic vesicles known as macropinosomes, which resulted in a transient increase of cellular fluid uptake.

EXPERIMENTAL SECTION Materials and reagents. Vaccinia virus (VACV) tiantan strain and poliovirus (PV) were obtained from Wuhan Institute of Virology, Chinese Academy of Sciences. Vesicular stomatitis virus (VSV) was purchased from Wuhan University. Vero cells (African green monkey kidney cells) were purchased from Peking Union Medical College Hospital. EZ-Link SulfoNHS-LC-Biotin was purchased from Thermo. Streptavidin modified quantum dots 605 (SA-QDs605) were purchased from Jiayuan Quantum Dots Company (Wuhan, China). 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-vaccinia virus mouse polyclonal antibody was purchased from Immune Technology Corp (USA). FITC-dextran was purchased from NANOCS. Cypher5 and NAP-5 columns were purchased from GE Healthcare. Chlorpromazine and amiloride were purchased from Melone Pharmaceutical Company (Dalian, China). Phorbol myristate acetate (PMA), filipin and Cyto D were purchased from Sigma. All other chemical reagents were supplied by Beijing Chemical Reagent Company. Preparation of labeled VACV-TT. Vero cells were maintained in DMEM containing 10% FBS at 37 °C until they reached 70%-80% confluency. The VACV-TT was propagated in monolayer cultures of 400 µM Azide-Cho modified Vero cells at 37 °C in the presence of 2% FBS.7 The propagation lasted for 24 h, after which, 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.8 After three rounds of freezeing-thawing, the cell debris was removed. The phospholipids of viral envelope were labeled by 5 µM DBCO-Fluor 568 at 37 °C for 1 h. The viruses were purified with a sucrose density gradient as previously described.7 For labeling the viral envelope with streptavidin-modified QDs (SA-QDs), the purified control viruses were incubated with EZ-Link SulfoNHS-LC-Biotin at room temperature for about 1 h and purified by a NAP-5 column (GE Healthcare). Secondly, the bioti-

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nylated viruses were added to cells at 4 °C for 15 min. The cells were washed three times, and then incubated with 2 nM SA-QDs at 4 °C for 15 min. After washing three times, we could get the QDs-labeled viruses on the cell surface.17 For labeling the viral envelope with the pH-sensitive Cypher 5, purified viruses were incubated with the amine-reactive dye in a carbonate buffer (pH 9.3) at 37 °C and gently rocked for 1 h. Unbound dye was removed via buffer exchange into 50 mM Hepes buffer (pH 7.4, 145 mM NaCl) using Nap5 columns.2 Virus titers were determined using plaque assays on adherent Vero cells.6 Flow cytometry and plaques assay analysis of viral infectivity. To analyze the effect of inhibitors on virus entry and infection by plaques assays, Vero cells were serum starved for 90 min. Then, the monolayers of Vero cells were treated with inhibitors: Cyto-D (2 µg/mL), chlorpromazine (2.5 µg/mL), filipin (2.5 µg/mL) or amiloride (400 µM) for 30 min, followed the infection with VACV for 30 min at 37 °C in the presence of the inhibitors. At this point, the cells were washed with trypsin to remove unbound viruses, then the monolayers of Vero cells were covered with a DMEM containing 0.5% agar and 2% FBS. After 2 days, plaques were stained with crystal violet after fixation with 4% PFA. Enlarge one plaque of each monolayer in order to show the ﬂuorescent cell more clearly in ﬂuorescence microscope with a 4 × objective. To analyze the viral infectivity by flow cytometry, Serum starved Vero cells were pretreated with inhibitors: sodium azide (20、 100、500 mM) plus 2-deoxyglucose (50、250、1250 mM), antimycin A (0.002、0.02、0.2 µM), wortmannin (0.25、0.5、 1 µM), genistein (25、50、100 µM) or amiloride (50、100、 200、400、800 µM) for 30 min. Then the cells were incubated with VACV-TT (MOI = 10) in DMEM containing 2% FBS for an additional 12 h in the presence of inhibitors, and analyzed by flow cytometry for GFP expression. Fluid phase uptake assay.18 Vero cells were seeded in 6-well culture dish (1 × 105 cells per well), and then cells were serum starved for 20 h before virus adsorption with VACV-TT or PV (MOI = 10) on ice for 30 min. After washing, cells were pulsed for 30 or 60 min with 0.5 mg/mL FITC-conjugated 10kDa dextran. As experimental control, cells were treated with 200 nM PMA at 37 °C for 30 or 60 min. Non-internalized dextran was removed by acid washing (0.1 M sodium acetate, 50 mM NaCl, pH 5.5) and cells were detached and resuspended in 1% BSA, 1% FBS, 0.01% sodium azide in PBS. Dextran uptake was measured using a flow cytometer (BD Sciences). Transmission electron microscopy (TEM). 1 × 105 cells were plated onto 22 × 22-mm cover glasses in 30-mm plastic petri dishes and serum starved for 16 h. Vero cells were incubated with purified VACV-TT (MOI = 100) in 0.5 mL of DMEM for 15 min at 4 °C to allow virus binding. Subsequently, the cells were cultured at 37 °C for 0, 15, 30 or 60 min before fixation. These cells were then fixed in 2% glutaraldehyde in PBS. Ultrathin sections of these cells were examined by JEM-1400 transmission electron microscopy. Scanning electron microscopy (SEM). 1 × 105 cells were cultured in 30-mm plastic petri dishes. Then cells were serum starved for 16 h and infected with purified VACV (MOI = 100) for 30 min at 37 °C. Cells were fixed in 4% (W/V) PFA in 0.1


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M PB (pH 7.4) overnight at RT. Specimens were analyzed with a Hitachi field emission scanning electron microscope.

the endocytosis pathway rather than membrane fusion, and the viral envelope separated from virion in cytoplasm after the entry.

Fluorescence imaging of cells infected with dual-labeled viruses. Cells, cultured in glass-bottom dishes, were grown to 70% confluence. Then the cells were incubated with asprepared viruses for 15 min at 4 °C to allow virus binding, the unbound virions were removed by washing with PBS containing 1% glucose, after which the cells were imaged by a Leica laser confocal fluorescence microscope (TCS SP5). DBCOFluor 568 was excited with 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. FITCphalloidin was excited with a 488 nm laser, emitting 500–520 nm fluorescence. Live-cell fluorescence imaging. Cells, cultured in glassbottom dishes, were grown to 70% confluence, and then the cell membrane was labeled by incubating with 1 µM of lipophilic dye DiO in PBS for 5 min at 4 °C. After being washed with PBS, the cells were incubated with biotinylated VACV (MOI = 5) for 15 min at 4 °C to allow virus binding, the unbound virions were removed by washing with PBS containing 1% glucose. Then SA-QDs605 (2 nM) was added to cells at 4 °C. After 15 min, the unbound SA-QDs605 was removed. Fresh phenol red-free DMEM (Invitrogen) containing 1% glucose and 50 mM Hepes was added to cells. Fluorescence images were acquired with a spinning-disk confocal microscope (Andor Revolution XD), which was equipped with an Olympus IX 81 microscope, an EMCCD (Andor iXon DV 885K single photon detector), a Nipkow disk type confocal unit (CSU 22, Yokogawa), and a cell culture system (INUBG2-PI). DiO and SA-QDs605 were excited with 488 and 405 nm lasers, respectively. The fluorescence signals were separated with 525/50 and 617/73 nm band-pass emission filters, respectively. For multicolor imaging, fluorescence signals were detected separately with the EMCCD by the corresponding different channels.

RESULTS AND DISCUSSION DISCUSSION VACV-TT enters host cells via endocytosis instead of membrane fusion. One of the remarkable differences between the membrane fusion and endocytosis is if the virions take off their envelopes when they bind to the host cell membrane. To determine which way was taken by VACV-TT, the viral envelope and genome were individually labeled with DBCO-Fluor 568 or Cy5-Tz. And then, they were used to infect Vero cells and visualize through laser scanning confocal microscopy (LSCM) at different time. If the envelope enwrapped the virion, the fluorescence signal of DBCO-Fluor 568 on the membranes should co-localize with that of Cy5-Tz from the genome, otherwise, the two signals should separate from each other. As could be seen, the fluorescence of DBCO-Fluor 568 co-localized with Cy5-Tz on the cell surface (co-localization efficiency = 90.3%) at 0 mpi (min post infection) (Figure 1a1a4). Then more and more virions entered the cells, but most of the two fluorescence signals still co-localized at the very beginning (Figure S1). As the infection time extending, the colocalization efficiency in host cells decreased to 49.5% at 30 mpi and 28.9% at 60 mpi (Figure 1b-1b4, 1c-1c4). So it was reasonably to speculate that VACV-TT entered Vero cells via

Figure 1. The viral envelope phospholipids (DBCO-Fluor 568, green) and nucleic acids (Cy5-Tz, red) co-localize in host cells. Vero cells were incubated with dual-labeled VACV-TT at 37 ℃ for 0 min (a), 30 min (b) 60 min (c). After being washed by PBS, The DNA of cells was stained with Hoechst 33342 (blue). Colocalization efficiency of DBCO-Fluor 568 and Cy5-Tz was calculated by Image J software. Scale bar, 50 µm. PDM image (a1, b1, c1), intensity correlation plots (ICP) of green and red signals (a2-a3, b2-b3, c2-c3) and histograms of tMr, tMg, ICQ values (a4, b4, c4) were obtained from a, b and c.

In order to further confirm this speculation, we performed another experiment, in which the envelope of VACV-TT was labeled with CypHer 5, meanwhile, all its proteins were modified by AHA and labeled by DBCO-Fluor 568. The CypHer 5 is a pH-sensitive dye that is fluorescent brightly in neutral and acidic pH environment (pH ≤ 7) but becomes essentially nonfluorescent in pH ≥ 9 condition (Figure 2a, 2d).2 Therefore, if the viral envelope fused with the plasma membrane during entry, it would stay on the cell membrane and be sensitive to the external pH change. On the contrary, if the viral envelope entered the cells together with the virion, that is, VACV-TT enters cells via endocytosis; the sensitivity of internalized membranes to external pH change would decrease, because living cells are able to buffer its internal pH. When the duallabeled virions were initially added to Vero cells at 4 °C to synchronize the binding, we found that the fluorescence signal of viral envelope indeed varied with the external pH change (left and middle panels in Figure 2b). Subsequently, the cells were infected with the dual-labeled virus at 37 °C for 15 min, and then pulsed with pH 9.5 PBS, the signal of CypHer 5 was not visible from the virions remained on cell surface as expected, whereas it still could be seen from those virions entered into the cytoplasm (right panel in Figure 2b), indicating that viral envelope really entered host cells along with the virions during internalization. Meanwhile, internalized



VACV-TT particles were observed within endocytic vesicles instead of directly exposing to the cytoplasm (Figure 2c). These results again demonstrated that VACV-TT entered Vero cells through the endocytic pathway.

Figure 2. VACV-TT enters Vero cells by endocytosis. (a) The pH-sensitive Cypher 5 labeled VACV-TT envelope allows distinction between the populations of viruses outside versus inside the cell. Cypher 5 becomes non-fluorescent by briefly changing the pH in the medium to pH 9.5, resulting in the disappearance of envelope signals outside the cell (white envelope), whereas viruses that have already entered the cell are protected by the buffering capacity of living cells and are still observable (red envelope). (b) Fluorescence imaging of dual-labeled VACV-TT on living cells. The protein of VACV-TT was modified with AHA and labeled with DBCO-Fluor 568 (green), and the viral envelope was labeled by Cypher 5 (red), then labeled virions were incubated with Vero cells at 4 ℃ for 15 min (left and middle panels) or 37 ℃ for 15 min (right panels) and visualized by fluorescence microscopy. Scale bar, 50 µm. (c) TEM image of endocytic vesicles containing VACV-TT particles. Scale bar: 500 nm. (d) Fluorescence spectra of Cypher 5 in PBS buffer pH 7.2 or 9.5.

docytosis, the clathrin and caveolin of host cells was individually fused with enhanced green fluorescent protein (EGFP), meanwhile, the envelope of VACV-TT was labeled by quantum dots (QDs) with the emission wavelength at 605 nm (Figure S2a).17,19 By using of the excellent fluorescence brightness and photostability of QDs (Figure S2b and Movie S1), the movement of QDs-labeled virions was tracked through longterm single-virion imaging, and the results showed that the virions did not co-localize with clathrin or caveolin during internalization (Figure 3a and Movie S2, S3). Next, we performed the clathrin- or caveolin-mediated endocytosisinhibiting study by pretreating the host cells with corresponding inhibitor, chlorpromazine or filipin. The optimal concentrations of chlorpromazine and filipin were determined to be 2.5 µg/mL, in which condition the cell viability was kept more than 80% (Figure S3a, S3b). After the inhibitor incubation, VACV-TT was left to infect the cells. We found that the plaques formed in the inhibitor treated cells were similar with that in untreated cells (n = 3, p ＞ 0.05) (Figure 3b, 3c), suggesting that clathrin or caveolin inhibitor had no significant effect on VACV-TT infectivity. On the contrary, the infectivity of vesicular stomatitis virus (VSV), which has been proved entered host cells via clathrin-mediated endocytosis,20-21 was obviously affected by the clathrin inhibitor, and the infectivity in the presence of chlorpromazine reduced to 36.4% that of control group (n = 3, p ＜ 0.05) (Figure S4). Therefore, it could be concluded that the entry of VACV-TT did not rely on clathrin- or caveolin. VACV-TT entry undergoing actin rearrangements. Many viruses rely on the dynamic changes of actin cytoskeleton to promote their endocytosis,22 which is an energy- and actindependent process. To verify if the entry of VACV-TT was actin mediated, ATP was depleted2,23 at first, and then, followed with VACV-TT addition. The VACV-TT carrying GFP reporter gene was used in our study,24-25 so that the infectivity could be assessed according to the GFP expression. As could be seen, compared with untreated cells, the infectivity significantly reduced to 18.9 % (n = 3, p ＜ 0.01) in the presence of 500 mM sodium azide plus 1.25 M 2-deoxyglucose, or 45.1% (n = 3, p ＜ 0.05) in the presence of 0.2 µM antimycin A (Anti A) (Figure 4a), indicating that the entry of VACV-TT relied on cellular energy. Meanwhile, the infectivity of VACV-TT in the Cyto-D (a drug that causes actin depolymerization) treated cells was only about 5.3 % (n = 3, p ＜ 0.001) that of in control cells (Figure 4b, 4c), suggesting the requirement of actin cytoskeleton rearrangements during VACV-TT infection. Several critical cellular factors are often involved in actin cytoskeleton rearrangements, such as phosphoinositide-3 (PI(3))

Endocytosis of VACV-TT is clathrin- and caveolinindependent. Endocytic pathway can be divided into several sub-pathways that different from each other. So we were in the position to further determine which kind of endocytic subpathway the VACV-TT used. At first, to verify if VACV-TT was internalized through clathrin- and caveolin-mediated en-


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Figure 3. VACV-TT internalization does not require clathrin- or caveola-mediated endocytosis pathways. (a) Snapshots of the entry of VACV-TT into living cells. A movie depicting this process is also available (see Video S2 and Video S3 in the supplemental material). Vero cells, transfected with individual plasmids encoding GFP-fused caveola (top panels) or clathrin (bottom panels), were infected with QDs-labeled VACV-TT. White lines delineate the edges of GFP-expressing cells; white arrows delineate QDs-labeled VACV-TT. The time for each snapshot was taken and indicated in white at the bottom of each frame. (b) Plaque phenotype of VACV-TT. The monolayers of Vero cells were pretreated with inhibitors and subsequently infected with VACV-TT. The infectivity of virus was analyzed by plaques assays, and then plaques were either stained with crystal violet (top panels) or viewed under fluorescence (bottom panels) microscope. Scale bar, 400 µm. (c) The percentage of viral infectivity to drug-treated cells relative to untreated control cells was calculated from panel b. Data from three independent experiments were normalized as a percentage of the control. The data shown represent the mean values and standard deviations of the results.

Figure 4. VACV-TT infection can be modulated by actin, ATP, PI(3) kinase and tyrosine kinases. (a) Inhibition of ATP with inhibitors has significant impact on VACV-TT infection. Vero cells were pretreated with inhibitors: NaN3-2, deoxyglucose or anti A for 30 min at the indicated concentrations, and then incubated with VACV-TT for an additional 12 h period in the presence of inhibitors and analyzed by cytometry for GFP expression. Results are the percent GFP-positive cells normalized to values for control cells. (b) Plaque phenotype of VACV-TT. Vero cells were pretreated with Cyto D and subsequently infected with VACV-TT. The infectivity of virus was analyzed by plaques assays. Scale bar, 400 µm. (c) The percentage of infectivity of viruses to drug-treated cells relative to untreated control cells was calculated from panel b. Data from three independent experiments were normalized as a percentage of the control. *** p ＜ 0.001. The data shown represent the mean values and standard deviations of the results (d) Inhibition of PI(3) kinase and tyrosine kinases with inhibitors has significant impact on VACV-TT infection. The infectivity of virus was analyzed by cytometry for GFP expression. * p ＜ 0.05.



Figure 5. VACV-TT enters Vero cells via macropinocytosis. (a-b) Inhibition of macropinocytosis with amiloride has significant impact on VACV-TT infection. The infectivity of virus was analyzed by plaque assay (a) and cytometry (b). Data from three independent experiments were normalized as a percentage of the control. * p ＜ 0.05. (c) Mock (c1) and VACV-TT infected (c2-c4) Vero cells were fixed at 30 mpi and analyzed by SEM. VACV-TT particles were detected on filopodia of host cells areas. (d) Snapshots of the entry of VACV-TT in living cells. A movie depicting this process is also available (see Video S4 in the supplemental material). DiO labeled Vero cells were infected with QDs-labeled VACV-TT. White circles delineate QDs-labeled VACV-TT. The time at which each snapshot was taken and indicated at the top of each frame. Scale bar, 1 µm.

kinase and tyrosine kinases. Therefore, we subsequently tested the effect of these kinases on VACV-TT entry. The cells were pretreated with wortmannin, a specifically inhibitor of PI(3) kinase, or genistein, an inhibitor of tyrosine kinase,2,23 and then VACV-TT was added. The entry and infection of VACVTT were quantitatively analyzed through flow cytometry and plaque assay. The results showed that when 1 µM wortmannin or 100 µM genistein was added, the viral infectivity significantly decreased to 33.9% (n = 3, p ＜ 0.05) and 19.7% (n = 3, p ＜ 0.05 ) that of the control (Figure 4d). These results together indicated that the entry of VACV-TT was an actin mediated process that is ATP, tyrosine kinases and PI(3) kinase dependent. VACV-TT entered Vero cells through macropinocytosis. Macropinocytosis is one typical subway of endocytosis that is clathrin- and caviolin-independent while actin dependent. Based on the above results, it seemed probably that VACV-TT enters Vero cells through macropinocytosis. To confirm this speculation, the effect of amiloride on VACV-TT infection was investigated at first, because amiloride is a potent and specific inhibitor of Na+/H+ exchanger activity that is important for macropinosome formation.26-27 As expected, amiloride obviously reduced the infectivity of VACV-TT in a dose-dependent manner (Figure 5a-5b). In terms of morphological change, macropinocytosis is characterized by inducing actin-driven membrane ruffling at the cell surface to form large and irregular macropinosomes. As could be seen from the scanning electron microscopy (SEM) results, VACV-TT induced a dramatic increase in the number and length of filopodia (one of the forms of ruffling) on the cell surface within 30 min infection (Figure 5c2–5c4), while uninfected cells did not show such protrusions (Figure 5c1). To further verify this point, the cell membranes were labeled with DiO, and then

QDs-labeled virions were added. The real-time dynamic tracking results showed that some of the virions bound to filopodia, and then moved toward the cell body (Figure 5d and Movie S4), and some of the bound virions were “grabbed” by waving filopodia and folded back to cell (Movie S5), suggesting that these dynamic filopodia served as the effective way to recruit virions to susceptible cells. Another feature that distinguishes macropinocytosis from the other types of endocytosis is the enhanced cellular fluidphase uptake and co-localization with fluid phase markers, such as dextran.14,18 Therefore, an increase in fluid-phase uptake would be expected if VACV-TT entered Vero cells via macropinocytosis.18 To test this possibility, the uptake of fluorescent dextran (10-kDa) was analyzed in serum-starved Vero cells after VACV-TT infection or phorbol myristate acetate (PMA) treatment, which could promote macropinocytosis by inducing membrane ruffling.18 For comparison, the cells infected by poliovirus (PV), which was proved to enter cells not through macropinocytosis,2,28 were used as negative control. The flow cytometry assay results showed that the PMA treatment or VACV-TT infection significantly increased the dextran uptake to more than 122.2% or 141.8% at 30 min and 164.1% or 176.2% at 60 min, respectively (Figure 6b) whereas PV infection showed no significant increase. Subsequently, VACV-TT uptake in the presence of fluid-phase marker was examined by confocal laser scanning microscopy (CLSM). We found that about 74% of VACV-TT co-localized with FITCdextran (Figure 6a). These results further demonstrated that VACV-TT entered Vero cells through macropinocytosis.


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Figure 6. VACV-TT enters Vero cells via macropinocytosis. (a) VACV-TT infected Vero cells were pulsed with FITC-dextran. After fixation, VACV-TT particles were labeled with an antibody against envelope protein H3 followed by a Dylight 594-conjugated secondary antibody. The DNA of Cells was stained with Hoechst 33342 (blue). Scale bar, 25 µm. (b) Mock and VACV-TT infected Vero cells were pulsed for 30 or 60 min at 37 °C with FITC-dextran. As a control, cells treated with PMA or infected with PV were analyzed in parallel. Dextran uptake was quantified by flow cytometry and normalized to unstimulated cells (mean percentage ± SD of three independent experiments). (c-d) Global analysis of the entry of VACV-TT into Vero cells using TEM and SDCLM. (c1 and d1) VACV-TT bond to the surfaces of cells and the cell filopodia extended. (c2 and d2) A VACV-TT particle internalized within endocytic vesicles. (c3 and d3) Endocytic vesicles containing VACV-TT particles appeared to fuse. (c4, d4 and c5, d5) Large vesicles containing several virions were observed. White arrows delineate VACV-TT. Scale bar, 200 nm and 5 µm.

Finally, to globally investigate the entry process of VACVTT, transmission electron microscope (TEM) and spinning disk confocal laser microscopy (SDCLM) imaging were used to observe the host-pathogen interaction. We found that, at the beginning, the virions attached to the cell surface, following the extension of cell filopodia (Figure 6c1, 6d1), which was the effective way to recruit virions for entry. Subsequently, virions were internalized within vacuoles, which were formed by the filopodia folding back to the cell surface (Figure 6c2, 6d2). Then, the VACV-TT detached from the cell surface (Figure 6c3, 6d3), and localized within endocytic vesicles ranging from 200 to 500 nm, which were interpreted as early macropinosomes (EMs).29 At later time, EMs gradually fused together (Figure 6c4, 6d4), resulting in the formation of large vesicles about 400 to 1,000 nm in diameter and contained several viral particles (Figure 6c5, 6d5), which were interpreted as late macropinosomes (LMs).29 These phenomena were also consistent with the features of macropinocytosis.

In conclusion, we systematically studied the mechanisms of VACV-TT entering Vero cells on the basis of the viral labeling methods we developed. It was found that VACV-TT entered cells in an ATP and actin dependent but clathrin- and caveolin-independent manner. The virions moved toward the cell body along the filopodia induced by the virus infection, and then were “grabbed” by waving filopodia and folded back to cell. The viral envelopes did not separate from virions before the virion internalization, and were gradually taken off after they entered into cells. The entry process could be influenced by PI(3) kinases, tyrosine kinases and the inhibitors of macropinocytosis. After the entry, VACV-TT co-localized with FITC-dextran, one of the fluorescent markers for macropinocytosis. Therefore, it could be concluded that, as a typical enveloped virus, VACV-TT entered Vero cells by macropinocytosis instead of plasma membrane fusion. This finding is of significant importance for VACV-TT based vaccine vectors and oncolytic virus study.

CONCLUSIONS

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Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Co-localization analysis of viral envelope phospholipids and nucleic acids; TEM and CLSM images of QDs-labeled VACV-TT; Detection of cells viability and viral infectivity (PDF)

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

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Notes The authors declared that no competing interest exists.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 21372028, No. 21422502 and No. 81571813).

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