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Imaging Early Steps of Sindbis Virus Infection by Total Internal Reflection Fluorescence Microscopy

Imaging Early Steps of Sindbis Virus Infection by Total Internal Reflection Fluorescence Microscopy Hindawi Publishing Corporation Advances in Virology Volume 2011, Article ID 535206, 7 pages doi:10.1155/2011/535206 Research Article Imaging Early Steps of Sindbis Virus Infection by Total Internal Reflection Fluorescence Microscopy Youling Gu, Yuanzheng Yang, and Yuechueng Liu Department of Pathology, University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, Ok 73190, USA Correspondence should be addressed to Yuechueng Liu, yuechueng-liu@ouhsc.edu Received 11 July 2011; Revised 7 September 2011; Accepted 8 September 2011 Academic Editor: Amiya K. Banerjee Copyright © 2011 Youling Gu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sindbis virus (SINV) is an alphavirus that has a broad host range and has been widely used as a vector for recombinant gene transduction, DNA-based vaccine production, and oncolytic cancer therapy. The mechanism of SINV entry into host cells has yet to be fully understood. In this paper, we used single virus tracking under total internal reflection fluorescence microscopy (TIRFM) to investigate SINV attachment to cell surface. Biotinylated viral particles were labeled with quantum dots, which retained viral viability and infectivity. By time-lapse imaging, we showed that the SINV exhibited a heterogeneous dynamics on the surface of the host cells. Analysis of SINV motility demonstrated a two-step attachment reaction. Moreover, dual color TIRFM of GFP-Rab5 and SINV suggested that the virus was targeted to the early endosomes after endocytosis. These findings demonstrate the utility of quantum dot labeling in studying the early steps and behavior of SINV infection. 1. Introduction SINV has long been used as an experimental model for studying encephalitis because it causes encephalomyelitis in Sindbis virus (SINV) is an alpha virus and a member of neonatal mice [9]. Moreover,sinceSINVhas abroad host the Toga virus family that also includes Semliki Forest range and can deliver efficient gene expression, vectors based and Ross River viruses [1]. It was first isolated in 1952 on the virus have been developed for vaccine production from mosquitoes found in the Sindbis area in Egypt [2]. and gene therapy purposes [10, 11]. In cancer therapy, SINV The decoded SINV genome revealed a single-strand RNA has been tested as a potential oncolytic reagent. It has been of 11.7 kb in size [3]. Upon cell entry, the viral RNA strand shown that a SINV strain (Toto1101) derived from the wild- serves as a messenger RNA to translate four nonstructure type SINV was effective targeting and killing tumor cells of genes that are essential for viral replication. In late stages of ovarian, colon, prostate, and liver cancer patients, leading to infection, structural genes are translated and synthesized tumor regression in animal models [12, 13]. from a subgenomic RNA. The resulting viral particles con- In recent years, quantum-dot- (Qdot-) based fluores- taining nucleocapsids of genomic RNA and capsid proteins, cence labeling has become increasingly employed for imaging packaged with envelope proteins E1/E2, are formed and bud- cellular events including single-molecule tracking and live- ded off as mature virus [4, 5]. The receptors that are involved cell imaging [14–16]. Qdots are semiconductor nanocrystals in SINV entry into host cells remain poorly characterized. with a broad excitation and emission spectra. One advan- One of such receptors is the 67 kD high-affinity laminin tage of Qdot is its resistance to photobleaching, allowing receptor (LR), which has been shown to mediate SINV prolonged exposure to excitation light. In the current study, infection of BHK cells [6, 7]. In addition, it has been reported we labeled SINV with biotin and conjugated the virus with that heparan sulfate, a major cell surface component, is Qdot 605. Using single-virus tracking, we were able to dissect directly involved in SINV infection of cultured cells [6, 8]. the behavior and dynamics of individual SINV during the Little is known regarding viral attachment and movement on initial stages of infection, which showed a two-step, receptor- host cell surfaces during early stages of SINV infection. mediated cell attachment process. 2 Advances in Virology 2. Materials and Methods ing with PBS to remove unbound virus, the cells were further incubated with streptavidin-conjugated Qdot 605 2.1. Cell Culture and SINV Preparation. BHK-21 cells were (Invitrogen) for 60 minutes and washed 3 times with cold cultured in MEM supplemented with 10% FBS and antibi- PBS. The cells were fixed with 3.7% formaldhyde in PBS for otics. The cells were maintained in a humidified 37 C 30 minutes at room temperature. Fluorescent images were incubator with 5% CO . For the construction of SINV-GFP acquired and digitized for analysis. For live cell experiments, expressing the green fluorescent protein (GFP), GFP cDNA the infection was performed on ice or at 4 Cfor 1hour (from pS65T-C1, Clonetech) was first cloned into a shuttle ◦ before shifting to a 35 C chamber for microscopy. vector pH2J1Y which was modified from pH3 2J1 (kindly For TIRFM, we used an Olympus IX71 equipped with a provided by Dr. Guangpu Li, University of Oklahoma, 60X (n.a. = 1.45) TIRF objective heated to 37 C. Cells were Ok, USA). The plasmid pH2J1Y contained a linker with cultured in a home-made glass bottom dish and maintained additional multiple cloning sites, Kpn I, Sal I, EcoR V, at 35 C in an environmental control chamber (Olympus, Hind III, and Nhe I, which was inserted between the Xba USA) supplemented with 5% CO . TIRF illumination was I and Bam H1 sites of pH3 2J1. The cDNA encoding the achieved using a 20 mW 488 nm laser source for excitation GFP was excised by Nhe I and Bam H1 and ligated into and a long-pass dichroic beam splitter (500 nm). GFP/YFP pH2J1Y at same sites. An Apa I-Xho I fragment of pH2J1Y was viewed with an emission filter of 525/20 nm, and harboring the GFP cDNA was subcloned into the SINV Qdot 605 was viewed with an emission filter of 600/40 nm. vector pToto/3 2J. To construct SINV-C -YFP, in which the Emission filters were controlled with a Lambda 10 (Sutter 62–106 amino acid sequence of the capsid protein was Instrument, Novato, CA, USA) high-speed filter wheel. replaced with EYFP, an Sph I and an Mlu I sites flanking the Images were captured with a CCD camera (Quantix 57, replaced capsid sequence were introduced into pToto/3 2J ◦ Photometrics, Tucson, Ariz, USA) air cooled to −25 C. The by PCR mutagenesis. EYFP with Sph I and Mlu I cloning camera was controlled by IPlab 3.9.4 (Scanalytics, Fairfax, Va, sites was amplified by PCR using pEYFP-C1 as a template. USA) and analyzed with the IPlab software. For expression Recombinant SINV was produced by transfection of BHK21 of GFP-Rab5, BHK-21 cells were transfected using Lipo- cells using capped RNAs derived from sp6 RNA polymerase fectamine 2000 (Invitrogen) and pGFP-Rab5 (Dr. Guangpu transcription of Xho I-linearized plasmid templates. Viral Li, University of Oklahoma Health Sciences Center). The stocks were obtained by harvesting the culture supernatants cells expressing GFP-Rab5 were imaged under TIRFM 24–48 30 hr after infection and aliquots were stored at −80 C until hours after transfection. use. Viral titers were determined by plaque assay on BHK21 monolayers using crystal violet staining. 3. Results 2.2. Biotinylation of SINV. SINV-GFP was collected in serum 3.1. Labeling SINV with Qdot 605. SINV was first conjugated free MEM diluted with saline (1 : 1) at ∼4 × 10 pfu/mL to biotin linked through a 29APEG arm. In orderto and was labeled with biotin at room temperature or on achieve a near 100% labeling of SINV, an excessive amount ice. Briefly, the virus was mixed with 10 mM stock NHS- of NHS-biotin at 300 µM was used in the reaction. This PEG4-Biotin (29 A spacer arm, Pierce) in PBS (pH 8.0) was necessary for ensuring that the observed infection was at a final concentration of 300 µMfor 30minatroom indeed by the biotinylated SINV, not by the native and temperature. The reaction was terminated by adding glycine un-modified viruses. The conjugation reaction, however, 8 6 to 0.1 M and incubating for 15 min at room temperature. For reduced the infective titer of the virus from 4×10 to 10 – biotinylation on ice, SINV was incubated with NHS-PEG4- 7 10 pfu/mL. Such a reduction was mostly due to the excessive Biotin as described above for 90 minutes on ice. The reaction chemical modification by NHS-biotin since viral titer was was terminated by addition of glycine and further incubation less affected when the concentration of NHS-biotin was at room temperature for 20 minutes. We did not notice any reduced (data not shown). Western blot analysis showed that significant differences in the efficiency of biotinylation and both the E1/E2 envelope proteins were labeled (Figure 1(a)). the infectivity of the resulting biotinylated SINV produced Moreover, the excessive crosslinking caused some E1/E2 under the two labeling conditions. For western blot analysis proteins shifting to slightly higher apparent molecular weight of the labeled SINV, aliquots of SINV were heated at 100 C (Figure 1(a)). When the biotin-SINV was preincubated with for3mininsamplebuffer containing 1% SDS, 10 mM Qdot 605, a medium-sized quantum dot of 5–12 nm diame- EDTA, 10 mM DTT, 15% glycerol, 20 mM Tris-HCl, pH ter, the virus-Qdot conjugate was still active infecting BHK- 6.8, and 0.01% bromophenol blue. The SINV proteins were 21 cells with a small reduction in infection activities (<2 separated by SDS-PAGE and transferred onto nitrocellulose fold). However, if we preincubated the cells with SINV for 1 membrane. An alkaline phosphatase-conjugated streptavidin ◦ hour at 4 C and then added Qdot, the reduction in infectivity (1 : 5,000, Sigma) was used to visualize biotinylated SINV became minimum. Specific binding to host cells by Qdot- envelope proteins. labeled SINV was evident under fluorescence microscopy (Figure 1(b)). To determine if the Qdot-SINV was able to 2.3. SINV Infection, Fluorescence Microscopy, and Image enter cells and carry out viral replication, we followed the Analysis. Biotinylated SINV was used to infect BHK-21 cells expression of recombinant GFP encoded by the viral vector. at MOI (multiplicity of infection) of 5–20. The virus was BHK-21 cells were scored for GFP and Qdot 605 fluorescence incubated with the cells at 4 C for 1 hour. After wash- 14 hours after infection, following the completion of first Advances in Virology 3 (a) (b) GFP Qdot Phase contrast Overlay (c) Figure 1: Biotinylation and Qdot labeling of SINV. SINV was labeled with NHS PEG4-biotin as described in Section 2. (a) Western blot analysis of biotinylated SINV using alkaline-phosphatase-conjugated streptavidin showing efficient labeling of the E1/E2 envelope proteins (indicated by ). Lanes 1: control unlabeled SINV and 2: biotinylated SINV. Molecular weight markers (kD) are labeled. (b) Biotinylated SINV labeled with Qdot 605 specifically bound target cells. Arrows indicate Qdot-labeled SINV. Bar = 25 µm. (c) Biotinylated SINV labeled with Qdot 605 was active infecting target cells and expressing GFP. Images were taken 14 hours after infection. Arrowheads indicate Qdot signals. Bar = 25 µm. round of infection. Examination of randomly selected cells we used time-lapsed microscopy to record the movement of revealed that >70% cells expressing GFP (n = 500) were also the virus in real time under TIRF. In order to synchronize positive for Qdot 605 (Figure 1(c)), which suggested that the viral attachment and to inhibit viral entry, we performed Qdot-labeled SINV was able to enter the cells and continue the incubation reactions at 4 Coronice.Thiswould to replicate and express viral genome. presumably arrest SINV at surface attachment stage. The cell culture was then shifted to a 35 C environmental control 3.2. Dynamics of Qdot-Labeled SINV on BHK Cell Surface. To chamber for microscopy. We first recorded the movement study the dynamics of SINV on host cell plasma membranes, of the SINV-C -YFP, which had a 44-amino-acid domain Nonlabeled SIN Biotin-SIN 4 Advances in Virology 1.4 Step 1 Step 2 1.2 0 s 10 s 20 s 30 s 40 s 50 s 60 s 70 s 0.8 ∗∗ ∗ ∗∗ ∗ ∗ ∗ 0.6 80 s 90 s 100 s 110 s 115 s 120 s 125 s 130 s ∗ ∗ ∗ ∗ ∗ 0.4 ∗ ∗ 0.2 0 50 100 150 Time (s) (a) 1.2 Step 1 Step 2 −5 s 25 s 30 s 35 s 40 s 45 s 50 s 55 s 0.8 0.6 60 s 65 s 70 s 75 s 80 s 85 s 95 s 100 s 0.4 0.2 0 20406080 100 Time (s) (b) a(60%) b(23%) c(7%) d(10%) (c) Figure 2: Single-particle tracking of a SINV virus binding to cell surface. Pseudocolor images showing that SINV attachment involved a two-step binding process. (a) A SINV-C -YFP (arrow) was tracked under TIRF mode for 130 seconds. Its movement rate was measured manually frame-by-frame. Boxed images indicate no measurable movement by the virus. (b) Tracking of a SINV labeled with Qdot 605. (c) Four types of representative trajectories of Qdot-labeled SINV movement in BHK plasma membranes. a: immobile; b: mobile but confined in small areas (<0.5 µm diameter); c: mobile through long distances (>1 µm); d: mobile through medium distances (0.5–1 µm). Scale bar = 10 µm. Movement rate (µm/2 s) Movement rate (µm/2 s) Advances in Virology 5 0 1020304050607080 82 84 86 88 90 92 94 96 98 100 110 120 130 140 150 160 170 Overlay Figure 3: Dual color TIRFM of SINV and Rab-5 after endocytosis. A SINV virus after entering the cell was tracked (red). After approximately 90 seconds, it was associated with GFP-Rab5 (green). The association lasted ∼60 second before the GFP-Rab5 dissociated and the SINV moved deeper into the cell. Time in seconds is labeled. Arrowheads indicate the association of Rab5. The last image shows an overlay composite picture of the cell. of the capsid protein replaced with EYFP. This virus was step involved a highly mobile receptor, and a second step shown to retain the same infectivity as the wild-type virus involved the immobilization of the receptor/virus, perhaps generated from pToto/3 2J (data not shown). As shown in prior to viral internalization (Figure 2). Figure 2(a), the virus moved rapidly in a random fashion at rates ranging from 0 to 600 nm/second. We were able 3.3. Tracking SINV after Cell Entry. SINV has been shown to identify two types of virus-receptor association based to undergo clathrin-dependent endocytosis after receptor on the rate of movement. One was brief and transient, binding [17]. However, the fate of the virus following which was exemplified by rapid movement of the virus. The endocytic reaction remains to be fully delineated. By tracking other type of virus-receptor interaction appeared to be firm single SINV particles labeled with Qdot 605, we were attachment, accompanied by the immobilization of the virus able to determine that SINV was transported via Rab5- (Figure 2(a)). When we tracked SINV labeled with Qdot 605, containing early endosomes. BHK cells expressing GFP-Rab5 the virus also exhibited the two types of cell association as were infected with Qdot-labeled SINV, and the virus was the SINV-C -YFP. The movement of attached Qdot-SINV tracked at 35 C under TIRF. By taking the advantage of the appeared similar to that of SINV-C -YFP, with a max rate superior optical sectioning capability of TIRFM, we were of ∼500 nm/second (Figure 2(b)). The SINV moved rapidly able to identify an internalized SINV based on its presence with a random trajectory. There were frequent associations in the same optical plane with Rab5 under TIRF. As shown and disassociations between the virus and cell surface. in Figure 3, SINV was targeted to Rab5-containing early Most interactions appeared brief and transient. Full viral endosomes after internalization. The co-localization of SINV attachment became evident when the virus was firmly bound with Rab5 lasted from 15 to 60 seconds, which indicated a to the cell surface and became nearly immobile (Figure 2(b)). rapid maturation of the early endosomes to late endosomes. This finding suggested that Qdot did not significantly impact Interestingly, about 40% of the SINV did not colocalize with on the property of virus-receptor interactions. To investigate GFP-Rab5 (data not shown). further into the behavior of Qdot-labeled SINV during cell surface attachment, we collected images from multiple cells (n = 200) and recorded 100 frames at 2 sec per frame rate for 4. Discussion each cell. As illustrated in Figure 2(c), we observed 4 typical movements of SINV on cell surface. Nearly 60% of the viral Quantum dots have been used in a wide range of applications particles were virtually immobile, indicating that they were including visualization of hepatitis C virus infection in likely bound to receptors nonspecifically or they might be human liver [18], tracking single SV40 virus [19], and functionally impaired from Qdot labeling. The other 40% of imaging HER2 on tumor cells in real time [20]. Using Qdot- the SINV particles showed a heterogeneous lateral motility based single-virus tracking, we were able to begin dissecting along the membranes. While some (23%) were confined in a the early events involving SINV infection. We have shown small area (<0.25 µm ), others (17%) were moving beyond that, during a productive infection, SINV employed a two- several µm in distance. The rate of SINV movement was step binding reaction to achieve high-affinity binding with also heterogeneous, ranging from 0 to 500 nm a second its receptors. The first binding reaction resulted in a bound (Figure 2). It appeared that the virus often had preference virus that was highly mobile and dynamic on the host cell for certain sites and would move in the vicinity. Moreover, surface. This binding often did not lead to viral internal- movement would dramatically slow down once the virus ization and could last from seconds to several minutes. settled on a particular site (Figure 2). These results suggested The second step was high-affinity binding and attachment, a two-step attachment process for the motile SINV. A first which was accompanied by drastically reduced viral mobility. 6 Advances in Virology The immobilization of the virus might be a necessary step and Technology (HR07-056) and Sarcoma Foundation of before viral endocytosis. After internalization, Qdot-SINV America. appeared to be transported via early endosomes containing Rab5 (Figure 3), which was consistent with previous reports References on Semliki Forest virus [21]. However, approximately 40% [1] J. H. Strauss and E. G. Strauss, “The alphaviruses: gene expres- of the Qdot fluorescence did not colocalize with Rab5-GFP sion, replication, and evolution,” Microbiological Reviews, vol. (data not shown). One possibility for such a significant 58, no. 3, pp. 491–562, 1994. amount of Qdot-SINV without GFP-Rab5 co-localization [2] R. M. Taylor, H. S. Hurlbut, T. H. Work, J. R. Kingston, was that they involved Rab5-negative endosome domains, and T. E. Frothingham, “Sindbis virus: a newly recognized similar to those reported for Semliki Forest virus [21]. arthropodtransmitted virus,” The American Journal of Tropical Alternatively, due to Qdot’s highly stable and strong fluo- Medicine and Hygiene, vol. 4, no. 5, pp. 844–862, 1955. rescence, it was difficult to record low levels of Rab5-GFP [3] E. G. Strauss, C. M. Rice, and J. H. Strauss, “Complete fluorescence, especially after long exposure (>2minutes). nucleotide sequence of the genomic RNA of Sindbis virus,” Thus, the association of the Qdot-SINV with Rab5-negative Virology, vol. 133, no. 1, pp. 92–110, 1984. compartment remains to be further investigated. [4] J. A. Lemm and C. M. Rice, “Roles of nonstructural polypro- One advantage for quantum dots is that they are highly teins and cleavage products in regulating Sindbis virus RNA resistant to photobleaching. This allows prolonged excita- replication and transcription,” Journal of Virology, vol. 67, no. tion of the sample of interest, which in some instance re- 4, pp. 1916–1926, 1993. quires minutes to hours of observation. In our experience [5] Y. Shirako and J. H. Strauss, “Regulation of Sindbis virus RNA replication: uncleaved P123 and nsP4 function in minus- using a 20 mW 488 nm laser, the Qdot 605 retained nearly strand RNA synthesis, whereas cleaved products from P123 are 100% fluorescence intensity after 3 minutes of continuous required for efficient plus-strand RNA synthesis,” Journal of illumination under TIR. In comparison, the EYFP showed Virology, vol. 68, no. 3, pp. 1874–1885, 1994. a dramatic reduction of fluorescence emission after only [6] W. B. Klimstra, K. D. Ryman, and R. E. Johnston, “Adaptation 30 seconds of excitation and lost >90% of the intensity of Sindbis virus to BHK cells selects for use of heparan sulfate after 3 minutes. Consequently, tracking the SINV-C -YFP as an attachment receptor,” Journal of Virology, vol. 72, no. 9, was limited to <2 minutes, making it difficult to study the pp. 7357–7366, 1998. virus after internalization. With Qdot-labeled SINV, we were [7] K. S. Wang, R. J. Kuhn, E. G. Strauss, S. Ou, and J. H. Strauss, able to follow the virus for up to an hour, which greatly “High-affinity laminin receptor is a receptor for Sindbis virus expanded data collection on viral infection process. Another in mammalian cells,” Journal of Virology,vol. 66, no.8,pp. benefit of quantum dots is that they have broad excitation 4992–5001, 1992. spectra, which allows simultaneous excitation of multiple [8] A. P. Byrnes and D. E. Griffin, “Binding of Sindbis virus to cell fluorophores. As shown in Figure 3,wewereabletoperform surface heparan sulfate,” Journal of Virology,vol. 72, no.9,pp. 7349–7356, 1998. dual TIRF imaging using a single excitation source. [9] S. Lustig, A. C. Jackson, C. S. Hahn, D. E. Griffin, E. G. The limitation of quantum dots is their relatively large Strauss, and J. H. Strauss, “Molecular basis of Sindbis virus sizes, which range from 10 to 20 nm. When conjugated neurovirulence in mice,” Journal of Virology,vol. 62, no.7,pp. to biomolecules such as the E1/E2 envelope proteins of 2329–2336, 1988. SINV, they could potentially interfere with the normal [10] S. C. Derrick,A.L.Yang, andS.L.Morris, “Vaccination functions of the proteins. The greatest loss of viral infectivity with a Sindbis virus-based DNA vaccine expressing antigen was due to the initial biotinylation reaction. It appeared 85B induces protective immunity against Mycobacterium that some viruses were rendered inactive due to excessive tuberculosis,” Infection and Immunity, vol. 73, no. 11, pp. biotinylation. Interestingly, binding of streptavidin-Qdot 605 7727–7735, 2005. had less adverse effect on SINV infection, which was further [11] H. V. Huang, “Sindbis virus vectors for expression in animal improved by adding the Qdot after SINV attachment to cells,” Current Opinion in Biotechnology, vol. 7, no. 5, pp. 531– the cell surface. The relatively benign hindering effect upon 535, 1996. Qdot binding to SINV suggested that, at an average 5– [12] C. Li, Y. Gu, D. Andrade, and Y. Liu, “Susceptibility of 12 nm diameter, Qdot 605 did not significantly affect the colorectal cancer cells to sindbis virus infection,” Journal of Experimental Therapeutics and Oncology, vol. 8, no. 2, pp. 167– infectivity of SINV. We believe this is likely due to the fact 175, 2009. that Qdot 605 bound with SINV mostly at an 1 : 1 ratio. [13] J. C. Tseng, B. Levin, A. Hurtado et al., “Systemic tumor target- Nevertheless, conditions for the conjugation process can be ing and killing by Sindbis viral vectors,” Nature Biotechnology, further optimized to minimize hindering, which will make vol. 22, no. 1, pp. 70–77, 2004. quantum-dot-based tracking a more widely used tool for [14] S. R. Nelson,M.Y.Ali,K.M.Trybus, andD.M.Warshaw, investigating molecular details of viral infection. “Random walk of processive, quantum dot-labeled myosin Va molecules within the actin cortex of COS-7 cells,” Biophysical Acknowledgments Journal, vol. 97, no. 2, pp. 509–518, 2009. [15] F. Pinaud, X. Michalet, L. A. Bentolila et al., “Advances in fluo- The authors thank Dr. Guangpu Li (University of Oklahoma rescence imaging with quantum dot bio-probes,” Biomaterials, Health Sciences Center) for providing the pToto/3 2J plasmid vol. 27, no. 9, pp. 1679–1687, 2006. and pGFP-Rab5 vector. This work was supported by grants [16] E. Saint-Michel, G. Giannone, D. Choquet, and O. Thoumine, from the Oklahoma Center for the Advancement of Science “Neurexin/neuroligin interaction kinetics characterized by Advances in Virology 7 counting single cell-surface attached quantum dots,” Biophys- ical Journal, vol. 97, no. 2, pp. 480–489, 2009. [17] L. DeTulleo and T. Kirchhausen, “The clathrin endocytic pathway in viral infection,” EMBO Journal, vol. 17, no. 16, pp. 4585–4593, 1998. [18] Y. Liang, T. Shilagard, S. Y. Xiao et al., “Visualizing hepatitis C virus infections in human liver by two-photon microscopy,” Gastroenterology, vol. 137, no. 4, pp. 1448–1458, 2009. [19] P. Kukura,H.Ewers,C.Muller ¨ , A. Renn, A. Helenius, and V. Sandoghdar, “High-speed nanoscopic tracking of the position and orientation of a single virus,” Nature Methods, vol. 6, no. 12, pp. 923–927, 2009. [20] H. Tada, H. Higuchi, T. M. Wanatabe, and N. Ohuchi, “In vivo real-time tracking of single quantum dots conjugated with monoclonal anti-HER2 antibody in tumors of mice,” Cancer Research, vol. 67, no. 3, pp. 1138–1144, 2007. [21] A. Vonderheit and A. Helenius, “Rab7 associates with early endosomes to mediate sorting and transport of Semliki forest virus to late endosomes,” PLoS Biology, vol. 3, no. 7, pp. 1225– 1238, 2005. 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Imaging Early Steps of Sindbis Virus Infection by Total Internal Reflection Fluorescence Microscopy

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Hindawi Publishing Corporation Advances in Virology Volume 2011, Article ID 535206, 7 pages doi:10.1155/2011/535206 Research Article Imaging Early Steps of Sindbis Virus Infection by Total Internal Reflection Fluorescence Microscopy Youling Gu, Yuanzheng Yang, and Yuechueng Liu Department of Pathology, University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, Ok 73190, USA Correspondence should be addressed to Yuechueng Liu, yuechueng-liu@ouhsc.edu Received 11 July 2011; Revised 7 September 2011; Accepted 8 September 2011 Academic Editor: Amiya K. Banerjee Copyright © 2011 Youling Gu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Sindbis virus (SINV) is an alphavirus that has a broad host range and has been widely used as a vector for recombinant gene transduction, DNA-based vaccine production, and oncolytic cancer therapy. The mechanism of SINV entry into host cells has yet to be fully understood. In this paper, we used single virus tracking under total internal reflection fluorescence microscopy (TIRFM) to investigate SINV attachment to cell surface. Biotinylated viral particles were labeled with quantum dots, which retained viral viability and infectivity. By time-lapse imaging, we showed that the SINV exhibited a heterogeneous dynamics on the surface of the host cells. Analysis of SINV motility demonstrated a two-step attachment reaction. Moreover, dual color TIRFM of GFP-Rab5 and SINV suggested that the virus was targeted to the early endosomes after endocytosis. These findings demonstrate the utility of quantum dot labeling in studying the early steps and behavior of SINV infection. 1. Introduction SINV has long been used as an experimental model for studying encephalitis because it causes encephalomyelitis in Sindbis virus (SINV) is an alpha virus and a member of neonatal mice [9]. Moreover,sinceSINVhas abroad host the Toga virus family that also includes Semliki Forest range and can deliver efficient gene expression, vectors based and Ross River viruses [1]. It was first isolated in 1952 on the virus have been developed for vaccine production from mosquitoes found in the Sindbis area in Egypt [2]. and gene therapy purposes [10, 11]. In cancer therapy, SINV The decoded SINV genome revealed a single-strand RNA has been tested as a potential oncolytic reagent. It has been of 11.7 kb in size [3]. Upon cell entry, the viral RNA strand shown that a SINV strain (Toto1101) derived from the wild- serves as a messenger RNA to translate four nonstructure type SINV was effective targeting and killing tumor cells of genes that are essential for viral replication. In late stages of ovarian, colon, prostate, and liver cancer patients, leading to infection, structural genes are translated and synthesized tumor regression in animal models [12, 13]. from a subgenomic RNA. The resulting viral particles con- In recent years, quantum-dot- (Qdot-) based fluores- taining nucleocapsids of genomic RNA and capsid proteins, cence labeling has become increasingly employed for imaging packaged with envelope proteins E1/E2, are formed and bud- cellular events including single-molecule tracking and live- ded off as mature virus [4, 5]. The receptors that are involved cell imaging [14–16]. Qdots are semiconductor nanocrystals in SINV entry into host cells remain poorly characterized. with a broad excitation and emission spectra. One advan- One of such receptors is the 67 kD high-affinity laminin tage of Qdot is its resistance to photobleaching, allowing receptor (LR), which has been shown to mediate SINV prolonged exposure to excitation light. In the current study, infection of BHK cells [6, 7]. In addition, it has been reported we labeled SINV with biotin and conjugated the virus with that heparan sulfate, a major cell surface component, is Qdot 605. Using single-virus tracking, we were able to dissect directly involved in SINV infection of cultured cells [6, 8]. the behavior and dynamics of individual SINV during the Little is known regarding viral attachment and movement on initial stages of infection, which showed a two-step, receptor- host cell surfaces during early stages of SINV infection. mediated cell attachment process. 2 Advances in Virology 2. Materials and Methods ing with PBS to remove unbound virus, the cells were further incubated with streptavidin-conjugated Qdot 605 2.1. Cell Culture and SINV Preparation. BHK-21 cells were (Invitrogen) for 60 minutes and washed 3 times with cold cultured in MEM supplemented with 10% FBS and antibi- PBS. The cells were fixed with 3.7% formaldhyde in PBS for otics. The cells were maintained in a humidified 37 C 30 minutes at room temperature. Fluorescent images were incubator with 5% CO . For the construction of SINV-GFP acquired and digitized for analysis. For live cell experiments, expressing the green fluorescent protein (GFP), GFP cDNA the infection was performed on ice or at 4 Cfor 1hour (from pS65T-C1, Clonetech) was first cloned into a shuttle ◦ before shifting to a 35 C chamber for microscopy. vector pH2J1Y which was modified from pH3 2J1 (kindly For TIRFM, we used an Olympus IX71 equipped with a provided by Dr. Guangpu Li, University of Oklahoma, 60X (n.a. = 1.45) TIRF objective heated to 37 C. Cells were Ok, USA). The plasmid pH2J1Y contained a linker with cultured in a home-made glass bottom dish and maintained additional multiple cloning sites, Kpn I, Sal I, EcoR V, at 35 C in an environmental control chamber (Olympus, Hind III, and Nhe I, which was inserted between the Xba USA) supplemented with 5% CO . TIRF illumination was I and Bam H1 sites of pH3 2J1. The cDNA encoding the achieved using a 20 mW 488 nm laser source for excitation GFP was excised by Nhe I and Bam H1 and ligated into and a long-pass dichroic beam splitter (500 nm). GFP/YFP pH2J1Y at same sites. An Apa I-Xho I fragment of pH2J1Y was viewed with an emission filter of 525/20 nm, and harboring the GFP cDNA was subcloned into the SINV Qdot 605 was viewed with an emission filter of 600/40 nm. vector pToto/3 2J. To construct SINV-C -YFP, in which the Emission filters were controlled with a Lambda 10 (Sutter 62–106 amino acid sequence of the capsid protein was Instrument, Novato, CA, USA) high-speed filter wheel. replaced with EYFP, an Sph I and an Mlu I sites flanking the Images were captured with a CCD camera (Quantix 57, replaced capsid sequence were introduced into pToto/3 2J ◦ Photometrics, Tucson, Ariz, USA) air cooled to −25 C. The by PCR mutagenesis. EYFP with Sph I and Mlu I cloning camera was controlled by IPlab 3.9.4 (Scanalytics, Fairfax, Va, sites was amplified by PCR using pEYFP-C1 as a template. USA) and analyzed with the IPlab software. For expression Recombinant SINV was produced by transfection of BHK21 of GFP-Rab5, BHK-21 cells were transfected using Lipo- cells using capped RNAs derived from sp6 RNA polymerase fectamine 2000 (Invitrogen) and pGFP-Rab5 (Dr. Guangpu transcription of Xho I-linearized plasmid templates. Viral Li, University of Oklahoma Health Sciences Center). The stocks were obtained by harvesting the culture supernatants cells expressing GFP-Rab5 were imaged under TIRFM 24–48 30 hr after infection and aliquots were stored at −80 C until hours after transfection. use. Viral titers were determined by plaque assay on BHK21 monolayers using crystal violet staining. 3. Results 2.2. Biotinylation of SINV. SINV-GFP was collected in serum 3.1. Labeling SINV with Qdot 605. SINV was first conjugated free MEM diluted with saline (1 : 1) at ∼4 × 10 pfu/mL to biotin linked through a 29APEG arm. In orderto and was labeled with biotin at room temperature or on achieve a near 100% labeling of SINV, an excessive amount ice. Briefly, the virus was mixed with 10 mM stock NHS- of NHS-biotin at 300 µM was used in the reaction. This PEG4-Biotin (29 A spacer arm, Pierce) in PBS (pH 8.0) was necessary for ensuring that the observed infection was at a final concentration of 300 µMfor 30minatroom indeed by the biotinylated SINV, not by the native and temperature. The reaction was terminated by adding glycine un-modified viruses. The conjugation reaction, however, 8 6 to 0.1 M and incubating for 15 min at room temperature. For reduced the infective titer of the virus from 4×10 to 10 – biotinylation on ice, SINV was incubated with NHS-PEG4- 7 10 pfu/mL. Such a reduction was mostly due to the excessive Biotin as described above for 90 minutes on ice. The reaction chemical modification by NHS-biotin since viral titer was was terminated by addition of glycine and further incubation less affected when the concentration of NHS-biotin was at room temperature for 20 minutes. We did not notice any reduced (data not shown). Western blot analysis showed that significant differences in the efficiency of biotinylation and both the E1/E2 envelope proteins were labeled (Figure 1(a)). the infectivity of the resulting biotinylated SINV produced Moreover, the excessive crosslinking caused some E1/E2 under the two labeling conditions. For western blot analysis proteins shifting to slightly higher apparent molecular weight of the labeled SINV, aliquots of SINV were heated at 100 C (Figure 1(a)). When the biotin-SINV was preincubated with for3mininsamplebuffer containing 1% SDS, 10 mM Qdot 605, a medium-sized quantum dot of 5–12 nm diame- EDTA, 10 mM DTT, 15% glycerol, 20 mM Tris-HCl, pH ter, the virus-Qdot conjugate was still active infecting BHK- 6.8, and 0.01% bromophenol blue. The SINV proteins were 21 cells with a small reduction in infection activities (<2 separated by SDS-PAGE and transferred onto nitrocellulose fold). However, if we preincubated the cells with SINV for 1 membrane. An alkaline phosphatase-conjugated streptavidin ◦ hour at 4 C and then added Qdot, the reduction in infectivity (1 : 5,000, Sigma) was used to visualize biotinylated SINV became minimum. Specific binding to host cells by Qdot- envelope proteins. labeled SINV was evident under fluorescence microscopy (Figure 1(b)). To determine if the Qdot-SINV was able to 2.3. SINV Infection, Fluorescence Microscopy, and Image enter cells and carry out viral replication, we followed the Analysis. Biotinylated SINV was used to infect BHK-21 cells expression of recombinant GFP encoded by the viral vector. at MOI (multiplicity of infection) of 5–20. The virus was BHK-21 cells were scored for GFP and Qdot 605 fluorescence incubated with the cells at 4 C for 1 hour. After wash- 14 hours after infection, following the completion of first Advances in Virology 3 (a) (b) GFP Qdot Phase contrast Overlay (c) Figure 1: Biotinylation and Qdot labeling of SINV. SINV was labeled with NHS PEG4-biotin as described in Section 2. (a) Western blot analysis of biotinylated SINV using alkaline-phosphatase-conjugated streptavidin showing efficient labeling of the E1/E2 envelope proteins (indicated by ). Lanes 1: control unlabeled SINV and 2: biotinylated SINV. Molecular weight markers (kD) are labeled. (b) Biotinylated SINV labeled with Qdot 605 specifically bound target cells. Arrows indicate Qdot-labeled SINV. Bar = 25 µm. (c) Biotinylated SINV labeled with Qdot 605 was active infecting target cells and expressing GFP. Images were taken 14 hours after infection. Arrowheads indicate Qdot signals. Bar = 25 µm. round of infection. Examination of randomly selected cells we used time-lapsed microscopy to record the movement of revealed that >70% cells expressing GFP (n = 500) were also the virus in real time under TIRF. In order to synchronize positive for Qdot 605 (Figure 1(c)), which suggested that the viral attachment and to inhibit viral entry, we performed Qdot-labeled SINV was able to enter the cells and continue the incubation reactions at 4 Coronice.Thiswould to replicate and express viral genome. presumably arrest SINV at surface attachment stage. The cell culture was then shifted to a 35 C environmental control 3.2. Dynamics of Qdot-Labeled SINV on BHK Cell Surface. To chamber for microscopy. We first recorded the movement study the dynamics of SINV on host cell plasma membranes, of the SINV-C -YFP, which had a 44-amino-acid domain Nonlabeled SIN Biotin-SIN 4 Advances in Virology 1.4 Step 1 Step 2 1.2 0 s 10 s 20 s 30 s 40 s 50 s 60 s 70 s 0.8 ∗∗ ∗ ∗∗ ∗ ∗ ∗ 0.6 80 s 90 s 100 s 110 s 115 s 120 s 125 s 130 s ∗ ∗ ∗ ∗ ∗ 0.4 ∗ ∗ 0.2 0 50 100 150 Time (s) (a) 1.2 Step 1 Step 2 −5 s 25 s 30 s 35 s 40 s 45 s 50 s 55 s 0.8 0.6 60 s 65 s 70 s 75 s 80 s 85 s 95 s 100 s 0.4 0.2 0 20406080 100 Time (s) (b) a(60%) b(23%) c(7%) d(10%) (c) Figure 2: Single-particle tracking of a SINV virus binding to cell surface. Pseudocolor images showing that SINV attachment involved a two-step binding process. (a) A SINV-C -YFP (arrow) was tracked under TIRF mode for 130 seconds. Its movement rate was measured manually frame-by-frame. Boxed images indicate no measurable movement by the virus. (b) Tracking of a SINV labeled with Qdot 605. (c) Four types of representative trajectories of Qdot-labeled SINV movement in BHK plasma membranes. a: immobile; b: mobile but confined in small areas (<0.5 µm diameter); c: mobile through long distances (>1 µm); d: mobile through medium distances (0.5–1 µm). Scale bar = 10 µm. Movement rate (µm/2 s) Movement rate (µm/2 s) Advances in Virology 5 0 1020304050607080 82 84 86 88 90 92 94 96 98 100 110 120 130 140 150 160 170 Overlay Figure 3: Dual color TIRFM of SINV and Rab-5 after endocytosis. A SINV virus after entering the cell was tracked (red). After approximately 90 seconds, it was associated with GFP-Rab5 (green). The association lasted ∼60 second before the GFP-Rab5 dissociated and the SINV moved deeper into the cell. Time in seconds is labeled. Arrowheads indicate the association of Rab5. The last image shows an overlay composite picture of the cell. of the capsid protein replaced with EYFP. This virus was step involved a highly mobile receptor, and a second step shown to retain the same infectivity as the wild-type virus involved the immobilization of the receptor/virus, perhaps generated from pToto/3 2J (data not shown). As shown in prior to viral internalization (Figure 2). Figure 2(a), the virus moved rapidly in a random fashion at rates ranging from 0 to 600 nm/second. We were able 3.3. Tracking SINV after Cell Entry. SINV has been shown to identify two types of virus-receptor association based to undergo clathrin-dependent endocytosis after receptor on the rate of movement. One was brief and transient, binding [17]. However, the fate of the virus following which was exemplified by rapid movement of the virus. The endocytic reaction remains to be fully delineated. By tracking other type of virus-receptor interaction appeared to be firm single SINV particles labeled with Qdot 605, we were attachment, accompanied by the immobilization of the virus able to determine that SINV was transported via Rab5- (Figure 2(a)). When we tracked SINV labeled with Qdot 605, containing early endosomes. BHK cells expressing GFP-Rab5 the virus also exhibited the two types of cell association as were infected with Qdot-labeled SINV, and the virus was the SINV-C -YFP. The movement of attached Qdot-SINV tracked at 35 C under TIRF. By taking the advantage of the appeared similar to that of SINV-C -YFP, with a max rate superior optical sectioning capability of TIRFM, we were of ∼500 nm/second (Figure 2(b)). The SINV moved rapidly able to identify an internalized SINV based on its presence with a random trajectory. There were frequent associations in the same optical plane with Rab5 under TIRF. As shown and disassociations between the virus and cell surface. in Figure 3, SINV was targeted to Rab5-containing early Most interactions appeared brief and transient. Full viral endosomes after internalization. The co-localization of SINV attachment became evident when the virus was firmly bound with Rab5 lasted from 15 to 60 seconds, which indicated a to the cell surface and became nearly immobile (Figure 2(b)). rapid maturation of the early endosomes to late endosomes. This finding suggested that Qdot did not significantly impact Interestingly, about 40% of the SINV did not colocalize with on the property of virus-receptor interactions. To investigate GFP-Rab5 (data not shown). further into the behavior of Qdot-labeled SINV during cell surface attachment, we collected images from multiple cells (n = 200) and recorded 100 frames at 2 sec per frame rate for 4. Discussion each cell. As illustrated in Figure 2(c), we observed 4 typical movements of SINV on cell surface. Nearly 60% of the viral Quantum dots have been used in a wide range of applications particles were virtually immobile, indicating that they were including visualization of hepatitis C virus infection in likely bound to receptors nonspecifically or they might be human liver [18], tracking single SV40 virus [19], and functionally impaired from Qdot labeling. The other 40% of imaging HER2 on tumor cells in real time [20]. Using Qdot- the SINV particles showed a heterogeneous lateral motility based single-virus tracking, we were able to begin dissecting along the membranes. While some (23%) were confined in a the early events involving SINV infection. We have shown small area (<0.25 µm ), others (17%) were moving beyond that, during a productive infection, SINV employed a two- several µm in distance. The rate of SINV movement was step binding reaction to achieve high-affinity binding with also heterogeneous, ranging from 0 to 500 nm a second its receptors. The first binding reaction resulted in a bound (Figure 2). It appeared that the virus often had preference virus that was highly mobile and dynamic on the host cell for certain sites and would move in the vicinity. Moreover, surface. This binding often did not lead to viral internal- movement would dramatically slow down once the virus ization and could last from seconds to several minutes. settled on a particular site (Figure 2). These results suggested The second step was high-affinity binding and attachment, a two-step attachment process for the motile SINV. A first which was accompanied by drastically reduced viral mobility. 6 Advances in Virology The immobilization of the virus might be a necessary step and Technology (HR07-056) and Sarcoma Foundation of before viral endocytosis. After internalization, Qdot-SINV America. appeared to be transported via early endosomes containing Rab5 (Figure 3), which was consistent with previous reports References on Semliki Forest virus [21]. 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Nelson,M.Y.Ali,K.M.Trybus, andD.M.Warshaw, investigating molecular details of viral infection. “Random walk of processive, quantum dot-labeled myosin Va molecules within the actin cortex of COS-7 cells,” Biophysical Acknowledgments Journal, vol. 97, no. 2, pp. 509–518, 2009. [15] F. Pinaud, X. Michalet, L. A. Bentolila et al., “Advances in fluo- The authors thank Dr. Guangpu Li (University of Oklahoma rescence imaging with quantum dot bio-probes,” Biomaterials, Health Sciences Center) for providing the pToto/3 2J plasmid vol. 27, no. 9, pp. 1679–1687, 2006. and pGFP-Rab5 vector. This work was supported by grants [16] E. Saint-Michel, G. Giannone, D. Choquet, and O. Thoumine, from the Oklahoma Center for the Advancement of Science “Neurexin/neuroligin interaction kinetics characterized by Advances in Virology 7 counting single cell-surface attached quantum dots,” Biophys- ical Journal, vol. 97, no. 2, pp. 480–489, 2009. [17] L. DeTulleo and T. 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