Progression of Respiratory Syncytial Virus Infection Monitored by

Elizabeth L. Bentzen, Frances House, Thomas J. Utley, James E. Crowe,, and David W. Wright*. Department of Chemistry, Vanderbilt University, Station B...
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Progression of Respiratory Syncytial Virus Infection Monitored by Fluorescent Quantum Dot Probes

2005 Vol. 5, No. 4 591-595

Elizabeth L. Bentzen,† Frances House,‡ Thomas J. Utley,‡ James E. Crowe, Jr.,‡ and David W. Wright*,† Department of Chemistry, Vanderbilt UniVersity, Station B 351822, NashVille, Tennessee 37235-1822, and Pediatrics and Microbiology and Immunology, Vanderbilt UniVersity School of Medicine, NashVille, Tennessee 37232-2581 Received November 22, 2004; Revised Manuscript Received March 7, 2005

ABSTRACT We report the use of quantum dots (QDs) to identify the presence and monitor the progression of respiratory syncytial virus (RSV) infection over time by labeling the F and G proteins. In addition, co-localization of these viral proteins was shown using confocal microscopy. The implications of these results are that QDs may provide a method for early, rapid detection of viral infection and open the door for future studies of the intricate spatial features cell trafficking of viral proteins.

Colloidal semiconductor quantum dots (QDs) are finding increased uses in a variety of practical medical applications. They have been used as immunofluorescent probes to detect the Her2 breast cancer marker,1 as signal transduction components in immunoassays for microbial toxins,2 and as labels for dynamic studies of cancer cell motility and correlation of metastatic potential.3 Recently, near-IR QDs have been used in the surgical suite to map sentinel lymph nodes in a large animal cancer model.4 Many of the inherent properties offered by QDs, including broad absorption spectra, narrow emission spectra, and excellent photostability, are driving their expanded use. Despite their proven utility, there have not been widespread reports of their use for the detection or imaging of viruses. Respiratory infections are among the leading causes of medical presentation in the United States. In humans, the most common respiratory viruses are influenza virus, parainfluenza, metapneumovirus, and respiratory syncytial virus (RSV). RSV is the leading cause of lower respiratory tract infections in infants and young children, resulting in an estimated 90,000 hospitalizations of children under the age of 5.5 Increasingly, RSV is becoming a concern for elderly and immunocompromised populations.6 Ribavirin is an antiviral that is available for treatment of RSV,7 but treatment is effective only when given early in the course of infection and presently there is no approved vaccine.8 * Corresponding author. Tel.: 1-615-322-2636. Fax: 1-615-343-12354. E-mail: [email protected]. † Department of Chemistry, Vanderbilt University, Nashville, TN, 37235-1822. ‡ Pediatrics and Microbiology and Immunology. 10.1021/nl048073u CCC: $30.25 Published on Web 03/29/2005

© 2005 American Chemical Society

Figure 1. Viral structure of respiratory syncytial virus. The structure shows the location of the F and G proteins at the surface of the virion particle in the lipid bilayer envelope. Antibodies to these two surface glycoproteins were used to label RSV infected HEp-2 cells.

RSV is an enveloped negative-sense, single-stranded RNA paramyxovirus that produces cell fusion resulting in multinucleated giant cells, referred to as syncytia, when HEp-2 monolayer HEp-2 cell cultures are inoculated.9 The structure of the RSV virion consists of a lipid membrane envelope studded with the fusion (F) and attachment (G) proteins supported by matrix proteins surrounding a nucleocapsid core composed of the genomic RNA, nucleoprotein (N), phosphoprotein (P), and polymerase proteins (Figure 1). The F protein mediates the fusion of the viral membrane with the host cell membrane delivering the nucleocapsid of the virion particle into the host cell cytoplasm. The G surface glycoprotein aids in attachment of the virion to a host cell. Once the nucleocapsid has been released into the host cell, the viral genes initially are transcribed to produce mRNA

Figure 2. Replication cycle of RSV. The RSV virion attaches to the host cell via the G protein. The F protein then mediates fusion of the viral membrane into the host cell membrane. The RSV virion nucleocapsid is released into the host cell cytoplasm. The host cell ribosomes are used to translate the RSV mRNAs transcribed by the viral polymerase to produce viral proteins; the full-length RSV genomic RNA is replicated at later stages of infecton. These newly synthesized viral proteins and genomic RNA are assembled and a new virion buds from the plasma membrane at the surface of the infected cell. Additionally, F protein on the infected cell and the newly budding virions may fuse with neighboring cells to form syncytia.

and newly translated viral proteins, and eventually the fulllength RSV genomic RNA is replicated. These newly synthesized viral proteins and genomic RNA molecules are assembled into virions that bud from the surface of the infected cell, producing a virus particle to continue the cycle of infection. The fusion protein on the surface of the cell or perhaps newly budding virions may also simply fuse with neighboring cells to form syncytia (Figure 2). Clusters of syncytia may coalesce to form a plaque, an area of cells in a monolayer culture that displays cytopathic effects, with centers devoid of viable cells due to virus-induced lysis.10 Considering the infection cycle of RSV, the virus seems an ideal subject for study of viral diagnostic methods using QD probes. The fusion (F) and attachment (G) proteins are incorporated into the surface of the host cell, making them ideal antigenic markers for the presence of RSV. Further, viral replication provides an inherent amplification of magnitude of these markers as a function of time. Capitalizing on these aspects of virus infectivity, we report the use of QDs to identify the presence, and monitor the progression of RSV infection. Strategies for the sequential labeling of the F and G protein were optimized from literature methods.11 In a typical experiment, 80-90% confluent monolayer cell cultures of HEp-2 cells were infected with 50 PFU (plaque forming units) of RSV wild-type strain A2 per well in 96 well microtiter plates. The infected HEp-2 cell monolayer cultures were overlaid with methylcellulose and incubated at 37 °C with 5% CO2 for the duration of the study. At desired intervals, a plate of infected cells was fixed with cold 80% methanol and stored at 4 °C for at least 1 h. At each time point examined, the F protein was labeled first. Cells were 592

blocked with 2% BSA in Dulbeccos’ PBS (Mg2+ and Ca2+ free) for 1 h. After blocking, the cells were incubated for 30 min at room temperature with a 1:104 dilution of a primary antibody mixture of two purified F protein specific mouse monoclonal antibodies (clones 1269 and 1214; designated Fmix),12 previously reported to bind to different antigenic sites of the F protein, for 30 min. After washing with PBS the cells were incubated in a 1:1000 dilution of biotinylated secondary antibodies (polyclonal goat anti-mouse) for 1 h. Again, excess antibody was removed with PBS. The cells were labeled with 10 nM 605 nm streptavidin quantum dots for 30 min. The cells were washed with PBS and then labeled for the G protein. The G protein was labeled by blocking the 605 nm streptavidin QDs with 200 µg/mL biotin and employing the same primary13 and secondary antibody strategy using 10 nM 525 nm streptavidin QDs. Subsequently, labeled cells were imaged. Using confocal laser scanning microscopy, the F and G proteins of RSV were examined four days after infection.14 Confocal microscopic images showed that the F and G proteins were found predominantly on the surface of infected cells with significant co-localization as seen in the composite image of both the 525 and 605 nm channel (Figure 3). Both of these observations are consistent with the previously determined pathophysiology of RSV infection.9,10,15 Although there is significant co-localization of F and G protein in these images, the orthogonal slices (XY and XZ) of the confocal image also reveal areas of segregation of F protein (605 nm QDs) and G protein (525 nm QDs) at opposing surfaces of the cell. We have observed this phenomenon repeatedly in HEp-2 cell monolayer cultures that are nonpolarized and exhibit cytopathological effects. In contrast, polarized cells preferentially bud RSV virions from the apical surface.16,17 To ensure that this observation was not due to the sequential labeling of the target proteins, quantum dots conjugated to species-specific secondary antibodies were used to label the F and G proteins simultaneously, and the same observation was made (Supporting Information). Control images clearly indicate that there is no bleedthrough of the 525 nm QDs into the 605 nm channel or vice versa (Supporting Information) with the use of QDs. To ensure that the biotinylated secondary antibody for the F protein was saturated with streptavidin QDs and that co-localization was in fact being observed, a second incubation with streptavidin QDs was performed as control. We could not detect a significant difference in the fluorescence intensity of the initial F protein labeling and that following re-incubation (Supporting Information). However, there was a substantial increase in fluorescent intensity after the subsequent labeling of the G protein when the same colored QDs were used. These observations suggest that under these labeling conditions, the primary biotinylated antibodies were indeed saturated after the first QD labeling/biotin blocking step and that the observed co-localization of the F and G proteins is not an artifact of the labeling strategy. The sensitivity of quantum dot probes for the identification of RSV infection was also examined as a function of the initial viral concentration (PFUs) used to infect a given well Nano Lett., Vol. 5, No. 4, 2005

Figure 3. Confocal microscopic image showing co-localization of the F and G proteins in RSV infected HEp-2 cell monolayer cultures four days post infection. The composite image (A2) shows orthogonal slices XZ(A1) and YZ(A3), suggesting that the F and G proteins are predominately located on the surface of the infected cells. In addition to the co-localization the images show segregated areas of F and G protein at opposing surfaces. Images B (605 nm channel) and C (525 nm channel) are of the same syncytium.

Figure 4. Fluorescence intensity of the F protein (9) labeled with 605 nm streptavidin QDs 24 h after infection of HEp-2 cell monolayer cultures with varying concentrations of RSV wild-type strain A2. The graph indicates that in a 96-well microtiter plate the monolayer is saturated with infectious particles at 110 pfu per well and that the limit of detection is 35-50 pfu at 24 h. At 42 h post infection there is a linear response of F protein production from 1 to 110 pfu (inset).

(Figure 4). At 18-24 h post infection, wells infected by RSV and labeled with QDs for the F protein show a linear increase in detected F protein that saturates at initial concentrations

of infecting virus higher than 110 PFUs, as the monolayer becomes saturated with virus particles. The limit of detection from this curve is between 35 and 50 PFUs at 24 h. At times later than 42 h post infection, the range of the linear response of F protein production is expanded to 1 to 110 PFUs as a result of inherent amplification of viral proteins during replication. Prior to 24 h, the response differs due to the time needed for the virus to commandeer the host cell machinery and bud new virions. All subsequent infections were performed with PFUs below the saturation level of 24 h. Having demonstrated that QDs can serve as immunofluorescent labels for key RSV proteins, the progression of viral infection was followed by monitoring the expression of F and G proteins over time. At each time point, QD labeled cells (as described above) were imaged using a Nikon Diaphot epifluorescent microscope equipped with phase/ fluorescent/camera and a Quixell automated micromanipulator. An optimized filter set for 605 and 525 nm QDs (Chroma Inc.) was used, and images were captured with a Nikon D100 digital camera. Images of cells 1, 24, 48, 72, and 96 h after infection (Figure 5) indicate an increase in the amount of both the F and G proteins over the course of infection. Furthermore, both the F and G proteins could be detected visually as early as 1 h after infection. In addition, the F protein was labeled using a common organic fluoro-

Figure 5. Fluorescent images of the F protein labeled with 605 nm streptavidin quantum dots at 1 h (A1), 24 h (B1), 48 h (C1), 72 h (D1), and 96 h (E1) after infection. The G protein was subsequently labeled using 525 nm streptavidin quantum dots on the same infected monolayer, images are shown for the same time intervals. Nano Lett., Vol. 5, No. 4, 2005

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Figure 6. (A) Graph of fluorescence intensity of the F protein (9) measured at 605 nm and the G protein (b) measured at 525 nm over the course of RSV infection of HEp-2 cell monolayer cultures. The graph indicates that early in the RSV infection F and G proteins are located on the surface during attachment of the virion. At 18 h there is a reduction of F and G proteins due to the fusion of the virion into the host cell. During the remainder of the infection F and G protein production increases as the host cell continues to produce new viral proteins and new virions. After 84 h the F and G production begins to decline due to lack of viable cells. B) Western blot of RSV F and N protein over the course of a 4 day infection. Only the N protein at 96 h is detectable, indicating that at this low multiplicity of infection the amount of viral proteins made are below the level of detection for western blot. (C) Real time quantitative RT-PCR data showing the production of the mRNA for the N(b) and F(9) proteins of RSV at this low moi over the course of infection. 594

phore, fluorescein isothiocyanate (FITC), over the same period (Supporting Information). While FITC conjugated antibodies could be used to detect the F protein at time points later than 24 h, they required exposures 46 times that of the QD labeled cells for clear imaging and were almost completely photodegraded within 15 min. In addition to epifluorescent microscopy, the fluorescence intensity of the labeled cells was examined using a Synergy HT fluorometer with time-resolved excitation at 250 nm and emission filters 590(18 (605 nm quantum dots) and 528(10 (525 nm quantum dots) at constant gain. The fluorescence intensities indicated that there is an increase in F and G protein production over the course of infection (Figure 6). While F and G protein production fluctuated, the overall trend was that the amount of each protein was increasing over time. Early in the RSV infection, F and G proteins likely were detected when they were still present on the surface of the virion after attachment to the host cell. At 18 h post infection there was a reduction of F and G proteins due to the fusion of the virion into the host cell. During the remainder of the infection, F and G protein production increased as the host cell continued to manufacture and bud new virions and infect other cells. After 84 h the F and G protein production began to decline due to cell death. This sequence is consistent with previous quantitative mRNA analysis performed on RSV-infected cells in which an increase in mRNA for F and G was observed 18-24 h after infections as virus began to shed from infected cells.9 Alternative methods were used to compare the efficacy of quantum dot labeling of the RSV infected cells. Under similar conditions, the RSV F protein could not be detected by Western blot analysis, using an established sensitive chemiluminescent detection protocol (Figure 6B), for at least 96 h following infection.18 Repeated attempts to identify RSV F proteins with antisera to the extracellular or cytoplasmic tail domains with a purified murine monoclonal antibody did not reveal protein bands. As a control, the RSV N protein, which is transcribed first and most abundantly, was only detectable 96 h post infection. These results suggest that at the low multiplicity of infection (moi) used in these experiments (0.0032), the quantities of viral protein produced over the four day period are below the limits of detection for Western blot analysis. Real time, quantitative RT-PCR was also used to corroborate the increase in the expression of the RSV F and G proteins on the cell surface labeled with QDs (Figure 6A) by measuring the mRNA of the F and N proteins at low moi.19 The comparative Ct (threshold) method (2-∆∆Ct method) was used as the quantitation approach. In this method the Ct values from samples of interest were compared to a control Ct value, both of which had been normalized to an endogenous housekeeping gene.20 The real time RT-PCR data indicated a trend in the production of mRNA of the F and N protein (Figure 6C) that was similar to that of the fluorescence of QD labeled F protein over the course of a four day infection (Figure 6A). It should be noted that as the low moi of this experiment approaches the limit of detection for standard RT-PCR protocols,21 quantum dot labels are more Nano Lett., Vol. 5, No. 4, 2005

sensitive than RT-PCR methods, particularly at early time points during infection. Recently, several nanotechnologies, including atomic force microscopy (AFM),22 carbon nanotubes,23 and magnetic nanoparticles24 have been used for viral detection. While these methodologies provide detection and characterization of virion surfaces, they offer minimal insight into understanding the molecular level of viral infections. Our studies indicate applications that make it possible to use multiple probes to investigate the spatial distribution of several viral proteins simultaneously throughout the stages of infection. The implications of these results are that QDs may provide a method for early, rapid detection of viral infection and open the door for future studies of the intricate spatial features and cell trafficking of viral proteins. Acknowledgment. D.W.W. thanks the NSF for financial support through an NSF CAREER award (CHE-0304124). Additionally, we thank Sandra J. Rosenthal and Quantum Dot Corporation for helpful discussions during the course of this work. Confocal images using a Zeiss LSM 510 Meta were performed in part through use of the VUMC Cell Imaging Shared Resource (supported by NIH grants CA68485, DK20593, DK58404, HD15052, DK59637, and EY08126).

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Supporting Information Available: Co-localization controls, confocal imaging controls for channel crossover and alternate labeling strategy, comparison of Qdot and FITC labels, and photostability study. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Wu, X.; Liu, H.; Liu, J.; Haley, K. N.; Treadway, J. A.; Larson, J. P.; Ge, N.; Peale, F.; Bruchez, M. P. Nat. Biotech. 2002, 21, 41-46. (2) Goldman, E. R.; Balighian, E. D.; Mattoussi, H.; Kuno, M. K.; Mauro, J. M.; Tran, P. T.; Anderson, G. P. J. Am. Chem. Soc. 2002, 124, 41-46. (3) Parak, W. J.; Boudreau R.; Le Gros, M.; Gerion, D.; Zanchet, D.; Micheel, C. M.; Williams, S. C.; Alivisatos, A. P.; Larabell, C. AdV. Mater. 2002, 14, 41-46. (4) Kim, S.; Lim, Y. T.; Soltesz, E. G.; De Grand, A. M.; Lee, J.; Nakayama, A.; Parker, J. A.; Michaljevic, T.; Laurence, R. G.; Dor, D. M.; Cohn, L. H.; Bawendi, M. G.; Frangioni, J. V. Nat. Biotech. 2002, 14, 41-46. (5) Davidson, T. Respiratory Syncytial Virus. In Gale Encyclopedia of Medicine, Olendorf, D., Jeryan, C., Boydon, K., Fyke, M. K., Eds.; Gale Group: Detroit, MI, 1999; Vol. 4, pp 2478-2480. (6) Falsey, A. R.; Walsh, E. E. Clin. Microbiol. ReV. 2000, 13, 371384. (7) Hruska, J. F.; Bernstein, J. M.; Douglas, R. G., Jr.; Hall, C. B. Antimicrob. Agents Chemother. 1980, 17, 770-775. (8) Durbin, A. P.; Karron, R. A. Vaccines 2003, 37, 1668-1677. (9) Collins, P. L.; Chanock, R. M.; Murphy, B. R. Respiratory Syncytial Virus. In Fields’ Virology, 4th Ed.; Fields, B. N., Knipe, D. M., Howley, P. M., Griffin, D. E., Eds.; Lippincott Williams & Wilkins: Philadelphia, 2001; Vol.2, pp 1443-1485. (10) Hull, J.; Hacking, D. J. Infec. 2002, 45, 18-24. (11) A checkerboard analysis was performed in order to determine the optimal dilutions/concentrations of primary antibody, secondary antibody and quantum dots. 96 well plates of HEp-2 cells were infected with RSV A2 and incubated at 37 °C with 5% CO2 for four days to allow plaque formation. The columns (1-12) on each plate were used to test dilutions of 1:104, 1:105, 1:106, and 1:107of the primary antibody. The rows (A-H) of each plate were used to test dilutions of 1:1000 and 1:2000 of the secondary antibody. Concentrations 0.1 nM, 1 nM, and 10 nM quantum dots were tested on each of the primary and secondary antibody dilutions. The plates were then inspected with a fluorescent microscope to determine the optimal combination of antibodies and quantum dots for labeling of infected

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cells. The antibody dilution of 1:104 for the primary followed by 1:1000 for the secondary labeled with 10 nM quantum dots was selected. While we were able to detect visually the 1 nM quantum dots on the 1:104 primary, 1:1000 secondary antibodies using the 10 nM allowed for more options in the expansion of future labeling experiments. Beeler, J. A.; van Wyke Coelingh, K. J. Virol. 1989, 63, 29412950. The primary antibody for the G protein is commercially available and supplied as a purified antibody specific for the G protein of RSV A2. HEp-2 cells that had been infected for four days were fixed on glass coverslips using 0.1% formaldehyde for 15 min were incubated with Fmix and G antibodies for 1 h. Cells were then washed and incubated with 605 nm QDs conjugated to goat anti-mouse antibodies and 525 nm QDs conjugated to goat anti-rabbit antibodies for 1 h. After this final incubation, cells were washed and mounted to glass slides using aqu Poly/Mount (Polysciences Inc.). Ba¨chi, T. J. Cell Biol. 1988, 107, 1689-1695. Zheng, L.; Peeples, M. E.; Boucher, R. C.; Collins, P. L.; Pickles, R. J. J. Virol. 2002, 76, 5654-5666. Roberts, S. R.; Campans, R. W.; Wertz, G. W. J. Virol. 1995, 69, 2667-2673. HEp-2 cell monolayer cultures at 90% confluence were inoculated with RSV strain A2 at an moi of 0.0032, proportionate to that of 50 pfu in a 96-well plate. At the time indicated, cells were washed twice with PBS followed by collection by scraping the dish in PBS. The samples were centrifuged at 1500× g for 5 min; the cell pellet was lysed with 50 mM Tris HCl, 150 mM NaCl, and 1% Triton X-100 at pH8.0 with Sigma protease cocktail for 10 min on ice. The nuclei were removed by centrifugation at 13 000 rpm (Sorvall Biofuge Pico) for 5 min at 4 °C. Samples were stored at -80 °C until all time points were collected. Bio-Rad protein assay was performed to ensure equal loading onto a NuPAGE 4-12% nonreducing bis-tris gel, then transferred to a PVDF membrane. Membrane was probed with either MAb19 (R-RSV F), C797 (R-RSV N), or Sigma anti-actin (A5060), followed by a secondary conjugation to HRP. The Western blot was developed using SuperSignal West pico chemiluminescent substrate (Pierce). Stripping was achieved with 0.1 M glycine HCl pH 2.7 for 30 min then washing with PBS. Indicated above is the time point in hours or (-) in which no RSV was present. HEp-2 cell monolayer cultures at 90% confluence were inoculated with RSV strain A2 at an moi of 0.0032, proportional to that of 50 pfu in a 96-well plate. At the time indicated, cells were collected by washing with PBS followed by addition of 0.05% Trypsin and 0.53 mM EDTA. Opti-MEM medium was added, then the suspension was centrifuged at 300× g for 5 min. The cell pellet was then resuspended in RNeasy RLT buffer and stored at -80 °C until all time points were collected. RT-PCR analysis was performed as previously described (Kallewaard et al.). In brief, total RNA was collected then archived using the high capacity cDNA archive kit (ABI). Samples for each time point were collected in triplicate; we performed RTPCR in duplicate for each of these samples measuring GAPDH, RSV F, or RSV N RNA levels. Data on the RNA levels were collected as cycle threshold (CT), or the level at which the fluorescence measured was 10 standard deviations above background. Viral RNA was normalized by subtracting the CT for GAPDH from that of the viral RNA, yielding the ∆CT. The duplicate ∆CT for each of the three samples per time point was averaged separately, for RSV F and RSV N, then each of the three ∆CT averaged to determine a single value at each time point with standard deviation. At time 0, viral RNA could not be detected for 50 cycles, after normalizing to GAPDH the ∆CT for time zero was calculated to be 37.4, or the threshold of detection. We then calculated the ∆∆CT, which is the threshold (37.4) minus the ∆CT, for both RSV F and RSV N at each time point. Kallewaard, N. L. et al. Virology 2005, 331, 73-81. Gueudin, M.; Vabret, A.; Petitjean, J.; Gouarin, J.; Freymuth, F. J. Virol. Methods 2003, 109, 39-45. Hu, A.; Colella, M.; Tam, J. S.; Rappaport, R.; Cheng, S. J. Clin. Microbiol. 2003, 41, 149-154. Nettikadan, S. R.; Johnson, J. C.; Vengasandra, S. G.; Muys, J.; Henderson, E.; Nanotechnology 2004, 15, 383-389. Patolsky, F.; Zheng, G.; Hayden, O.; Lakadamyall, M.; Zhuang, X.; Lieber, C. M. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 14017-14022. Perez, J. M.; Simeone, J.; Saeki, Y.; Josephson, L.; Weissieder, R. J. Am. Chem. Soc. 2003, 125, 10192-10193.

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