1Defence Medical and Environmental Research Institute, DSO National Laboratories, 2Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, 3Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School
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Zhang, S., Tan, H. C., Ooi, E. E. Visualizing Dengue Virus through Alexa Fluor Labeling. J. Vis. Exp. (53), e3168, doi:10.3791/3168 (2011).
The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.
The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer’s protocol for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.
1. Alexa Fluor labeling of dengue virus
2. Purifying Alexa Fluor labeled dengue virus
3. Representative Results
An example of the yield of dengue virus labeled with AF594 dye is shown in Figure 2. Normally, less than 10-fold drop from the initial titer should be observed following successful labeling. However, it should be noted that all buffers have to be prepared fresh for the labeling to be successful and the Alexa Fluor succinimidyl esters should be used immediately upon reconstitution as they hydrolyze into nonreactive free acids in aqueous solutions8.
Next, the labeled virus has to be checked for sufficient fluorescence before use in experiments. A simple immunofluorescence assay was done on Vero cells and the degree of labeling can be estimated from the co-localization of the labeled virus with anti-E protein antibody staining. Several cells were examined and a typical confocal image is shown in Figure 3. Co-localization analysis of the images using the LSM Zen software demonstrated overlap coefficients ranging from 0.65 to 0.8, suggesting that approximately 65 to 80% of the virions were labeled with the dye.
Figure 1.Overall scheme depicting the Alexa Fluor dye labeling of dengue virus procedure. First, the relevant buffers and purified dengue virus are prepared. The Alexa Fluor dye is reconstituted and added to the dengue virus diluted in labeling buffer. The reaction is then stopped 1 hour later with the addition of stop reagent. Subsequently, the labeled virus is purified through a size exclusion column to remove free dye. Finally, the labeled virus is re-titrated by plaque assay and tested for fluorescence.
Figure 2. Mean number of viable virions (pfu/ml) as determined on a plaque assay before and after AF594 labeling. An aliquot of the AF594 labeled dengue virus is thawed and re-titrated by plaque assay and it typically shows less than 10-fold drop from the starting titer. Error bars indicate standard deviation of duplicates.
Figure 3. Co-localization of AF594 labels with dengue virus E proteins in Vero cells. cells grown on coverslips the day prior were infected with AF594 labeled dengue at MOI of 1 for 10 minutes at 37°C. The cells were subsequently fixed and labeled with anti-E antibody, and examined for colocalization of E protein (green) and AF594 labeling (red). Fluorescent signals were visualized under 63X magnification using Zeiss LSM 710 confocal microscope. Scale bar is 10μm. Yellow indicate areas of colocalization, as shown in the inset.
Although AF594 dye was used in this report, a wide range of fluorophores in the Alexa Fluor succinimidyl esters series is available with similar labeling chemistry. This could extend the labeling application beyond imaging. Flow cytometry can be used as an alternative to confocal microscopy for estimating the degree of labeling for fluorophores that can be excited and detected by the FACS machine.
Alexa Fluor dyes are small molecules that react with free amino groups, primarily arginine and lysine9, normally outward facing residues of proteins. In our laboratory, conjugation of dengue virus with 100μM of Alexa Fluor 594 dye provided sufficient brightness for imaging with minimal loss in the viral titer. Different brightness may be achieved by varying the concentration of dye used. However, increasing the dye concentration can reduce virus viability7,10. One possible limitation is the interference in receptor binding due to the blockade of access by the fluorophores. Therefore, depending on application, an optimal level of labeling should be determined to ensure a balance between the degree of labeling and functional abrogation10. Do note that the concentration can also be affected by the post-packaging reactivity of the Alexa Fluor succinimidyl esters8.
The direct labeling of dengue virus with Alexa Fluor dye presented here does not require any additional labeling steps to visualize the virus, thus removing the possibility of non-specific staining from indirect antiviral antibodies. It also allows for real-time tracking of post-internalization events in live cell imaging. This method is relatively simple, and because the conjugation is stable, it can be used to produce and store batch-labeled virus for multiple experiments as opposed to lipophillic fluorescent dyes, such as long-chain carbocyanine1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine (DiD) or styryl dyes, which cannot be stored in the cold for more than 3 days.
We have nothing to disclose.
This work has been funded by the National Medical Research Council, Singapore.
|Sodium hydroxide||Merck & Co., Inc.||106498|
|AF594 succinimidyl esters||Molecular Probes, Life Technologies||A20004|
|PD-10 column||GE Healthcare||17-0851-01|
|4-well plate||Nalge Nunc international||176740|
|Microscope slide||Sail Brand||7105|
|10x PBS||BUF-2040-10X1L||1st Base|
|Confocal microscope||Carl Zeiss, Inc.||LSM 710|
|To prepare M-199 growth medium, add 50ml of FBS, 5ml of sodium pyruvate and 5ml of non-essential amino acids to 500ml of M-199, sterile filter.|
|To prepare M-199 maintenance medium, add 15ml of FBS, 5ml of sodium pyruvate and 5ml of non-essential amino acids to 500ml of M-199, sterile filter.|