1Lady Davis Institute for Medical Research, Sir Mortimer B. Davis Jewish General Hospital, 2Department of Microbiology and Immunology, McGill University, 3Department of Medicine, Division of Experimental Medicine, McGill University
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Vyboh, K., Ajamian, L., Mouland, A. J. Detection of Viral RNA by Fluorescence in situ Hybridization (FISH). J. Vis. Exp. (63), e4002, doi:10.3791/4002 (2012).
Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8.
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.
1. Probe Preparation
In vitro transcription reaction:
|Linearized DNA||23 μl|
|1X DIG RNA labeling mix (Roche)||4 μl|
|1X Transcription buffer||8 μl|
|20U RNase OUT||1 μl|
|80U T7 RNA polymerase||4 μl|
2. Cell Fixation
3. Fluorescence in situ Hybridization (FISH)
Hybridization Mix (for one coverslip, 50 μl):
|Amount of stock||Material (at final concentration)|
|25 μl||Formamide, 50% (deionized; any source)|
|5 μl (of 10 mg/ml)||tRNA, 1 mg/ml (Sigma-Aldrich, Inc)|
|5 μl (of 20X)||SSPE, 2X|
|5 μl (of 50X)||Denharts, 5X|
|0.125 μl (of 5 units)||RNase OUT|
|5 μl (25 ng of 5 ng/μl)||Probe|
|5 μl||H2O DEPC, to make the final volume 50 μl|
4. Immunofluorescence Staining
5. Representative Results
An example of viral RNA staining can be seen in Figure 2. The viral RNA for HIV-1 is seen throughout the cytoplasm displayed diffusely for the most part, although small cytoplasmic punctae are not uncommon. The specificity can be seen by comparing positive cells to surrounding cells that display no fluorescence. As mentioned in the introduction, HIV-1 that lacks the regulatory Rev protein produces RNA which is retained in the nucleus: this can be visualized as a bright signal in the nucleus, and a lack of RNA fluorescence signal in the cytoplasm. Overexpression of cellular proteins is also capable of shifting the distribution of the HIV-1 viral RNA. In Figure 2, the overexpression of proteins involved in endosomal vesicle trafficking (e.g., Rab7-interacting lysosomal protein (RILP)11 or N-terminally deleted RILP (RILPN)11, and proteins that interfere with dynein motor function [e.g. p50/dynamitin1,12], interfere with the normal steady-state localization of the viral genomic RNA as determined by FISH analyses. RILP expression leads to re-localization of the viral genomic RNA to the microtubule organization center (MTOC) 13; RILPN disperses late endosomes within the cytoplasm due to the inability to bind to p150Glued of the dynein motor complex 14 and p50/dynamitin blocks the large subunit of dynein motor and releases late endosomes but also the viral genomic RNA and HIV-1 structural proteins to the cell periphery1 (Figure 2). The manipulation of the steady-state localization of the viral genomic RNA, a key contributor to the infectiousness of virus particles, would be key to the eventual development of therapeutics. In our hands, viral RNA appears in the cell as early as 3 hours post-transfection and infection10 and becomes readily observable by this FISH technique by 12 hours.
Figure 1. Flow chart of the protocol for FISH/IF co-analyses. Cells are grown on coverslips and then fixed with paraformaldehyde. Treatments of glycine and Triton-X are performed before the cells are either used for FISH/IF or stored in 70% ethanol for storage. Cells dehydrated for storage are rehydrated in 1X DPBS (DEPC-treated PBS) before continuing on to FISH/IF. Cells are treated with DNase I and left overnight to hybridize with probe. Once hybridization is complete, the cells are washed with formamide, as well as with SSPE, and a final 1X DPBS rinse. Blocking solution is applied to the cells, followed by incubation with the primary antibodies. After washing, secondary antibodies are applied. Two final washes in 1X DPBS complete the staining procedure where upon the coverslips are dried and mounted on slides for imaging.
Figure 2. Representative results using FISH/IF co-analyses. A) HIV-1 is transfected into HeLa cells and in some cases with constructs that cause the overexpression of cellular proteins (RILP, p50/Dynamitin and RILPΔN (an amino-terminal deletion mutant)). FISH/IF co-analyses was performed: RNA is identified in green with either G3BP or LAMP1 (red) to differentiate. B) HIV-1 is expressed in HeLa and collected at different time points. Viral RNA expression is tracked in green while the cellular protein, hnRNP A1, is stained in red. Size bars are 10 μm. Images modified from references 1 (select figures from panel A; RILP, p50/Dynamitin, and RILP deltaN) and 17 (panel B).
FISH/IF co-analyses is a reliable method to visualize viral RNA in cells which has now been refined 9,15,16. Over the course of several years, we have developed a refined method of staining for RNA. This technique can be used on a wide array of cell types provided the probe is specific to the target RNA17. By labeling the probe with DIG, we are capable of visualizing the RNA by simple staining. When staining for RNA and other proteins are combined, FISH/IF co-analyses become a powerful tool to observe cellular structures and protein/RNA localization.
The specificity of the RNA detection is quite high. This is illustrated in Figure 2. In some of the original work that focused on viral genomic RNA localization, FISH established that a lack of Rev trapped viral RNA in the nucleus18. Since this, abundant new information on viral genomic RNA localization has been obtained using the technique outlined here. By overexpressing cellular proteins, specific populations -and not all- of the viral RNA can be forced to localize to different regions of the cell. RILP overexpression, which resembles the phenotype obtained when hnRNP A2 is depleted by siRNA in HIV-1-expressing cells13, causes the RNA to visibly accumulate at the MTOC. The viral genomic RNA can be pushed to the cell periphery by disabling the minus-end motor protein, dynein: p50/Dynamitin overexpression or knockdown of the dynein heavy chain1 results in the release of viral genomic RNA from intracellular domains to the cellular periphery (Figure 2). A mutant of RILP, RILPΔN, which no longer binds the dynein motor, disperses endosomes (tagged by LAMP1) into the cytoplasm because they are no longer actively localized at juxtanuclear domains. These results point to the notion of a plastic population of HIV-1 genomic RNA and in particular, that different pools of HIV-1 genomic RNA exist with sometimes identifiable roles in the viral replication cycle. These same notions have already been identified for the protein encoded by this mRNA, Gag 19.
There are limitations to the uses of FISH/IF co-analyses that are related to antibody choice and availability. The optimization of the best combination of primary and secondary antibodies, and their concentrations, will take time to optimize (see Table 1 & 2). Antibodies made from hybridomas or produced by major companies can have concentrations which vary anywhere from 1:2 to 1:2000, but be sure to follow the guidelines provided by the manufacturer for best results. The host in which the antibody is produced must also be taken under consideration. Mixing sheep with goat antibodies should also be avoided in primary and secondary antibodies as this combination results in high background due to cross-species recognition (data not shown). For Alexa-Fluor secondary antibodies, the concentration can be used at 1:500 for almost any antibody in the set. The other drawback to FISH/IF co-analyses as described in this report is it captures the RNA at its location at steady-state, after cells have been fixed and permeabilized using paraformaldehyde and detergent (Figure 1).
However, RNA imaging in live cells has been achieved through a variety of means 20-22, mostly using variations of viral RNA genomes that are tagged by fluorescent proteins such as GFP. However, additional means to identify RNA localization in live cells continue to surface. These new methods involve the tagging RNA by means of Spinach 23, SNAP 24, or MTRIP 2. These techniques also suffer from a few drawbacks including the requirement to permeabilize cells before adding substrates or that a moiety must be engineered to tag the mRNA in order detect the mRNA by microscopy. Indeed, the major advantage of FISH analyses outlined in this report lies in the fact that the native RNA is unaltered leading to the most physiologically relevant results.
No conflicts of interest declared.
The authors thank past and present members of the lab for contributions to the development of the methodology outlined here and to Alan Cochrane for advice. L.A. is a recipient of a Canadian Institutes of Health Research (CIHR) Doctoral Fellowship and AJM is supported by a Fraser, Monat and MacPherson Career Award. This work is supported by a grant from the CIHR (grant #MOP-56974).
|18mm coverslips||VWR international||48380 046|
|16% paraformaldehyde||Polysciences, Inc.||18814||Dilute to 4% (wt/vol) in 1X DPBS|
|Triton-X||OmniPur, EMD Millipore||9400|
|Micro Elute Gel Extraction Kit||Roche Group||D6294-02|
|DIG RNA Labelling Mix||Roche Group||11277073910|
|Transcription T7 RNA Polymerase||Invitrogen||18033-019|
|Quick Spin Columns||Roche Group||11814427001|
|1X DPBS||GIBCO, by Life Technologies||14190-250|
|Fluorescent Antibody Enhancer Set for DIG Detection #4 (blocking solution)||Roche Group||1768506|
|Alexa Fluor secondary antibodies||Invitrogen||See Table 2|
|Hybridization Oven||Boekel Scientific||Model 24100|
|Immunomount||Thermo Fisher Scientific, Inc.||9990402|
|Table 1. Identification of specific reagents and equipment|
|Table 2. Combinations of secondary antibodies and anti-DIG listed with catalogue numbers and concentrations|