Determining localization of Dengue viral protein, NS1, in eukaryotic host cells by immunofluorescence

ABSTRACT
HEK 293T cells were co-transfected with Dengue viral protein, NS1, and various intracellular markers.  NS1 was conjugated pre-transfection with protein reporter, HIS/V5, while intracellular markers were conjugated to GFP.  Fluorescent labeled antibodies are used to detect the transfected proteins and are visualized by confocal microscopy under immunofluorescence.  Interaction between NS1 and intracellular structures can be hypothesized when the proteins are in close proximity however additional tests need to be performed to confirm this. 
INTRODUCTION!
Dengue is a single strand, positive-sense RNA flavivirus with four serotypes transmitted to humans by mosquito vectors.  Severe pathology in humans consists of malaise, fever, hemorrhaging, and can lead to shock and death.  Understanding the cause of its pathogenicity is key to development of dengue treatment and vaccine.
NS1 protein is a well-studied protein in the virology field. In the dengue virus, the non-structural proteins are named NS1 through NS5.  NS2 and NS3 genes are found to code for protease and helicase.  NS5 is the RNA dependent RNA Polymerase.  NS1 and NS4 have unknown functions.  Despite its unknown function, NS1 is an important protein because it can be used in diagnosing viral infection.  NS1 levels in serum are elevated at viral infection as it is secreted as a soluble hexamer from infected host cells.  As previously mentioned, the function and method of NS1 secretion is also unknown, however a hypothesis is proposed; monomeric NS1 (mNS1) protein is glycosylated and prepared in Endoplasmic Reticulum (ER) vesicle packets (GPI-NS1).  From the ER it is either passed to the Golgi where it takes hexameric form (sNS1) and then secreted as a soluble protein, sNS1-LP, or is associated with lipids and delivered to the cell surface by cholesterol/lipid rafts where it becomes a membrane associated protein. 
This experiment uses immunofluorescence to visualize a protein marker, HIS/V5, conjugated to NS1 in host cells.  Proteins of certain organelle-associated proteins are conjugated with protein markers to visualize in conjunction with NS1 protein.  Fluorescent-labeled antibodies against these proteins are added to detect the proteins’ presence in the cell.  If NS1 is synthesized and secreted as hypothesized, we expect co-localization of NS1 in the host cell with ER, Golgi, and in the plasma membrane as it is being prepared for release from the cell.

MATERIALS AND METHODS
            A 60% confluent culture of HEK293T cells on cover slips were transfected using Polyfect, a transfection kit optimized for transfection with small lipid particles.  Prior to transfection, genes for NS1 protein were conjugated to genes for HIS/V5 protein marker while known intracellular protein genes were conjugated to green fluorescent protein (GFP) genes.  See Table 1 for list of conjugated proteins used.  Polyfect reagent and conjugated proteins were incubated together to form complexes between the lipid and the proteins were then added drop-wise to cell cultures.  Gene expression in cells took place over 24 hours at 37ºC.  After transfection, cells were fixed with 3.7% paraformaldehyde.
            For the immunostaining fixed cells were first blocked with 5% BSA, then monoclonal antibodies were added: mouse anti-V5 IgG to detect NS1 protein and rabbit anti-GFP IgG to detect cell proteins and incubated for 48 hours at 4ºC.  Cells were then washed 3 to 5 times with phosphate buffered saline (PBS).  Secondary antibodies conjugated to fluorochromes were then added.  See Table 1 for list of antibodies used.  Cells were incubated with secondary antibodies for 45 minutes, protected from light, at room temperature. Cells were washed 3 to 5 more times with PBS to remove excess antibody and reduce background.
            The cover slips were then removed and dried.  A nuclear stain, DAPI, was applied and then the cover slips were attached to glass slides with low viscosity enamel for viewing.  Confocal fluorescent microscopy was used to visualize cells.

Table 1: Conjugated proteins used for cell treatment
Protein and conjugate pairings
Primary Antibodies
Secondary Antibodies with fluorescent conjugate (color at excitation)
NS1-V5 + ARF1-GFP
Mouse anti-V5 +
Rabbit anti-GFP
Goat anti-mouse Alexafluor 555 (red) +
Goat anti-rabbit Alexafluor 488 (green)
NS1-V5 + FAPP1-GFP
Mouse anti-V5 +
Rabbit anti-GFP
Goat anti-mouse Alexafluor 555 (red) +
Goat anti-rabbit Alexafluor 488 (green)
NS1-V5 + SEC31-GFP
Mouse anti-V5 +
Rabbit anti-GFP
Goat anti-mouse Alexafluor 555 (red) +
Goat anti-rabbit Alexafluor 488 (green)




RESULTS
ARF1 is a protein that is associated with the golgi.  ARF1 exits the golgi and either returns to the ER or shuttles proteins to the plasma membrane.  NS1 proteins will be associated with vesicle proteins as it is moved from ER to golgi for hexamerization and from golgi to plasma membrane for secretion.  If NS1 becomes a membrane associated protein it will be transferred by cholesterol/lipid raft to the cell membrane.

Figure 1: NS1-V5 and ARF-GFP labeled HEK 293T cells
(L-R): DAPI & ARF-GFP & NS1-V5 | ARF-GFP | NS1-V5
In Figure 1 the ARF proteins are indicated with green color and NS1 is indicated with red color.  In the merged image co localization is visualized by yellow color produced by overlapping green and red light.


FAPP1 is a protein that localizes in the golgi where lipid synthesis takes place.  After GPI-NS1 leaves the ER in vesicles, it is either associated with lipids and taken to the cell surface to become a membrane associated protein or processed in the golgi as a hexamer to become a soluble secreted protein.  Two NS1 treatments were performed.  Figure 2 shows FAPP1 in green and NS1 in red where the host cell has been premeablized before fluorescent antibodies were applied.  In Figure 3, cells were not permeablized before antibody application in order to visualize the NS1 in the plasma membrane as a membrane associated protein or congregating before secretion. 




Figure 2: NS1-V5 and FAPP1-GFP (permeable)
DAPI & FAPP-GFP & NS1-V5 | FAPP1-GFP | NS1-V5
In Figure 2 golgi associated protein, FAPP1 is green.  There is a lot of background protein in the cell but the golgi is prominently shown as a large green mass.  NS1 has entered the permeablized cell but is not co-localizing with FAPP1 proteins as evidenced by the lack of yellow color in the merged image. 


Figure 3: NS1-V5 and FAPP1-GFP (non permeablized)
DAPI & FAPP-GFP & NS1-V5 | FAPP1-GFP | NS1-V5
In Figure 3 golgi protein FAPP1 is indicated by green color and NS1 is indicated by red color.  These cells were not permeablized before antibody addition.  Therefore we do not expect co localization of the two proteins as there are not golgi proteins in the plasma membrane. 


The third cellular protein used is SEC31-GFP.  SEC31 protein localizes in vesicles exiting the ER.  After mNS1 is glycosylated in the ER (GPI-NS1) it is taken by vesicles to the golgi for hexamerization and association with lipids.  Two NS1 treatments were performed.  Figure 4 shows SEC31 in green and NS1 in red where the host cell has been premeablized before fluorescent antibodies were applied.  In Figure 5, cells were not permeablized before antibody application in order to visualize the NS1 in the plasma membrane as a membrane associated protein or congregating before secretion. 


Figure 4: NS1-V5 and SEC31-GFP (permeable)
DAPI & SEC31-GFP & NS1-V5 | SEC31-GFP | NS1-V5
In Figure 4 merged image, there is one cell with a large amount of cytoplasm and strong NS1 signal.  The yellow dots indicate areas of co-localization of NS1 in SEC31-GFP stained vesicles.  Compare to the cell to the left in the merged image where there is no co-localization.


Figure 5: NS1-V5 and SEC31-GFP (non-permeablized)
DAPI & SEC31-GFP & NS1-V5 | SEC31-GFP | NS1-V5
In Figure 5 the non permeablized cells show no co-localization [yellow] areas.  This is expected because vesicles are located intracellular, in the cytoplasm between the ER and the golgi, but the labeled NS1 is restricted to the plasma membrane of the cell, where there are no vesicles.


Figure 6: HEK293T Cells Infected with Dengue Virus
A positive control was performed.  Figure 6 shows HEK293T cells that were infected with Dengue virus instead of transfected with viral protein.  DAPI is also applied for nuclear identification and is shown in blue.  ER is shown in green and NS1 protein is red.  This control demonstrates NS1 protein is present in infections. 

DISCUSSION AND CONCLUSION
In the non-permeablized cells there was no co localization of the NS1 protein and the labeled cell structures which was an expected result.  In non permeablized cells, antibodies are unable to get into the cell so only NS1 on the plasma membrane is labeled.  This demonstrates that NS1 is not only found intracellularly but on the surface and extracellularly as it is secreted.  In the permeablized cells there was co localization of the NS1 proteins with the cellular structures, as evidenced by the yellow color produced by the overlapping red and green light.  However, not much co localization is seen with NS1 and FAPP1.  Since FAPP1 is associated with the golgi this could indicate NS1 does not go to the golgi for synthesis as much as it goes to other areas.  However, NS1 is a known secreted protein and would require some assembly at the golgi for this to happen.  The lack of visible co localization could be from too much background from either fluorochrome masking the others’ color or not enough protein aggregated in one place to be visible.  Errors in transfection causing this problem is not likely because the transfection was preformed at an off site lab proficient in viral proteins.  Controls indicate the antibodies are satisfactorily labeling proteins as well.  A human error or factors concerning excitation of fluorochromes and the background proteins, as previously mentioned, may have caused this aberration.  This experiment may be repeated to confirm the lack of association since other results confirm a secretion pathway from ER to golgi to cell surface. 

This experiment shows only the localization of NS1 protein and does confer actual interaction between the organelle and the viral protein.  To confirm interaction, additional assays would have to be performed such as an Eastern blot on transfected cells to detect lipid or carbohydrate modified NS1 protein.


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