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.


* * * AND NOW, DANCE PARTY ! * * *


they all try to keep up, while we fuck this shit up! 

No comments:

Post a Comment

INSTANT TIME MACHINE!

CERCA TROVA:

TAG MANIA

music 30 days of music Aerial friday5 20Q hawaii samadhi yoga vinyl bikeventure la dispute school touché amoré book concert live show silversun pickups bicycle lists metric science cooking destiny poweryoga hawaii rooster teeth EmM food microbiology movie movies phantogram shopping spring break spring break 2015 2010 Book review Stephen king cardistry koji reads tissu work beach books ceremony coffee cover deafheaven movie review pyhiflygirls recol sick tycho virology aerial problems art b9 bacteria bus clothes comics drug church earl grey emily haines interpol lyra mlt no sleep records other realms pacific rim raveonettes republik roman literature sephora socks sspu summer sviib taking back sunday tattoo vales wiwt 6131 YYY amyz arcade fire balance and composure batman blood blood bank brambles carnavas casket lottery catullus cheese chinese christmas coffeesundays contortion culture abuse death cab for cutie dengue destiny 2 exercise grouplove gymnastic linguist handstand jawbreaker lily allen makeup me first and the gimme gimmes memories minecraft mlt adventures modest mouse molecular motion city soundtrack needle work nightwing octopus recipe records review saga scattegories small brown bike sunbather thursday tigers jaw tiny cats weezer xbox one yummiestars #hrf2015 2014 2018 7 inch accident adult swim singles afi album review anakin anamanaguchi angels and airwaves asm atul gawande beach house beer best of bike biomusicology birds in a row blast bruises cadence caitlin doughty campy candy hearts cards carpe noctem cayetana charcoal checklist manifesto chemistry chicago chinese new year clones of the queen concussion dads dc comics deathwish delerium descendents descender dragonette dress earth wind and fire evenings felix cartal fight club fishing fletcher c johnson ghost feet glow flow goodtime boys heart shaped box hematology hip key how to with joel and adam hungry ear hurricane interstellar jack white jjak joe hill john wick katy perry kintsugi lab last look live lucky hell lululemon manoa martin solveig megwin melissa mls adventures modern baseball mr mercedes nails ncbi nerves never come undone nicole atkins no doubt ofelia orange bang pixies poetry pool portal prawn ps4 research rick and morty rohnert park rsd rsd2018 rtpodcast sandwich school of seven bells sequencing shameless shave ice shimmering stars sirens six feet under sleep society of s sorority noise star wars static trap steam stereochemistry strava strfkr sysmex the donnas the killers the modern waikiki the saddest landscape the winter passing there is no learning curve third man records thrice tokyo milk topshelf records tour de france tove lo training for aerial on the ground tron unpopular opinion waiola xcm xur yeah yeah yeahs #hrf2016 #hrf2017 #myhour #tdf2015 #teampretzeltank '68 007 100% 11-22-63 16s 1970s 1999 2011 2015 2017 35mm 3hive 7seconds 90s :) @8_semesters @wild_twinkies Booker T. and the M.G.'s CD Carrie Fishbones GITS Godzilla Groundhog Day HST L-cuts L4D2 MCR Pink vans RIP SEM Streptococcus The Great Wall UH a favor house atlantic a girl walks home alone at night abraham lincoln vampire hunter achievement hunter aculpture aesop rock agar aji fry akari ala moana alam alfa beach allister alt/air amanda tastes ambient american hustle amoeba music antics arsenic art of play atlas genius autoclave b sides backstage bai san bakasana baking ballet bandcamp barbra streisand bargain basement battle lines battlewagon bayonetta beach slang beastie boys beats begiragons big bang theory big grams biochem bitter clarity uncommon grace black dress blaisdell blink 182 blondie bob bond boscia brad meltzer brand new brave new world brazil brew bridge 9 bro force broke for free bronchial wash burrito buxom buy local calligraphy calzone campy group campylobacter cancer candida candida albicans carrot carrot cake carsick cars cartwheel cascade casket girls catbird cave in cdc chairlift charlie day cheap monday chet faker chirashi chloe cho dang choir of the mind chores chozo christmas movies cinelli cinerama circuits circulus cisco kid clarity cloning cls coexist coffee stout coheed and cambria coincidence coli com truise comicon common reactor conference constantine cotq country comfort credence clearwater revival cremation crow csf cults cursive daiei dan and dave dan deacon daniel dae kim dashboard david rees day by day records dead weather deadlines dealersgrip death deep blue deer tick defender class delicious demetri martin deodato depression cherry detective dee devart devrim kay dhm diamond youth dick grayson dim mak dinosaur pile up disco discordia discount dance discovery distance/closure divination diy dl dna dntel does it work dog don quijote donnie darko doodles double ankle hang doubles down the cliff dreamcar dreams dscvry duolingo dupe dustin nguyen e. coli ebola edge edward benz 27 times el pintor el-p electric president electricity elle king emb embroidery enbalming enjoy your burrito epidemiology eqs eraas erik kwakkel esr event evil needle exam exotica eye shadow facebook fall behind me family fashion fat bob fatale father/daughter records fck feather fiction firefly fireworks fitz and the tantrums flash flash fact flight facilities flight of the conchords flower drum song forever 21 forgettable forgetters formula x fountains of wayne frameworks franz ferdinand fresh cafe friday the 13th fuck yes fuji fulton funeral game of thrones gate gatsby genetics ghost bc ghost in the shell ghost key ghostly giant glass glass animals good charlotte google gpc grant imahara grayson green river gtd guardians of the galaxy guest blogger gym class heroes h&m habits haemophilus influenzae haim hair half gramme holiday halloween handwriting haribo harlem harmonica harold harrow hawaii problems heavy circles helena bertinelli hell bunny hello hellogoodbye hematocrit heroquest high water marks history history channel hl2 hohokum holiday mart homework houdini howl howl gaff gaff hummus hxc hyper light drifter i love crazy tags iasip ice cream idea's music and books identity crisis in all dishonesty in and out of control in gratitude inside insomnia insound instagram instax instrumental interpro into the wayside intrepidity ipod iwant iyo udon jack johnson japan jaspas jejuni jellys jeniferever jimmy fallon jisuk jld jock journey joy departed joy division jpb julian casablancas julien baker justice league justice league dark kaimana kakaako kalapana kapiolani kat von d keep talking and nobody explodes kegg kent keratin kickstarter kiki kimchi kimchijeon kingsman kitsune kittygeddon klaxons knives don't have your back koolau ballroom korean koundinyasana lab problems lab report labyrinth ear lali puna last gang records latte art layers of fear lazaretto le castle vania leahi left 4 dead 2 leggings leotard leptospirosis les savy fav let me in lgc library light we made like like drive in lil gothams lipstick lipstories liquid nitrogen lismore lit little richard llea lol lolita loma prieta longboard lookbook looming love luster luck lust lust lust lymbyc systym magic magic island magnolia cafe mah jong maiden heist mail day mais2 major league makapuu maldi-tof mandarin martin denny marvel matty matheson mavica me earl and the dying girl media medieval memento memento bento mental block gaming meta metamorphoses mf doom mgmt michael crichton micr490L midweek mika milk tea mind of a chef miss manners modcloth mogwai mohawk monty python monuments men moon moose blood morbid mortdecai motown mr robot mr tea musubi my chemical romance my sharona naked juice narcissus ncis ncisla neo classical nerd nerdist new eagle cafe new politics new scene new year nickleback nicole atikins nightmare before christmas nijiya nimitz no backup plans no better noisey nonfiction novo biocin now now oats obituary oblivion old navy ombre omori boy on the spot on! air! library! only in youth opera origa osaka loop line ost outdoor voices outkast overcooked ovid pacific rim 2 pai gow pam pancakes pandemic paramore paranormal activity part time job party past is prologue past present felix pat mcgrath labs patreon patrick kindlon pcr pe'ahi peanut butter and jelly peb pecan tree penny dreadful peony perennial flux performance perfume peter pan collar pfam photog physics pikul pita pizza plants plastic metal platelets playdead pointe poker pole polefiction politics popo popoitis portugal the man post office potpourri pow wow hawaii prey primer prince prismatic procrastination protein pu er pun punk pusheen pyogenes pyromaniac quiche r dorothy wayneright rage ramblings ramen ramones random random weirdos rap record shop dude red hot chili peppers red machine redbone reflective replacements revision rhcp ringtone ripd roller skates rooms of the house roxy rozwell kid rumu run for cover run the jewels sailor moon sakura mochi salem's lot salonpas samurai ice cake san francisco sapling satoshi kon saves the day scans scar tissue scarjo scary science fiction scorpion scrabble scrubs sds page sean kingston searching for a pulse sed rate seeking a friend for the end of the world self defense family self published separation sequins serein settled seven psychopaths sewing sex pistols sgl shadowplay shaloha sheezus shipping shoes shout out louds show shrimp sia sierra nevada sikdorak silly girl sinclair library skateboard skyfall sleeve scribbles slipshift smart db smashing pumpkins smoke gets in your eyes snl somnio soundcloud speech and debate sphere spill spyral stamps staph staphylococci star trek starcrawler stellastarr* still corners straddle ups string db suckerpunch sudafed suetonius suicide squad suis la lune sunday morning at a funeral sunny day real estate superhot surya namaskar sushi sushi king swedish swim swoon taco tuesday taiyaki tantalus target taste tea taunt tcb technical writing tempura tf2 tguk thank jose thanksgiving the angels the artist the audition the bougies the byrds the calm blue sea the chase the get up kids the hives the kingsmen the kinks the las the pipettes the quick the raconteurs the russian the supremes the things we think we're missing the voices the weight of the world the wolfpack the xx there's nothing thieves like us threadless three cheers for sweet revenge ticket to ride tideland time travel tiny echo tiny me title fight tittibhasana toby todor kobakov tokyo godfathers tom moffat totoro touche amore tower records transfection transformation tricks trojans tumblr turnover turnstile tv twenty-two twitter two dots udon undercut uniprot unknowns unrelatableexperience vacation valve vampire vegetables verde verse vertigo vibrio vice video dump violet viva fest volary wakey wakey walking backwards man walmart waning star want to read war washed out wednesday addams welwing wharf cat what is free to a good home what we do in the shadows white house down white stripes whole foods wildhoney wildlife wired won tons word freak workaholics wu xia xbox 360 yoga ball yogagypsy yuck zatanna zero zippys zombie zones zotero