Name: confocal imaging of double-stranded rna and pattern recognition receptors in negative-sense rna virus infection
Uploaded: 2019-01-26T15:00:00
Description: Watch this Scientific Journal Video about Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection at JoVE.com

Vi vil gerne takke UTMB imaging core faciliteter og Maxim Ivannikov for mikroskopet bistand.
Dette arbejde blev støttet af Public Health Service give RO1AI093445 og RO1AI129198 tildelt til SP, UTMB engagement fonden P84373 til CH og T32 AI007526 til EM.

1. forberedelse af A549 celler og JUNV infektion
<ol><li>Frø 2 x 105 menneskelige lunge A549 epitelceller på poly-D-lysin (PDL) belagt glas coverslips i 12-godt plader på 24 timer før infektionen.</li>
	<li>Forberede delprøver af 150 mL af JUNV13 ved en multiplikation af infektion (MOI) af 1.0 plak danner enhed pr. celle fortyndet i Dulbeccos modificerede Eagle Medium (DMEM) medier suppleret med 2% føtal bovint serum (FBS) og 1% penicillin og streptomycin (P/S ).</li>
	<li>Fjern...

<ol>
	<li>Jensen, S., Thomsen, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+of+RNA+viruses:+a+review+of+innate+immune+receptors+involved+in+recognizing+RNA+virus+invasion.">Sensing of RNA viruses: a review of innate immune receptors involved in recognizing RNA virus invasion.</a> Journal of Virology. 86, 2900-2910 (2012).</li><li>Weber, F., Wagner, V., Rasmussen, S. B., Hartmann, R., Paludan, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-stranded+RNA+is+produced+by+positive-strand+RNA+viruses+and+DNA+viruses+but+not+in+detectable+amounts+by+negative-strand+RNA+viruses.">Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses.</a> Journal of Virology. 80, 6 (2006).</li><li>Triantafilou, K., Vakakis, E., Kar, S., Richer, E., Evans, G. L., Triantafilou, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualisation+of+direct+interaction+of+MDA5+and+the+dsRNA+replicative+intermediate+form+of+positive+strand+RNA+viruses.">Visualisation of direct interaction of MDA5 and the dsRNA replicative intermediate form of positive strand RNA viruses.</a> Journal of Cell Science. 125, 4761-4769 (2012).</li><li>Onomoto, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+role+of+an+antiviral+stress+granule+containing+RIG-I+and+PKR+in+viral+detection+and+innate+immunity.">Critical role of an antiviral stress granule containing RIG-I and PKR in viral detection and innate immunity.</a> PLoS One. 7, e43031 (2012).</li><li>Son, K. N., Liang, Z., Lipton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-stranded+RNA+is+detected+by+immunofluorescence+analysis+in+RNA+and+DNA+virus+infections,+including+those+by+negative-sense+RNA+viruses.">Double-stranded RNA is detected by immunofluorescence analysis in RNA and DNA virus infections, including those by negative-sense RNA viruses.</a> Journal of Virology. 89, 9383-9392 (2015).</li><li>Mateer, E. J., Paessler, S., Huang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+double-stranded+RNA+colocalizing+with+pattern+recognition+receptors+in+arenavirus+infected+cells.">Visualization of double-stranded RNA colocalizing with pattern recognition receptors in arenavirus infected cells.</a> Frontiers Cellular and Infection Microbiology. 8, 251 (2018).</li><li>Buchmeier, M. J., de la Torre, J. C., Peters, C. J., Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arenavirdae:+The+viruses+and+their+replication.">Arenavirdae: The viruses and their replication.</a> Fields Virology. 2, (2006).</li><li>Levis, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+interferon+in+Argentine+hemorrhagic+fever.">Endogenous interferon in Argentine hemorrhagic fever.</a> The Journal of Infectious Diseases. 149, 428-433 (1984).</li><li>Levis, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+endogenous+interferon+and+the+clinical+evolution+of+patients+with+Argentine+hemorrhagic+fever.">Correlation between endogenous interferon and the clinical evolution of patients with Argentine hemorrhagic fever.</a> Journal of Interferon Research. 5, 383-389 (1985).</li><li>Huang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+pathogenic+new+world+and+old+world+human+arenaviruses+induce+distinct+interferon+response+in+human+cells.">Highly pathogenic new world and old world human arenaviruses induce distinct interferon response in human cells.</a> Journal of Virology. 89, 7079-7088 (2015).</li><li>Huang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Junin+virus+infection+activates+the+type+I+interferon+pathway+in+a+RIG-I-dependent+manner.">Junin virus infection activates the type I interferon pathway in a RIG-I-dependent manner.</a> PLoS Neglected Tropical Diseases. 6, e1659 (2012).</li><li>Huang, C., Kolokoltsova, O. A., Mateer, E. J., Koma, T., Paessler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+pathogenic+new+world+arenavirus+infection+activates+the+pattern+recognition+receptor+protein+kinase+R+without+attenuating+virus+replication+in+human+cells.">Highly pathogenic new world arenavirus infection activates the pattern recognition receptor protein kinase R without attenuating virus replication in human cells.</a> Journal of Virology. 91, 20 (2017).</li><li>Emonet, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+from+cloned+cDNAs+and+in+vivo+characterization+of+recombinant+pathogenic+Romero+and+live-attenuated+Candid#1+strains+of+Junin+virus,+the+causative+agent+of+Argentine+hemorrhagic+fever+disease.">Rescue from cloned cDNAs and in vivo characterization of recombinant pathogenic Romero and live-attenuated Candid#1 strains of Junin virus, the causative agent of Argentine hemorrhagic fever disease.</a> Journal of Virology. 85 (4), 1473-1483 (2011).</li><li>Child, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonism+of+the+protein+kinase+R+pathway+in+human+cells+by+Rhesus+Cytomegalovirus.">Antagonism of the protein kinase R pathway in human cells by Rhesus Cytomegalovirus.</a> Journal of Virology. 92, 6 (2018).</li><li>Hobro, A. J., Smith, N. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+fixation+methods:+Spatial+and+compositional+cellular+changes+observed+by+Raman+imaging.">An evaluation of fixation methods: Spatial and compositional cellular changes observed by Raman imaging.</a> Vibrational Spectroscopy. 91, 31-45 (2017).</li></ol>

For positive-sense RNA og dsDNA virus opdages dsRNA let med udbredte J2 anti-dsRNA antistoffer. Dog menes negative-følelse RNA-vira til at producere dsRNA på et lavt eller under detektionsgrænsen ved hjælp af den samme antistof2. Mange aspekter af viral RNA og PRR interaktion er således i vid udstrækning uklart for negative-følelse RNA-vira. Vi forsøgte at pletten for dsRNA i arenavirus infektion ved hjælp af J2 antistof men fluorescens-signaler var ikke differentiable sammenlignes med fo...

Det første skridt af induktion af det medfødte immunforsvar er vært anerkendelse af dobbelt-strenget (ds) RNA af mønster anerkendelse receptorer (PRRs) såsom retinoid syre (RIG-jeg) lide receptorer (RLRs) RIG-jeg og melanom differentiering-associerede protein 5 (MDA-5)1. For positive-sense RNA-vira, kan dsRNA normalt være let påvises ved hjælp af den J2 eller K1 anti-dsRNA monoklonale antistoffer (Mab)2. Samspillet mellem dsRNA og PRRs i positiv-strenget RNA-vira, såsom picornavirus, har været karakteriseret ved hjælp af konfokalmikroskopi3. Men for negative-følelse RNA-virus, visualisering og karakterisering af PRR og dsRNA interaktion har været hæmmet af manglen på følsomme antistoffer til dsRNA. RNA fluorescerende in situ hybridisering (FISH) er blevet anvendt til visualisering af viral RNA og PRRs4. Ikke desto mindre fisk metode kræver kendskab til target RNA sekvens og ikke kan være forenelig med PRR Co farvning. For nylig, 9 d 5 Mab, som oprindelig blev udviklet til diagnosticering af pan-enterovirus infektion, blev fundet at være mere følsomme end J2 Mab og kan let opdage dsRNA i negative-følelse RNA-virus infektion5,6. Mab 9 d 5 er således en roman og nyttigt redskab til at studere viral replikation og samspillet mellem PRR og viral RNA for negative-følelse RNA-virus.
Arenaviruses er en familie af enkeltstrenget, negative-følelse RNA-vira, som omfatter flere menneskelige patogener, såsom Lassa virus (LASV), Junín virus (JUNV) og Machupo virus (MACV), der forårsager alvorlige hæmoragisk feber sygdomme i mennesker7. Kliniske data fra alvorlige og dødelige tilfælde af argentinske hæmoragisk feber forårsaget af New World arenavirus JUNV udviser usædvanlig høje niveauer af serum IFN-α8,9. Vi har vist, at den patogene NW arenaviruses (JUNV og MACV), men ikke den patogene Old World arenavirus, LASV, fremkalde en type jeg interferon (IFN) svar i humane monocyt-afledte dendritiske celler10. Desuden RIG-jeg er en af sensorerne mægle skrive jeg IFN svar i JUNV-inficerede celler11. Vi fandt også, at protein kinase Rasmussen (PKR) receptor, som traditionelt er kendt for dsRNA anerkendelse, aktiveres i patogene NW arenavirus infektion12. For yderligere at forstå mekanismen af virus-specifikke IFN svar under arenavirus infektion, vi sigter mod at udvikle en protokol for at visualisere interaktion mellem viral dsRNA og cytoplasmatisk PRRs.
Infektion eksperimenter med patogene JUNV, MACV og LASV skal udføres i biosikkerhed niveau 4 (BSL-4) faciliteter. Således ud over det formentlig lave niveau af dsRNA dannet i arenavirus infektion, er opfylder kravene til biosikkerhed en anden teknik udfordring når du udfører imaging studier til disse staerkt patogene virus. Ved at udnytte 9 d 5 antistof og Candid1 # vaccinestamme af JUNV, en Konfokal mikroskopi-baseret protokol er beskrevet i denne betænkning, som har været anvendt med succes til at visualisere dsRNA, virale proteiner og PRR samtidig i celler inficeret af arenavirus i BSL2 labs. Protokollen er også velegnet til visualisering af intracellulære distribution af dsRNA og PRR under patogene arenavirus infektion i BSL4 faciliteter.

<table>
	<tbody>
		<tr>
			<td>APEX Alexa Fluor 647 antibody labeling kit</td>
			<td>Invitrogen</td>
			<td>A10475</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>A4503</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Cell Signaling</td>
			<td>4083</td>
			<td>1:1,000 dilution</td>
		</tr>
		<tr>
			<td>Donkey-anti rabbit Alexa Fluor 594</td>
			<td>Invitrogen</td>
			<td>A-11058</td>
			<td>1:2,000 dilution; Lot #: 1454437</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Atlanta Bio</td>
			<td>S11150</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microscope slides</td>
			<td>Fisher</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat-anti mouse Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A-11029</td>
			<td>1:2000 dilution; Lot #: 1874804</td>
		</tr>
		<tr>
			<td>Human lung epithelial A549 cells</td>
			<td>ATCC</td>
			<td>CCL-185</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A412</td>
			<td>Stored at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Mouse MAb anti-JUNV NP</td>
			<td>BEI</td>
			<td>NA05-AG12</td>
			<td>Conjugated to Alexa-647 at 1:1,000 dilution</td>
		</tr>
		<tr>
			<td>Mouse MAb pan-Enterovirus 9D5 Reagent</td>
			<td>Millipore Sigma</td>
			<td>3361</td>
			<td>ready for use; diluted to 1:2; Lot #: 3067445</td>
		</tr>
		<tr>
			<td>PBS supplemented with Ca and Mg</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>PDL Coated coverslips</td>
			<td>Neuvitro</td>
			<td>H-12-1.5-pdl</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold antifade</td>
			<td>Invitrogen</td>
			<td>P10144</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit MAb MDA-5</td>
			<td>Abcam</td>
			<td>ab126630</td>
			<td>1:250 dilution; Lot #: GR97758-7</td>
		</tr>
		<tr>
			<td>recombiant Candid#1 strain of JUNV</td>
			<td>Lab generated</td>
			<td>Lab generated</td>
			<td>As previously described in reference 13.</td>
		</tr>
		<tr>
			<td>RIG-I mouse MAb conjugated to Alexa-594</td>
			<td>Santa Cruz</td>
			<td>sc-376845</td>
			<td>1:1000 dilution; Lot #: AO218</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T8787&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

confocal imaging of double-stranded rna and pattern recognition receptors in negative-sense rna virus infection

Dobbelt-strenget (ds) RNA er produceret som en replicative mellemting under RNA-virusinfektion. Anerkendelse af dsRNA af vært mønster anerkendelse receptorer (PRRs) såsom retinoid syre (RIG-jeg) lide receptorer (RLRs) RIG-jeg og melanom differentiering-forbundet protein 5 (MDA-5) fører til induktion af det medfødte immunforsvar. Dannelse og intracellulære fordeling af dsRNA i positiv forstand RNA-virusinfektion har været godt karakteriseret ved mikroskopi. Mange negative-følelse RNA-vira, herunder nogle arenaviruses, udløser det medfødte immunforsvar under infektion. Imidlertid var negative-følelse RNA-vira troede at producere lave niveauer af dsRNA, som hindrer en billeddannelse undersøgelse af PRR anerkendelse af viral dsRNA. Derudover skal infektion eksperimenter med højpatogen arenaviruses udføres i høj biosikkerhed niveau indeslutningsfaciliteter (BSL-4). Interaktion mellem viral RNA og PRRs for højpatogen RNA-virus er stort set ukendt på grund af de yderligere tekniske udfordringer, som forskere skal stå i BSL-4 faciliteter. For nylig, et monoklonalt antistof (Mab) (klon 9 d 5) oprindeligt brugt til registrering af pan-enterovirus er blevet fundet at specifikt opdage dsRNA med en højere følsomhed end de traditionelle J2 eller K1 anti-dsRNA antistoffer. Heri, ved at udnytte 9 d 5 antistof, vi beskriver en Konfokal mikroskopi protokol, der har været anvendt med succes til at visualisere dsRNA, virale proteiner og PRR samtidig i enkelte celler inficeret med arenavirus. Protokollen er også velegnet til billeddiagnostiske undersøgelser af dsRNA og PRR distribution i patogene arenavirus inficerede celler i BSL4 faciliteter.

Denne protokol blev anvendt til at studere distribution og colocalization mellem RLRs (RIG-jeg og MDA-5) og dsRNA i JUNV-inficerede celler. Som vist i figur 1 og figur 2, øger ophobningen af dsRNA over tid som virusinfektion skrider frem. Koncentreret MDA-5 (figur 1) og RIG-jeg (figur 2) signaler blev fundet colocalized med punktformet strukturer for NP og dsRNA.
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Watch this Scientific Journal Video about Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection at JoVE.com

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Dobbelt-strenget RNA produceret under RNA-virus replikering kan genkendes af mønster anerkendelse receptorer til at fremkalde en medfødte immunrespons. For negative-følelse RNA-vira uklart, samspillet mellem den laveste-niveau-dsRNA og PRRs. Vi har udviklet en Konfokal mikroskopi metode til at visualisere arenavirus dsRNA og PRR i individuelle celler.

Konfokal billeddannelse af dobbelt-strenget RNA og mønster anerkendelse receptorer i Negative-følelse RNA-virusinfektion

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...

Traditionel dobbelt-streng RNA detektionsanalyse normalt ikke er følsom nok til at opdage dobbelt-streng RNA dannet i negativ-sense RNA virus infektion. Derfor har samspillet mellem dobbeltstrengede RNA- og værtsmønstergenkendelsesreceptorer været stort set undvigende for disse vira. Ved hjælp af denne protokol er vi i stand til at visualisere og karakterisere samspillet mellem viralt nukleart protein, dobbelt-streng RNA og være vært for en PRRs på et enkeltcelleniveau for negativ-sense RNA-virus. Dette er en multiplex analyse, der giver mulighed for samtidig farvning af dobbeltstrenget RNA og to virale eller vært protein mål i de enkelte celler. I modsætning til RNA-FISH-analysen er denne teknik RNA-sekvens uafhængig og sædvanligvis kompatibel med antistofdetektion af proteinmål. Begynd 24 timer før infektion ved såning 200, 000 menneskelige lunge epitel A549 celler på Poly-D-Lysin belagt glas dække glider i 12-brønd plader og dyrke dem på 37

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