Name: using click chemistry to measure the effect of viral infection on host-cell rna synthesis
Uploaded: 2013-08-09T13:00:00
Description: Watch this Scientific Journal Video about Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis at JoVE.com

Vi takker RB Tesh for at levere musen polyklonale anti-RVFV antiserum og M. Griffin i UTMB Flowcytometri Core Facility for hjælp med flowcytometri. Dette arbejde blev støttet af 5 U54 AI057156 gennem det vestlige Regional Center of Excellence, NIH tilskud R01 AI08764301, og finansiering fra Sealy Center for Vaccine Development hos UTMB.BK blev støttet af James W. McLaughlin Fellowship Fund på UTMB.OL blev støttet af en Maurice R. Hilleman Early-Stage Career Investigator Award.

Analyse af transkription ved fluorescensmikroskopi 1.. Inficere celler med MP-12 og Label Gryende RNA med EU <ol><li> Place 12-mm runde dækglas i 12-brønds vævskulturplade, et dækglas per brønd. </li></ol> Bemærk: Hvis cellerne har problemer overholde dækglas, frakke med poly-L-lysin (MW ≥ 70.000), før anbringelse i brønde. Til dette formål nedsænkes dækglas i en steril 0,1 mg / ml opløsning i H2O af poly-L-lysin i 5 min. Skyl...

<ol>
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W., Christensen, C., Meldal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptidotriazoles+on+solid+phase:+[1,2,3]-triazoles+by+regiospecific+copper(i)-catalyzed+1,3-dipolar+cycloadditions+of+terminal+alkynes+to+azides.">Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(i)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides.</a> J. Org. Chem. 67, 3057-3064 (2002).</li><li>Kalveram, B., Lihoradova, O., Ikegami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NSs+protein+of+rift+valley+fever+virus+promotes+posttranslational+downregulation+of+the+TFIIH+subunit+p62.">NSs protein of rift valley fever virus promotes posttranslational downregulation of the TFIIH subunit p62.</a> J. Virol. 85, 6234-6243 (2011).</li><li>Kalveram, B., 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=Rift+Valley+fever+virus+NSs+inhibits+host+transcription+independently+of+the+degradation+of+dsRNA-dependent+protein+kinase+PKR.">Rift Valley fever virus NSs inhibits host transcription independently of the degradation of dsRNA-dependent protein kinase PKR.</a> Virology. , (2012).</li><li>Head, J. A., Kalveram, B., Ikegami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+Rift+Valley+fever+virus+NSs+encoding+a+partial+truncation.">Functional analysis of Rift Valley fever virus NSs encoding a partial truncation.</a> PLoS One. 7, e45730 (2012).</li><li>Lihoradova, O. A., 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=Characterization+of+Rift+Valley+Fever+Virus+MP-12+Strain+Encoding+NSs+of+Punta+Toro+Virus+or+Sandfly+Fever+Sicilian+Virus.">Characterization of Rift Valley Fever Virus MP-12 Strain Encoding NSs of Punta Toro Virus or Sandfly Fever Sicilian Virus.</a> PLoS Negl Trop Dis. 7, e2181 (2013).</li><li>Kalveram, B., Ikegami, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toscana+Virus+NSs+Protein+Promotes+Degradation+of+Double-Stranded+RNA-Dependent+Protein+Kinase.">Toscana Virus NSs Protein Promotes Degradation of Double-Stranded RNA-Dependent Protein Kinase.</a> J. Virol. , (2013).</li><li>Schmaljohn, C., Nichol, S., Knipe, D. M., 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=.">.</a> In Fields Virology. , 1741-1789 (2007).</li><li>Le May, N., 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=TFIIH+transcription+factor,+a+target+for+the+Rift+Valley+hemorrhagic+fever+virus.">TFIIH transcription factor, a target for the Rift Valley hemorrhagic fever virus.</a> Cell. 116, 541-550 (2004).</li><li>Caplen, H., Peters, C. J., Bishop, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutagen-directed+attenuation+of+Rift+Valley+fever+virus+as+a+method+for+vaccine+development.">Mutagen-directed attenuation of Rift Valley fever virus as a method for vaccine development.</a> J. Gen. Virol. 66, 2271-2277 (1985).</li><li>Ikegami, T., Won, S., Peters, C. J., Makino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+infectious+Rift+Valley+fever+virus+entirely+from+cDNA,+analysis+of+virus+lacking+the+NSs+gene,+and+expression+of+a+foreign+gene.">Rescue of infectious Rift Valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene.</a> J. Virol. 80, 2933-2940 (2006).</li><li>Vyboh, K., Ajamian, L., Mouland, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+viral+RNA+by+fluorescence+in+situ+hybridization+(FISH).">Detection of viral RNA by fluorescence in situ hybridization (FISH).</a> J. Vis. Exp. , e4002 (2012).</li><li>Reich, E., Goldberg, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actinomycin+and+nucleic+acid+function.">Actinomycin and nucleic acid function.</a> Prog. Nucleic Acid Res. Mol. Biol. 3, 183-234 (1964).</li><li>Holmes, K. L., Lantz, L. M., Russ, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugation+of+fluorochromes+to+monoclonal+antibodies.">Conjugation of fluorochromes to monoclonal antibodies.</a> Curr. Protoc. Cytom. Chapter 4 (Unit 4 2), (2001).</li></ol>

Den angivne protokol beskriver en metode til at måle effekten af ​​virusinfektion på værtscelle transskription via inkorporering af Uridin analoge EU til spirende RNA. Denne fremgangsmåde har flere fordele i forhold til tidligere metoder: det er hurtig, følsom, og det ikke er afhængige af anvendelsen af ​​radioaktive isotoper. Endvidere kan fremgangsmåden tilpasses til opnåelse af kvalitative data igennem fluorescensmikroskopi eller kvantitative data via flowcytometri med i det væsentlige de samme reage...

<html> Traditionelt har transkriptionel aktivitet blevet målt ved inkorporering af enten radioaktive (3 H-uridin) 1 eller halogenerede (BRU) 2 nukleosider i spirende RNA. Disse uridin analoger er taget op af celler, omdannet til uridin-trifosfat (UTP) gennem nukleosid bjærgning vej, og derefter indarbejdes i nysyntetiseret RNA. 3H-uridin kan påvises ved autoradiografi, hvilket kan være tidskrævende eller scintillation optælling, som kræver specialiseret udstyr. Endvidere kan anvendelse af radioaktive isotoper ikke være praktisk i en række laboratoriet indstillinger, herunder høj indeslutning biosikkerhed laboratorier. Bru kan påvises ved immunfarvning med anti-BRU-antistoffer, hvilket kan kræve barske permeabilisering at sikre adgang af antistoffet til kernen eller delvis denaturering af RNA, som RNA sekundær struktur kan være en sterisk hindring for antistofbinding. _content &quot;&gt; protokollen beskrevet her udnytter den nyligt udviklede klik kemi 3 at måle transkriptionel aktivitet i celler inficeret med RVFV stamme MP-12. klik kemi bygger på yderst selektiv og effektiv kobber (I)-katalyseret cycloadditionsreaktion mellem en alkyn og et azid del 4,5. I denne protokol, er spirende RNA i inficerede celler mærkes først gennem inkorporering af Uridin analog EU. I et andet trin, er indarbejdede EU opdaget ved reaktion med en fluorescerende azid. De vigtigste fordele ved denne metode er (1) at det ikke kræver anvendelse af radioaktive isotoper og (2) at det ikke kræver barske permeabilisering eller denaturering af RNA. Med en molekylvægt på mindre end 50 Da, adgang for de fluorescerende azid ikke hindres af utilstrækkelig permeabilization eller RNA sekundær struktur. Desuden er forskerne ikke begrænset i deres valg af primære antistoffer, når man kombinerer påvisning af RNA med en klasseical immunfarvning for virale proteiner. To forskellige fremgangsmåder er beskrevet i denne protokol: en til at visualisere transkription via fluorescensmikroskopi (trin 1-4), og én til kvantificering transkription gennem flowcytometri (trin 5-8). Begge disse metoder kombinere mærkning af spirende RNA med et intracellulært immunfarvning for virale proteiner, hvilket muliggør en korrelation mellem transkriptionel aktivitet og virusinfektion på en celle til celle-basis. Forfatterne har med succes brugt protokollen beskrevet her at bestemme transskriptionel aktivitet i celler inficeret med RVFV stamme MP-12 6 celler inficeret med forskellige MP-12 mutanter 7,8, 9 og celler inficeret med Toscana-virus (TOSV) 10.. Protokollen beskrevet her, kan let tilpasses til anvendelse med forskellige vira og har potentialet til at blive modificeret til at omfatte immunfluorescensfarvning af specifikke virale eller cellulære proteiner.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>5-ethynyl-uridine</td>
			<td>Berry &amp; Associates</td>
			<td>PY 7563</td>
			<td>dissolve in DMSO for 100 mM stock</td>
		</tr>
		<tr>
			<td>12-mm round coverslips</td>
			<td>Fisherbrand</td>
			<td>12-545-82</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>5 ml polystyrene tubes</td>
			<td>BD Biosciences</td>
			<td>352058</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Actinomycin D (ActD)</td>
			<td>Sigma</td>
			<td>9415</td>
			<td>dissolve in DMSO for 10 mM stock</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-mouse IgG</td>
			<td>Invitrogen</td>
			<td>A-11029</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Santa Cruz</td>
			<td>sc-2323</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>CuSO4</td>
			<td>Sigma</td>
			<td>C-8027</td>
			<td>dissolve in H2O for 100 mM stock</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D-9542</td>
			<td>dissolve in H2O for 5 mg/ml stock</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>11965092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Invitrogen</td>
			<td>16000044</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>fluorescent azide (Alexa Fluor 594-coupled)</td>
			<td>Invitrogen</td>
			<td>A10270</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>fluorescent azide (Alexa Fluor 657-coupled)</td>
			<td>Invitrogen</td>
			<td>A10277</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluoromount-G</td>
			<td>Southern Biotech</td>
			<td>0100-01</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-ascorbic acid</td>
			<td>Sigma</td>
			<td>A-5960</td>
			<td>dissolve in H2O for 500 mM</td>
		</tr>
		<tr>
			<td>paper filter</td>
			<td>VWRbrand</td>
			<td>28333-087</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>poly-L-lysine (MW &ge; 70000)</td>
			<td>Sigma</td>
			<td>P1274</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T-8787</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T-1503</td>
			<td>dissolve in H2O for 1.5 M stock, pH 8.5</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200056</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Fluorescence Microscope</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>e.g. Olympus IX71</td>
		</tr>
		<tr>
			<td>Flow Cytometer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>e.g. BD Biosciences LSRII Fortessa</td>
		</tr>
	</tbody>
</table>

using click chemistry to measure the effect of viral infection on host-cell rna synthesis

Mange RNA-vira har udviklet evnen til at inhibere værtscelle transskription som et middel til at omgå cellulære forsvar. Til undersøgelse af disse vira, er det derfor vigtigt at have en hurtig og pålidelig måde at måle transkriptionel aktivitet i inficerede celler. Traditionelt har transkription blevet målt enten ved inkorporering af radioaktive nucleosider såsom 3H-uridin efterfulgt af detektion via autoradiografi eller scintillationstælling eller inkorporering af halogenerede uridin analoger såsom 5-bromouridine (BRU) efterfulgt af detektion via immunfarvning. Anvendelse af radioaktive isotoper, imidlertid kræver specialiseret udstyr og er ikke mulig i en række laboratoriet indstillinger, mens påvisning af Bru kan være besværlige og kan lide af lav følsomhed. Den nyligt udviklede klik kemi, som indebærer en kobber-katalyseret triazole dannelse fra en azid og en alkyn, giver nu en hurtig og highly følsom alternativ til disse to metoder. Klik kemi er en totrins proces, hvor spirende RNA først mærkes ved inkorporering af uridin analogen 5-ethynyluridine (EU), efterfulgt af detektion af mærket med et fluorescerende azid. Disse azider er tilgængelige som forskellige fluoroforer, der giver mulighed for en bred vifte af muligheder for visualisering. Denne protokol beskriver en metode til at måle transkriptionel suppression i celler inficeret med Rift Valley fever virus (RVFV) stamme MP-12 under anvendelse af klik kemi. Samtidig er ekspression af virale proteiner i disse celler bestemt ved klassisk intracellulær immunfarvning. Trin 1 til 4 detalje en metode til at visualisere transkriptionel suppression via fluorescensmikroskopi, mens trin 5 til 8 detalje en metode til at kvantificere transkriptionel suppression via flowcytometri. Denne protokol er let at tilpasse til brug sammen med andre vira.

NSS protein RVFV (familie Bunyaviridae, slægten Phlebovirus) 11 inhiberer værtscelle generel transkription gennem to særskilte mekanismer: (I) ved at sekvestrere p44-underenheden af den basale transskriptionsfaktor TFIIH 11 og (ii) ved at fremme proteasomalaktivitet nedbrydning af p62 underenheden af TFIIH 6.. Den RVFV MP-12 vaccinestamme 13 er blevet anvendt til eksperimenterne beskrevet i denne protokol, da MP-12 stammen kan håndteres i en biosikkerhed ...

Watch this Scientific Journal Video about Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis at JoVE.com 

Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis (Video) | JoVE 

Denne metode beskriver brugen af ​​klik kemi til at måle ændringer i værtscelle transskription efter infektion med Rift Valley fever virus (RVFV) stamme MP-12. Resultaterne kan visualiseres kvalitativt via fluorescensmikroskopi eller opnået kvantitativt gennem flowcytometri. Denne metode kan tilpasses til brug med andre vira.

Brug Klik Chemistry at måle effekten af ​​Viral infektion på Host-celle RNA-syntese

Many RNA viruses have evolved the ability to inhibit host cell transcription as a means to circumvent cellular defenses. For the study of these viruses, ...

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Using Click Chemistry to Measure the Effect of Viral Infection on Host-Cell RNA Synthesis

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