Meget følsom analyse for måling af arenavirus-celle Attachment
Summary
The first step in the arenavirus life cycle is attachment of viral particles to host cells. We report a quantitative (q)RT-PCR-based assay for ultrasensitive detection and quantitation of arenavirus attachment events.
Abstract
Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.
Introduction
Arenavirus er en familie af kappeklædte, enkeltstrengede RNA-vira, der vedligeholdes primært i gnavere i naturen 1-3. Mens disse vira typisk etablere en asymptomatisk, vedvarende infektion i gnavere reservoirer 1,2, kan de forårsage alvorlig sygdom hos mennesker 3. Vigtige patogene arter omfatter den nye verden Junin virus (JUNV) 4 og den gamle verden Lassa virus 5, som er de ætiologiske agenter for argentinsk hæmoragisk feber i Sydamerika og Lassa feber i Afrika, hhv. Lymfocytisk choriomeningitis virus (LCMV), det prototypiske virus af familien 6, har en verdensomspændende distribution og er ansvarlig for en række sygdomstilstande, herunder aseptisk meningitis hos immunkompetente individer 6, alvorlige fødselsdefekter i det udviklende foster 7, eller høj dødelighed hos immunsupprimerede individer efter fast organtransplantation 8,9. Manglen på USAFood and Drug Administration (FDA) -godkendte vacciner eller effektive antivirale midler til at bekæmpe disse vira fremhæver det kritiske behov for at udvikle nye strategier til terapeutisk målrettet disse nye / re-nye patogener.
Den arenavirus genomet består af to enkeltstrengede RNA-segmenter, den store (L) (~ 7.2 kb) og lille (S) (~ 3,6 kb) segmenter 10. S-segmentet koder for den virale nukleoprotein (NP) og kappeglycoprotein (GP), mens L-segmentet koder for den virale polymerase (L) og matrix protein (Z). S genomiske RNA-segmenter (vRNA'er) L og er pakket i virioner 10-12. Det indledende trin af arenavirus infektion er binding af virale partikler til værtsceller via en interaktion mellem det virale GP og dens tilsvarende værtscellereceptorer som for JUNV, indbefatter den humane transferrin-receptor 1 (hTfR1) 13,14. Efter fastgørelse bliver JUNV partikler suges ind i cellen via clathrin endocytose 15.Partikler derefter trafikdata til den sene endosom, hvor den lave pH af dette rum udløser aktiveringen af GP 16,17. Når den er aktiveret, GP-kodet fusionspeptiddele indsatserne i den endosomale membran, indledning fusion mellem de virale og endosomale membraner. Vellykket fusion resulterer i frigivelse af virion-emballerede vRNA'er ind i cytoplasmaet, hvor de tjener som templates for genomisk replikation og transkription. Når tilstrækkelige mængder af virale strukturelle proteiner og vRNA'er genereres, nye partikler samle og knop fra den inficerede celle for at fuldføre livscyklus 18.
For at lette opdagelsen af nye strategier til terapeutisk målrette patogene arenavirus har vores gruppe fokuseret på identifikation af værten faktorer, der er afgørende for virus formering, men undværes for værten. I denne forbindelse definerer den præcise fase (r) af den virale livscyklus kontrolleres af sådanne vært faktorer er nødvendige for at styreudvikling af antivirale strategier. Heri beskriver vi en kvantitativ (q) RT-PCR-baseret assay, der kan anvendes til at måle arenavirus-cellebinding til at identificere, om udvalgte værtsproteiner og / eller antivirale molekyler påvirke denne indledende fase af viral indtrængen 19. Alternative metoder til at måle arenavirus-cellebinding har featured virioner mærket med biotin 20, fluorescerende farvestoffer 21, eller radioaktive isotoper 22. Ulemper ved disse fremgangsmåder kan indbefatte kravet om store mængder input-virus (f.eks høje infektionsmultipliciteter (MOI)), anvendelse af radioaktivitet, og / eller yderligere håndteringstrin at forberede input virus (fx koncentration og / eller mærkning af virus) . Især brugen af høje MOI gør det vanskeligt at skelne produktiv versus uproduktive vedhæftede begivenheder 15,23. Det QRT-PCR-baseret assay er beskrevet her, kan detektere vedhæftede begivenheder bruge så få som 20 plak forMing enheder (PFU) af virus, hvilket svarer til en MOI på 0,0004 for en 48-brønds plade. Derfor er den stærke følsomheden af analysen overvinder kravet om højt MOI samt eventuelle virus mærkning eller koncentrering. Endvidere kan assayet være i) indrettet til at måle virion endocytose samt frigivelse af virus-genomet ind i cytoplasmaet efter fusion og ii) skræddersyet til anvendelse med andre virus gennem udvikling af virusspecifikke QRT-PCR-reagenser (for eksempler, se 24-27).
Protocol
Representative Results
Discussion
Protokollen beskriver vi er ligetil og robust forudsat følgende kritiske overvejelser overholdes. For det første skal en streng pre-PCR-teknik anvendes (se 28 for en fremragende anmeldelse). Det er bydende nødvendigt, at alle reagenser og udstyr er fri for amplificeret DNA, der indeholder qPCR målsekvensen og at passende kontroller er på plads til at opdage en sådan forurening. For det andet, for at virus-cellebinding del af protokollen, virus, celler, og medierne skal holdes på 4 ° C for at forhindre…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Vi takker Markus Thali, Nathan Roy, Christopher Ziegler, Emily Bruce, og Benjamin konge for nyttige diskussioner og National Institutes of Health til støtte for disse undersøgelser gennem tilskud T32 AI055402 (JK), R21 AI088059 (JB), og P20RR021905 (JB) .
Materials
| DMEM | Life Technologies | 11965-118 | |
| Penicillin-Streptomycin | Life Technologies | 15140-163 | 100x |
| HEPES Buffer Solution | Life Technologies | 15630-130 | 100x |
| PBS pH 7.4 | Life Technologies | 10010-023 | 1x |
| Vero E6 cells | American Type Culture Collection | CRL-1586 | |
| Costar 48-well plate | Corning | 3548 | |
| RNeasy mini kit | Qiagen | 74106 | do not mix buffers RLT or RW1 with bleach |
| QIAshredder homogenizer | Qiagen | 79654 | |
| Taqman Universal PCR Master Mix | Life Technologies | 4326614 | |
| Multiscribe Reverse Transcriptase (50U/µl) | Life Technologies | 4311235 | |
| dNTP Mixture (10 mM; 2.5 mM each dNTP) | Life Technologies | N808-0260 | |
| GeneAmp 10X PCR Buffer II and 25 mM MgCl2 solution | Life Technologies | N808-0010 | |
| RNase Inhibitor (5000U/100 µl) | Life Technologies | N808-0119 | |
| RT primer 5’-AAGGGTTTAAAAATGGTAGCAGAC-3’ | Integrated DNA Technologies | 250 nmol DNA oligo | standard desalting |
| Forward qPCR primer 5’-CATGGAGGTCAAACAACTTCCT-3’ | Life Technologies | 4304971 | standard desalting |
| Reverse qPCR primer 5’-GCCTCCAGACATGGTTGTGA-3’ | Life Technologies | 4304971 | standard desalting |
| TaqMan Probe 5’-6FAM-ATGTCATCGGATCCTT-MGBNFQ-3’ | Life Technologies | 4316033 | HPLC purified |
| MicroAmp Fast Optical 96-well reaction plate (0.1 mL) | Life Technologies | 4346906 | |
| StepOnePlus Real-Time PCR System | Life Technologies | 4376598 | or other qPCR instrument |
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