Isolering af Viral Replication Rum-berigede underentreprise nukleare fraktioner fra Adenovirus-inficerede Normal humane celler
Summary
We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.
Abstract
During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.
Introduction
Adenovirus indeholde en dobbeltstrenget DNA-genom, der replikerer i den inficerede cellekerne. Når den virale DNA ind i kernen, det lokaliserer støder op til PML nukleare organer 1. Efter viral tidlig genekspression, er den nukleare arkitektur dramatisk reorganiseret, overtalelse dannelse af virale mikromiljøer, kaldet viral replikation Rum (RC) 2. Eftersom adenovirus (Ad) RC er steder, hvor virale genom replikation og ekspression af virale sene gener finder sted, de giver et miljø for rekruttering af alle de nødvendige virale og cellulære faktorer, der deltager i disse processer. Interessant, en række cellulære proteiner, der er ansvarlige for den cellulære antiviralt respons, såsom DNA beskadigelse respons, responset medfødte immunforsvar og tumor suppression co-valgt til disse virale steder 2. Derfor kan Ad RC betragtes regulatoriske hubs, der fremmer en effektiv viral replikation, mens samtidig at regulerecellulære antiviralt respons, hvilket indikerer, at disse strukturer er nøglen til forståelsen af virus-værts celle-interaktioner. Ikke desto mindre er de molekylære mekanismer i RC dannelse, deres sammensætning og tilhørende aktiviteter er dårligt forstået.
Adenoviral RC, samt RC fra andre DNA-vira, som replikerer i kernen er ikke forbundet til membraner, i modsætning til cytoplasmatiske RC 3. Endvidere vil sandsynligvis være sammensat udelukkende af proteiner og nukleinsyrer disse virusinducerede strukturer. RC dannet i celler inficeret med RNA-virus (betegnes normalt virale fabrikker) er blevet isoleret, at drage fordel af deres cytoplasmatiske lokalisering og membranbundne status, hvilket har lettet deres detaljerede morfologiske, funktionelle og biokemisk karakterisering 4.
Så vidt vi ved, har nukleare virale RC ikke er blevet isoleret, måske på grund af kompleksiteten af den nukleare arkitektur og fravær af intranucleær membranes som ville lette deres isolation. Deres undersøgelse har påberåbt i stedet på immunfluorescensmikroskopi, FISH og transmission elektronmikroskopi. Men på trods af komplikationer er forbundet med at isolere inde i kernen strukturer, andre nukleare områder som nucleoli og Cajal organer er blevet isoleret før 5,6. Da nucleoli og RC er begge sammensat af proteiner og nukleinsyrer, og har en diameter på mellem 0,5 – 5 um, vi den hypotese, at RC bør også være muligt at foretage en isolering. Derfor med henblik på at mere præcist at karakterisere den molekylære sammensætning og funktioner i tilknytning til RC, vi etableret en hidtil ukendt fremgangsmåde til isolering inde i kernen fraktioner beriget med RC. Til dette formål har vi fremstillet sub-nukleare fraktioner ved hjælp af hastighedsgradienter og saccharose puder svarende til procedurer, der anvendes til at isolere nucleoli 7 eller andre nukleare domæner 6 og etablerede et cellefrit system, der tillader undersøgelse af den molekylære sammensætning og associerede aktiviteterRC. Denne teknik bør derfor fremme forståelsen af virus-værts celle-interaktioner og repræsenterer et kraftfuldt værktøj, der bør også lette detaljeret analyse af RC fra andre virus, der replikerer i kernen og inducerer dannelsen af replikation rum af lignende dimensioner til dem, der dannes i adenovirus- inficerede-celler, såsom herpesvira, papillomvirus eller polyomaviruses.
Protocol
Representative Results
Discussion
In order to elucidate the molecular mechanisms that govern regulation of cellular activities by viral infection understanding the composition and activities associated with RC would be instrumental. Therefore, to make a detailed analysis of RC, we established a cell-free system that takes advantage of the size and biochemical composition of these virus-induced structures, to isolate subnuclear fractions enriched with RC using a simple procedure that relies on velocity gradients with sucrose cushions. Critical steps of th…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants from CONACyT-SEP (SEP-2008-84582; CB-2011-01-168497) and Promep-SEP for R.A.G.; P.H. received a scholarship from CONACyT (447442).
Materials
| DMEM | Gibco | 12100-046 | Warm in 37 ºC water bath before use |
| Fetal Bovine Serum | Gibco | 12484-028 | |
| Sucrose, Ultra Pure | Research Organics | 0928S | Prepare a 2.55 M stock solution and store at 4 ºC |
| Dounce homogenizer | Kontess Glass Company | 884900-0000 | |
| Branson 1800 Ultrasonic Bath | Branson | Z769533 SIGMA | Turn on 15 min before use. |
| Peroxidase AffiniPure F(ab')₂ Fragment Goat Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 115-036-003 | Use at a 1:10,000 dilution in PBS/0.03% non-fat milk |
| Goat anti-Mouse IgG1 Secondary Antibody, Alexa Fluor 488 conjugate | Life Technologies | A-21121 | Use at a 1:2,000 dilution in PBS |
| Silane-Prep Slides | Sigma | S4651-72EA | Open in a laminar flow cabinet |
| SuperSignal West Pico Chemiluminescent Substrate | Pierce ThermoScientific | 34080 |
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