Summary

Affinity очистки вируса гриппа рибонуклеопротеидных комплексов с хроматина инфицированных клеток

Published: June 03, 2012
doi:

Summary

Вирусы гриппа повторить их РНК-геном в связи с хост-клеточного хроматина. Здесь мы представляем метод, чтобы очистить нетронутыми вирусных комплексов рибонуклеопротеидных из хроматина инфицированных клеток. Очищенный вирусный комплексы могут быть проанализированы и Вестерн-блот и удлинения праймера белка и РНК, соответственно.

Abstract

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.

Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently.

Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the “nucleoplasmic” fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed “ch150”), then 500 mM NaCl (“ch500”) (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.

After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Protocol

Схема схема протокола показан на рис. 1 и таблицы реагентов приведены ниже. 1. Инфекция (16 – 24 ч) Семенной из клеток HeLa в 5 150 мм пластиковый культуре клеток блюд в среде Дульбекко изменения Орла (DMEM) высокого среднего глюкозы, например, что они достигнут при?…

Discussion

Хотя многие исследования недавно выявили отдельные белки или сотовой сети, участвующие в вирус гриппа инфекцию 8, функциональное значение большинства этих взаимодействий остается неясным. Учитывая абсолютную зависимость хроматина на основе функций для вируса гриппа синтез РНК…

Declarações

The authors have nothing to disclose.

Acknowledgements

Авторы хотели бы поблагодарить Нада Naffakh и Мари-Анн-Rameix Вельти (Institut Pasteur) в rWSN-PB2-Strep вируса.

Materials

Name of the reagent Company Catalogue number Comments
DMEM-high glucose Gibco 11965-092  
BSA Sigma A9418  
Protease inhibitor Mix G Serva 39101  
Benzonase Nuclease Novagen 71206 25 U/μl
DNase I, RNase-free ThermoScientific EN0523 50 U/μl
Dounce homogenizer Wheaton 432-1271 Use type “B” pestle
Strep-Tactin Sepharose IBA GmbH 2-1201-025 50% suspension column format can also be used
Desthiobiotin IBA GmbH 2-1000-002  

Referências

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Chase, G. P., Schwemmle, M. Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells. J. Vis. Exp. (64), e4028, doi:10.3791/4028 (2012).

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