Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Summary

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

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 characterized^1,2.

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

A schematic flowchart of the protocol is shown in Fig. 1 and a table of reagents is presented below.

1. Infection (16 - 24 h)

1. Seed out HeLa cells in 5 150 mm plastic cell culture dishes in Dulbecco's modified Eagle's medium (DMEM)-high glucose medium, such that they will reach approximately 80% confluency the following day (approximately 5 x 10⁸ cells). It is important that the cells do not reach 100% confluency.
2. On the next day, dilute the Strep-tagged influenza virus stock into phosphate-buffered saline (PBS) containing 0.3% bovine serum albumin (BSA) to a multiplicity of infection of 3.....

Discussion

While many studies have recently identified individual proteins or cellular networks involved in influenza virus infection⁸, the functional significance of the majority of these interactions remains unclear. Given the absolute dependence of chromatin-based functions for influenza virus RNA synthesis and the complex biophysical and biochemical nature of the nucleus⁹, new techniques will be required to elucidate these functions. The subnuclear fractionation we present here, coupled with affinity purif.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM-high glucose	GIBCO, by Life Technologies	11965-092
BSA	Sigma-Aldrich	A9418
Protease inhibitor Mix G	SERVA Electrophoresis	39101
Benzonase Nuclease	Novagen, EMD Millipore	71206	25 U/μl
DNase I, RNase-free	Thermo Fisher Scientific, Inc.	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