Department of Virology, Universitätsklinikum Freiburg
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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).
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.
A schematic flowchart of the protocol is shown in Fig. 1 and a table of reagents is presented below.
1. Infection (16 - 24 h)
The length of incubation depends on the phase of infection to be studied (see Discussion).
2. Subcellular Fractionation (3 h)
3. Strep-tag Purification (2 h)
4. Representative Results
The efficiency of the fractionation is best judged by Western blot analysis of the fractionated lysates using antibodies specific for subcellular markers (Fig. 2). Specifically, successful subnuclear fractionation should show little or no cellular RNA polymerase II (Pol II) in the nucleoplasmic, or soluble nuclear fraction4, and most should be extracted with 150 mM NaCl5. A degradation of Pol II is also reproducibly observed in influenza virus-infected cells compared to uninfected cells, consistent with the literature6.
Purification of vRNPs is best judged by silver or Coomassie staining, as shown for a cytoplasmic eluate in Fig. 3. In our experience, most Strep-PB2 is purified in as part of a fully-formed vRNP, i.e. little soluble polymerase is captured. This is reflected in the high NP:PA/PB1/PB2 ratio observed by silver or Coomassie staining. A lower ratio suggests buffer contamination with RNases, as several ubiquitous RNases (such as RNase A) are known to cleave viral RNA in such a way as to dissociate the polymerase from NP multimers7. Interestingly, treatment with Benzonase alone, while sufficient to digest viral RNA, appears to have no effect on the stochiometric ratios of vRNP proteins. Although we cannot explain this phenomenon, we observed disruption of vRNPs after digestion with other RNases (data not shown), suggesting that Benzonase may leave certain structurally important RNA regions intact. After purification of vRNPs from the cytoplasm and nucleoplasm (performed without nuclease digestion), 8 faint but discrete bands at high molecular weights can be observed by silver staining, which correspond to the predicted molecular weights of the 8 influenza genome segments (see ref. 3).
The relative amounts of vRNPs purified from each fraction at 9 h post infection are shown in Fig. 4. The distribution of vRNPs varies over the course of infection, with strongest accumulation in the ch150 fraction at early time points, and increased accumulation in the nucleoplasm and cytoplasm at late time points.
Figure 1. Flow chart of the subcellular fractionation. Adapted from ref. 3.
Figure 2. Western blot analysis of subcellular fractions from influenza virus-infected cells. 109 HeLa cells were infected with influenza virus strain WSN for 9 h prior to subcellular fractionation. Equal amounts of total protein were loaded in each lane and analyzed using the antibodies shown. RNA Polymerase II (Pol II) was detected using clone 8WG16, which recognizes all forms of the C-terminal domain (the hypophosphorylated band is most prominent). Adapted from ref. 3.
Figure 3. Silver stain analysis of purified vRNPs. HeLa cells were infected with rWSN (as a negative control) or rWSN-PB2-Strep for 9 h, followed by Strep purification from the cytoplasmic fraction. Asterisk denotes bands which are not visible after RNase digestion. Adapted from ref. 3.
Figure 4. Silver stain analysis of vRNPs purified from different cellular fractions. 109 HeLa cells were infected with rWSN-PB2-Strep or rWSN for 9 h followed by subcellular fractionation and Strep purification. Equal amounts of eluate from each fraction were loaded. Adapted from ref. 3.
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 purification of vRNPs, allows the characterization of crucial viral RNA factories which were previously inaccessible.
Many subcellular fractionation protocols have been previously described in the literature. We observed that most of these traditional methods lead to premature leakage of vRNPs from the nucleus, a phenomenon also described for Pol II10. By using a short incubation of pre-swelled cells with a low concentration of a weak detergent, NP-40, efficient plasma membrane lysis occurred without significant nuclear leakage. Interestingly, performing our subcellular fractionation using A549 and HEK293T cells rather than HeLa cells led to strong nuclear leakage of vRNPs in both cell lines. Lowering the NP-40 concentration to approximately 0.2% reduced this issue; however, due to some variability of these cell lines, optimal conditions should be determined empirically. Another possible altermative is the use of non-human, influenza virus-susceptible cell lines such as MDCK or Vero cells.
Our salt extraction protocol differs from traditional chromatin extraction procedures which often use lower salt concentrations and/or high EDTA concentrations. Our protocol separates chromatin into two specific fractions, a Pol II-high/histone-low fraction and its reciprocal. However, other chromatin extraction methods may also be compatible with the affinity purification of vRNPs. Since most chromatin extraction procedures have focused on DNA-protein interactions as compared to protein-protein interactions, new techniques are required to extract tightly chromatin-bound multi-protein complexes11.
In general, the length of infection should be determined empirically due to variations among virus strains and cell lines. Using the rWSN-PB2-Strep strain at an MOI of 3, we have performed subcellular fractionation at time points varying from 3 to 10 h post infection. In our experience, less than 3 h of infection yields too few purified vRNPs for useful Western blot analysis, while infection longer than 3 h results in strong cytopathic effect and a subsequent mixing of fractions.
The ability to isolate intact, tightly chromatin-bound viral complexes opens up several possibilities. While we utilized a recombinant virus expressing a Strep-tagged PB2, other affinity tags can also be used, as well as traditional immunoprecipitation targeting other viral proteins. Furthermore, while our analysis of vRNPs was limited to the characterization of their protein and RNA components, the purified complexes could also be used to perform functional in vitro synthesis assays. Finally, the separation of vRNPs into coarse functional species through subnuclear fractionation may also allow the closer study of several recently-described post-translational modifications of viral proteins required for efficient viral RNA synthesis12,13.
No conflicts of interest declared.
The authors would like to thank Nada Naffakh and Marie-Anne Rameix-Welti (Institut Pasteur) for the rWSN-PB2-Strep virus.
|DMEM-high glucose||GIBCO, by Life Technologies||11965-092|
|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|