Nuclear dynamics of influenza A virus ribonucleoproteins revealed by live-cell imaging studies

Abstract

The negative sense RNA genome of influenza A virus is transcribed and replicated in the nuclei of infected cells by the viral RNA polymerase. Only four viral polypeptides are required but multiple cellular components are potentially involved. We used fluorescence recovery after photobleaching (FRAP) to characterise the dynamics of GFP-tagged viral ribonucleoprotein (RNP) components in living cells. The nucleoprotein (NP) displayed very slow mobility that significantly increased on formation of transcriptionally active RNPs. Conversely, single or dimeric polymerase subunits showed fast nuclear dynamics that decreased upon formation of heterotrimers, suggesting increased interaction of the full polymerase complex with a relatively immobile cellular component(s). Treatment with inhibitors of cellular transcription indicated that in part, this reflected an interaction with cellular RNA polymerase II. Analysis of mutated influenza virus polymerase complexes further suggested that this was through an interaction between PB2 and RNA Pol II separate from PB2 cap-binding activity.

Fig. 1

Reconstitution of recombinant GFP-tagged influenza RNPs in 293T cells. (A) Living cells expressing GFP-tagged RNP subunits alone or in combinations as indicated (2P; polymerase protein pairs, as labelled, 3P; the full polymerase complex) were analysed directly for GFP expression by confocal microscopy. Scale bar 5 μm. (B) Cells were transfected with plasmids expressing a synthetic vRNA encoding CAT and three (2P-GFP) or four of the RNP polypeptides, either all untagged (WT) or with the indicated one tagged with GFP. Three days post-transfection cells were analysed for CAT accumulation by ELISA. The mean and range from two independent experiments is plotted relative to the amount produced by WT RNPs. (C, D) Cells were transfected with plasmids expressing the indicated polypeptides (P, PB2-GFP; 3P, PB2-GFP, PA and PB1; cRNP and vRNP, PB2-GFP, PA and PB1 SDD along with NP and either a cRNA or vRNA containing a CAT gene respectively) or left untransfected (−). 24 h post-transfection (C) cell lysates and samples of purified virus (lane 1, as a marker) were analysed by SDS-PAGE and western blotting with antisera against PA, PB1, PB2, NP and GFP; (D) total RNA was extracted and analysed by primer extension using radiolabelled oligonucleotides specific for negative (top panel) or positive sense (lower panel) CAT transcripts. Radiolabelled products were separated by urea-PAGE and detected by autoradiography. Primer extension products resulting from the indicated CAT RNA species are labelled.

Fig. 2

Nuclear mobility of viral polymerase proteins. 293T cells were transfected with plasmids expressing (A) PB2-GFP alone or (B) PB1-GFP and PA in combinations with other polymerase proteins and NP as indicated. 24 h post-transfection fluorescent cells were analysed by FRAP. The average recovery kinetics are plotted. (C, D) The mean DC ± SEM for each transfection combination are plotted. See Table 1 for information on the number of repetitions and statistical analyses.

Fig. 3

Distribution of DC values from individual cells. Histograms of DC values from cells transfected with the indicated plasmids are shown. Data are plotted on bin centres as the % of the total for each category. The same data are plotted in (A) and (B) but with a smaller bin size in (B) to resolve the spread of 3P DC values.

Fig. 4

Nuclear mobility of viral RNPs and NP. (A, B) 293T cells were transfected with plasmids expressing PB2-GFP alone (P) or in combinations with the other polymerase proteins (3P), NP and model genome segments as indicated or (C, D) GFP-NP alone, in the context of a WT segment 7 RNP (+3Pwt seg7) or in the context of a replication incompetent segment 7 RNP (+3Psdd seg7). 24 h post-transfection fluorescent cells were analysed by FRAP and (A, C) recovery kinetics were plotted, for comparison, alongside recovery curves for PB2-GFP alone and in the heterotrimeric polymerase context. (B, D) The mean DC ± SEM for each transfection combination are plotted. Asterisks and brackets indicate the probability (⁎⁎⁎ p < 0.001; Student's two-tailed t-test, assuming equal variance) of the compared data being equal.

Fig. 5

Role of cellular transcription in the nuclear dynamics of the viral polymerase. (A) 293T cells were mock-treated (−), treated with 5 μg/ml ActD for 1 h or with 5 μg/ml α-amanitin for 9 h, lysed and analysed by SDS-PAGE and Western blotting with sera specific for modified forms of the CTD of RNA Pol II as indicated. (B) 293T cells were transfected with expression vectors for the indicated GFP-tagged viral polymerase proteins (3P complex tagged with PB2-GFP) or GFP, 24 h post transfection treated or mock treated with RNA Pol II inhibitors as in (A) and fluorescent cells then analysed by FRAP. The fold change in mean DCs of the drug-treated samples relative to the corresponding untreated samples are plotted. A Student's two-tailed t-test, assuming equal variance, was used to compare DCs of drug-treated and untreated samples and returned p-values (⁎⁎p < 0.01, ⁎⁎⁎p < 0.001) describing the probability of the compared data being equal. (C) 293T cells were transfected with expression vectors for the indicated TAP-tagged viral polymerase proteins (3P complex tagged with PB1-TAP) and 42 h later cell extracts fractionated by IgG sepharose chromatography followed by SDS-PAGE and (top panel) silver staining; migration of TAP-tagged subunits indicated by black spots, (middle panel) western blotting for PB2 or (lower panel) serine 5 phosphorylated Pol II. (D) Bound Pol II from three independent experiments was quantified by densitometry and plotted as the mean ± SEM relative to WT 3P (100%). (E) The fold change in mean DC values of the indicated samples relative to WT 3P are plotted. The 2P value is from PB1-GFP + PA.