Temperature-sensitive lesions in two influenza A viruses defective for replicative transcription disrupt RNA binding by the nucleoprotein - PubMed (original) (raw)

Temperature-sensitive lesions in two influenza A viruses defective for replicative transcription disrupt RNA binding by the nucleoprotein

L Medcalf et al. J Virol. 1999 Sep.

Abstract

The negative-sense segmented RNA genome of influenza virus is transcribed into capped and polyadenylated mRNAs, as well as full-length replicative intermediates (cRNAs). The mechanism that regulates the two forms of transcription remains unclear, although several lines of evidence imply a role for the viral nucleoprotein (NP). In particular, temperature-shift and biochemical analyses of the temperature-sensitive viruses A/WSN/33 ts56 and A/FPV/Rostock/34/Giessen tsG81 containing point mutations within the NP coding region have indicated specific defects in replicative transcription at the nonpermissive temperature. To identify the functional defect, we introduced the relevant mutations into the NP of influenza virus strain A/PR/8/34. Both mutants were temperature sensitive for influenza virus gene expression in transient-transfection experiments but localized and accumulated normally in transfected cells. Similarly, the mutants retained the ability to self-associate and interact with the virus polymerase complex whether synthesized at the permissive or the nonpermissive temperatures. In contrast, the mutant NPs were defective for RNA binding when expressed at the nonpermissive temperature but not when expressed at 30 degrees C. This suggests that the RNA-binding activity of NP is required for replicative transcription.

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Figures

FIG. 1

Transcriptional activity and cellular accumulation of NP mutants. (A) The ability of the indicated mutants to support virus gene expression at 31 or 37°C was measured by transfecting CV1 cells containing the influenza virus RNA polymerase with plasmids pKT5, pKT5 S314-N, or A332-T and a synthetic influenza RNA segment containing an antisense CAT gene and then measuring the subsequent accumulation of CAT polypeptide. Data are plotted as the percentage (with the standard error) of the value obtained with the WT gene, and result from at least three independent determinations. (B) Cellular accumulation of WT and mutant NPs. CV1 cells were transfected with plasmids encoding WT NP (W), NP S314-N (S), NP A332-T (A), or plasmid vector (pKT0) not containing a heterologous gene (−), maintained at the indicated temperature for 6 h, detergent lysed, and examined for their NP content by SDS-PAGE and Western blotting with an anti-NP serum. The position of NP is indicated by the arrow.

FIG. 2

Cellular distribution of NP mutants. BHK cells (105) were transfected with 3 or 300 ng of plasmid and maintained for 4 h at 31 or 37°C as labelled. The cells were formaldehyde fixed, detergent permeabilized, and examined for NP content by indirect immunofluorescence with an anti-RNP serum.

FIG. 3

Expression and purification of WT and mutant NP fusion proteins. Lysates from E. coli cultures containing plasmids encoding WT MBP-NP (WT), MBP-NP A332-T (A332), MBP-NP S314-N (S314), or MBP were grown at the indicated temperatures and fractionated over amylose resin columns. Bound proteins were eluted by the addition of maltose, dialyzed, and analyzed by SDS-PAGE and stained with Coomassie brilliant blue dye. Also indicated are the sizes of molecular mass markers (in kilodaltons).

FIG. 4

Oligomerization of NP mutants. Aliquots of WT, S314-N, or A332-T NP translated in vitro at 37°C were analyzed by SDS-PAGE and autoradiography before (T) or after binding to MBP (−), WT MBP-NP (WT), or mutant MBP-NP fusion protein prepared at the indicated temperature.

FIG. 5

Ability of WT and mutant NP fusion proteins to bind influenza P proteins. (A) Subunit specificity. Radiolabelled lysates from Xenopus oocytes microinjected with synthetic mRNAs encoding the indicated P proteins (3P corresponds to a mixture of PB1, PB2, and PA mRNAs) were analyzed by SDS-PAGE and autoradiography before (T) or after precipitation with immobilized GST (G) or GST-NP (N). The positions of the P proteins are marked by arrows. The size of molecular mass markers are also indicated (in kilodaltons). (B) Association of WT and mutant MBP-NP fusion proteins with the polymerase complex. Radiolabelled lysates containing all three P proteins were analyzed as described above after precipitation with MBP (M), WT MBP-NP (WT), or mutant MBP-NP fusion proteins prepared at the indicated temperatures.

FIG. 6

RNA-binding activity of WT and mutant MBP-NP polypeptides. (A) Effect of growth and assay temperature on RNA-binding activity of WT MBP-NP. Proteins were grown (G) at the indicated temperatures and titrated for their ability to bind and retain a radiolabelled RNA on nitrocellulose filters at the indicated assay (A) temperature. (B) UV cross-linking assay. Equal amounts of MBP, WT, and mutant MBP-NP polypeptides (prepared at 30 or 37°C as labelled) were incubated with radiolabelled RNA at the indicated temperatures and subjected sequentially to UV irradiation, RNase digestion, SDS-PAGE, and autoradiography. The positions of MBP-NP, MBP, and the MBP-NP “stub” (as determined by staining of the gel with Coomassie brilliant blue [data not shown]) are indicated by arrows. (C) Thermal denaturation of RNA-binding activity. WT or mutant A332-T MBP-NPs (the latter synthesized at 30°C) were incubated with radiolabelled RNA at increasing temperatures, and aliquots were filtered through a nitrocellulose membrane to separate bound and free RNA. The bound values are plotted as a fraction of the amount retained at 37°C.

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Temperature-sensitive lesions in two influenza A viruses defective for replicative transcription disrupt RNA binding by the nucleoprotein - PubMed (original) (raw)