The RNA polymerase of influenza a virus is stabilized by interaction with its viral RNA promoter

Abstract

The RNA polymerase of the influenza virus is responsible for the transcription and replication of the segmented RNA viral genome during infection of host cells. Polymerase function is known to be strictly dependent on interaction with its RNA promoter, but no attempts to investigate whether the virion RNA (vRNA) promoter stabilizes the polymerase have been reported previously. Here we tested whether the vRNA promoter protects the polymerase against heat inactivation. We prepared partially purified recombinant influenza A virus RNA polymerase, in the absence of influenza virus vRNA promoter sequences, by transient transfection of expression plasmids into human kidney 293T cells. The polymerase was found to be heat labile at 40 degrees C in the absence of added vRNA. However, it was protected from heat inactivation if both the 5' and 3' strands of the vRNA promoter were present. By using the ability of vRNA to protect the enzyme against heat inactivation, we established a novel assay, in conjunction with a mutagenic approach, that was used to test the secondary structure requirement of the vRNA promoter for polymerase binding. Binding required a panhandle structure and the presence of local hairpin loop structures in both the 5' and 3' ends of vRNA, as suggested by the corkscrew model. The interaction of the vRNA promoter with the influenza virus RNA polymerase heterotrimeric complex is likely to favor a particular closed conformation of the complex, thereby ensuring the stability of the RNA polymerase within both the infected cell and the isolated virus.

FIG. 1.

ApG-primed transcription of 14-nt influenza virus vRNA model templates requires a RNA polymerase complex containing PB1, PB2, and PA. The results of in vitro transcription of a model 14-nt vRNA 3′ sequence in the presence of an equimolar concentration (2.5 μM) of a model 15-nt 5′ end sequence, evaluated using the [α-³²P]GTP incorporation assay with different preparations of RNA polymerase prepared in parallel from the indicated plasmids (see Materials and Methods) and fractionated by 20% PAGE in 7 M urea, are shown. a, 14-nt product; b, 13-nt product (see text).

FIG. 2.

Both 5′ and 3′ vRNA promoter arms are needed to initiate transcription by RNA polymerase prepared without endogenous influenza virus-like RNA. ApG-primed in vitro transcription using the [α-²P] GTP incorporation assay was performed in the presence of short oligonucleotides (0.2 μM) corresponding to the 5′ end (15 nt), 3′ end (14 nt), or both ends of wild-type vRNA as shown, and the polymerase was fractionated by 20% PAGE in 7 M urea. Lanes 1 to 4, RNA polymerase prepared from PB1, PB2, PA, NP, and pPOLI-GFP; lanes 5 to 8, RNA polymerase prepared from PB1, PB2, PA, and NP. a′, 14-nt product AGCAAAAGCAGGGU, b′, 13-nt product AGCAAAAGCAGGG derived from endogenous pPOLI-GFP; z, longer transcription products, estimated as 17 to 19 nt in length, derived from endogenous pPOLI-GFP; a, 14-nt major end product derived from the added 14-nt 3′ end of wild-type vRNA; b, 13-nt products derived from added vRNA template (see text); c, 12-nt product AGCAAAAGCAGG, present in transcripts derived from both pPOLI-GFP and the added 14-nt 3′ end of wild-type vRNA.

FIG. 3.

Heat stability of the influenza virus RNA polymerase. The results of assays of the heat stability of the RNA polymerase are shown. RNA polymerase, isolated from 293T cells transfected with the plasmids as indicated, was preincubated for 15 min at 40°C (even-numbered lanes) or not preincubated (odd-numbered lanes), evaluated by the [α-³²P]GTP incorporation assay in the presence of added 5′ and 3′ ends (each at 5 μM) of the vRNA promoter, and fractionated by 20% PAGE in 7 M urea. a′, 14-nt partial product AGCAAAAGCAGGGT, derived from the RNA of plasmid pPOLI-GFP; a, 14-nt product, derived from the added 14-nt 3′ end of wild-type vRNA; b, 13-nt minor transcript, probably derived from a contaminating 13-nt vRNA template; c, 12-nt product (see legend to Fig. 2). The nuclear extracts (see Materials and Methods) used in lanes 1 and 2 were prepared from mock-transfected (no added plasmids) 293T cells.

FIG. 4.

Both the 5′ and 3′ ends of the vRNA promoter are needed to obtain significant protection of the RNA polymerase from heat inactivation. RNA polymerase, obtained by transfection with PB1, PB2, and PA, was heated for 15 min at 40°C in the presence of the indicated oligonucleotides (5 μM), and transcription was subsequently evaluated with the [α-³²P]GTP incorporation assay with ApG in the presence of both 5′ and 3′ vRNA oligonucleotides (2.5 μM). Lane 1, no added RNA; lane 2, 5′ vRNA strand (15 nt); lane 3, 3′ vRNA strand (14 nt); lane 4, 5′ plus 3′ strand; lane 5, unheated control; a, 14-nt product; b, 13-nt minor product; c, 12-nt minor product.

FIG. 5.

Base pairing between the conserved 5′ strand and 3′ strand of the vRNA promoter is required to protect the RNA polymerase from heat inactivation. (A) The transcriptional activity remaining after heat inactivation of the polymerase in the presence of structures 1 to 11. Mutations are shown in bold; base pairs are shown by vertical lines. For sequences 3, 5, 7, 9, and 11, activity was expressed as a percentage of wild-type (sequence 1) activity (means and standard deviations of three independent experiments), by quantitation of the major bands a and a₁ to a₅; activity values for sequences 2, 4, 6, 8, and 10 were from single measurements. (B) RNA polymerase, obtained by transfection of 293T cells with PB1, PB2, and PA, was heated for 15 min at 40°C in the presence of the indicated sequences (1 μM), transcription was evaluated with the [α-³²P]GTP incorporation assay, and the polymerase was fractionated by 20% PAGE in 7 M urea. Lanes 1, 3, 7, 9, 11, 4, and 5 depict the results for the corresponding sequences shown in panel A. Bands b and c depict minor, 13-nt or 12-nt transcripts, shorter than the main 14-nt product, band a; the minor band, x, depicts a longer, 15-nt transcript, possibly formed by the nontemplated addition of an extra base. Bands a₁ to a₅ differ in gel mobility from one another because of their differing G content.

FIG. 5.

Base pairing between the conserved 5′ strand and 3′ strand of the vRNA promoter is required to protect the RNA polymerase from heat inactivation. (A) The transcriptional activity remaining after heat inactivation of the polymerase in the presence of structures 1 to 11. Mutations are shown in bold; base pairs are shown by vertical lines. For sequences 3, 5, 7, 9, and 11, activity was expressed as a percentage of wild-type (sequence 1) activity (means and standard deviations of three independent experiments), by quantitation of the major bands a and a₁ to a₅; activity values for sequences 2, 4, 6, 8, and 10 were from single measurements. (B) RNA polymerase, obtained by transfection of 293T cells with PB1, PB2, and PA, was heated for 15 min at 40°C in the presence of the indicated sequences (1 μM), transcription was evaluated with the [α-³²P]GTP incorporation assay, and the polymerase was fractionated by 20% PAGE in 7 M urea. Lanes 1, 3, 7, 9, 11, 4, and 5 depict the results for the corresponding sequences shown in panel A. Bands b and c depict minor, 13-nt or 12-nt transcripts, shorter than the main 14-nt product, band a; the minor band, x, depicts a longer, 15-nt transcript, possibly formed by the nontemplated addition of an extra base. Bands a₁ to a₅ differ in gel mobility from one another because of their differing G content.

FIG. 6.

Base pairs in the stem of the 5′ hairpin loop of the promoter are required to protect the RNA polymerase from heat inactivation. (A) Pseudo-wild-type (3) (see Fig. 5A, sequence 3, for full structure) and point (12 to 16) mutants tested. Only the first 10 residues of the 15-nt 5′ hairpin loop are shown; for the full duplex structure, see Fig. 5A. Sequences are numbered from their 5′ ends (1′, 2′, 3′, etc.). (B) The RNA polymerase preparation, reconstituted in vivo from PB1, PB2, and PA plasmids, was heated for 15 min at 40°C in the presence of the indicated sequences (1 μM) and controls. Transcription was evaluated by the [α-³²P]GTP incorporation assay with added pseudo-wild-type vRNA sequences (1.25 μM), and the polymerase was fractionated by 22% PAGE in 7 M urea. a, 14-nt transcription product; b, 13-nt product. Control lanes: No RNA, no added RNA; 3′, only the 3′ strand of the promoter was present; No heat, unheated enzyme. An independent experiment gave essentially similar results.

FIG. 7.

Base pairs in the stem of the 3′ hairpin loop of the promoter are required to protect the RNA polymerase from heat inactivation. (A) The wild-type vRNA (1) and mutants (17 to 24) tested. Only the 3′ hairpin loop (or potential loop) of a 14-nt 3′ strand is shown; all experiments used in addition an equimolar amount of the 15-nt wild-type 5′ strand of vRNA. Sequences are numbered from their 3′ ends. (B and C) RNA polymerase, reconstituted in vivo from PB1, PB2, and PA plasmids, was heated for 15 min at 40°C in the presence of the indicated sequences (1 μM), transcription was evaluated by the [α-³²P]GTP incorporation assay with ApG and added wild-type vRNA sequences (2.5 μM), and the polymerase was fractionated by 22% PAGE in 7 M urea. See legend to Fig. 6 for explanation of additional symbols. A duplicate experiment gave similar results.