Different de novo initiation strategies are used by influenza virus RNA polymerase on its cRNA and viral RNA promoters during viral RNA replication

Abstract

Various mechanisms are used by single-stranded RNA viruses to initiate and control their replication via the synthesis of replicative intermediates. In general, the same virus-encoded polymerase is responsible for both genome and antigenome strand synthesis from two different, although related promoters. Here we aimed to elucidate the mechanism of initiation of replication by influenza virus RNA polymerase and establish whether initiation of cRNA and viral RNA (vRNA) differed. To do this, two in vitro replication assays, which generated transcripts that had 5' triphosphate end groups characteristic of authentic replication products, were developed. Surprisingly, mutagenesis screening suggested that the polymerase initiated pppApG synthesis internally on the model cRNA promoter, whereas it initiated pppApG synthesis terminally on the model vRNA promoter. The internally synthesized pppApG could subsequently be used as a primer to realign, by base pairing, to the terminal residues of both the model cRNA and vRNA promoters. In vivo evidence, based on the correction of a mutated or deleted residue 1 of a cRNA chloramphenicol acetyltransferase reporter construct, supported this internal initiation and realignment model. Thus, influenza virus RNA polymerase uses different initiation strategies on its cRNA and vRNA promoters. To our knowledge, this is novel and has not previously been described for any viral RNA-dependent RNA polymerase. Such a mechanism may have evolved to maintain genome integrity and to control the level of replicative intermediates in infected cells.

FIG. 1.

Mutational analysis of the influenza virus model cRNA promoter using a primer-independent replication assay, an ApG-primed transcription assay, and a UV cross-linking assay in vitro. (A) Autoradiograph showing the full-length unprimed or ApG-primed transcription product (TP) synthesized by partially purified 3P/NP complex on a wild-type (lane 1) or mutant (lanes 3 to 11) model cRNA promoter, as indicated, in the presence of 1 mM ATP, 0.5 mM GTP, 0.5 mM UTP, and 0.1 μM [α ³²P]CTP, and analyzed on 25% PAGE in 6 M urea. The negative control (lane 2) represents replication on the wild-type (WT) cRNA promoter template in the absence of ATP. The weak signal in lane 11 has a slightly slower mobility due to the different sequence. Additional bands presumably representing early termination products (•) or transcripts with a single nontemplated nucleotide extension (asterisk) are indicated. The origin of the band labeled with an arrow head is unknown. (B) Autoradiograph of variously phosphorylated forms of full-length in vitro cRNA replication products separated on 25% PAGE in 6 M urea. The 15-nucleotide transcription products obtained by ApG-primed or unprimed in vitro replication as described in (A) were gel-eluted and left untreated (lanes 1 and 4) or treated with either calf intestinal phosphatase (CIP) (lanes 2 and 3), tobacco acid pyrophosphatase (TAP) (lane 5), or polynucleotide kinase (PNK) (lane 6) as indicated. (C) Detailed analysis of polymerase binding of the model cRNA promoter mutants 4U→A and 5C→A by a UV cross-linking competition assay. Lanes 1, 5, and 9, cross-linking to the wild-type ³²P-labeled 3′ end of cRNA in the presence of excess unlabeled 5′ strand. Lanes 2, 6, and 10, in the presence of 0.01 μM wild-type and mutant competitors. Lanes 3, 7, and 11, in the presence of 0.1 μM wild-type and mutant competitors. Lanes 4, 8, and 12, in the presence of 5 μM wild-type and mutant competitors.

FIG. 2.

Identification of nucleotide residues required for pppApG synthesis in the 3′ strand of both the cRNA and vRNA promoters. (A) Influenza virus model cRNA promoter in the corkscrew configuration (B) Influenza virus model vRNA promoter in the corkscrew configuration. The prime notation is used to identify nucleotides of the 5′ end of the promoter. (C) Autoradiograph showing pppApG synthesized by 3P with the wild-type (cWT) or mutant 3′ cRNA strand, as indicated, in the presence of the wild-type 5′ cRNA strand. (D) Autoradiograph showing pppApG synthesized by 3P with the wild-type (vWT) or mutant 3′ vRNA strand, as indicated, in the presence of the wild-type 5′ vRNA strand. (E) Autoradiograph showing pppApG synthesized by 3P with the wild-type (vWT) or mutant (single or double) 3′ vRNA strand, as indicated, in the presence of the wild-type 5′ vRNA strand. -ve, 3′ cRNA or vRNA promoter strands alone. *p, ³²P.

FIG. 3.

Identification of sequence requirements in the 5′ and 3′ arms of the cRNA promoter for internal pppApG synthesis by a “transplant” experiment. (A) Model wild-type (WT) or mutant 5′ vRNA strands with the 5′ cRNA-like elements transplanted one by one. The transplanted 5′ cRNA elements are boxed. 10′D, hinge A deleted in the 5′ strand. (B) Autoradiograph showing internal pppApG synthesis activity with the 5′ mutants shown in A in combination with the 3′ cRNA promoter strand containing a mutation at position 1(U→A). (C) Model wild-type (WT) or mutant 3′ vRNA strands with the 3′ cRNA-like elements transplanted one by one. The transplanted 3′ cRNA elements and a mutation at position 1 are boxed. 10I, hinge U inserted in the 3′ strand. (D) Autoradiograph showing internal pppApG synthesis activity with the 5′ wild-type cRNA promoter in combination with the 3′ mutants shown in C. Note that the promoter mutant used in lane 12 differs in sequence only at position 1 from the wild-type cRNA promoter used in lane 2. -ve, 3′ strand of the promoter alone. The position of pppApG is shown. *p, ³²P.

FIG. 4.

Internally synthesized pppApG from influenza virus cRNA can realign to terminal residues for extension into a trinucleotide in a template-directed manner. (A) Autoradiograph showing the trinucleotide pppApGpU and dinucleotides synthesized in the presence of 1 mM UTP, 1 mM ATP, and 0.02 μM [α-³²P]GTP with wild-type (WT) or mutant 3′ cRNA strand in the presence of the 5′ cRNA strand. The positions of the dinucleotides and pppApGpU are shown. (B) Characterization of bands A to F by treatment with T₂ RNase and PEI-cellulose TLC. The positions of pppA*p and pp*pGp are shown. -ve, 3′ cRNA strand alone. *p, ³²P.

FIG. 5.

Mutations or deletion of the 3′-terminal residue of the cRNA template are corrected to wild type in an in vivo minireplicon system. (A) Autoradiograph showing primer extension analysis of virus-like CAT RNA transcribed in an in vivo minireplicon system. 293T cells were transfected with four protein expression plasmids encoding the three subunits of the influenza virus RNA polymerase (either wild-type [3P] or inactive mutant [3P-ASM]) and the nucleoprotein (NP), together with a pPOLIcCAT plasmid encoding a CAT reporter gene flanked by either wild-type (WT) or mutant influenza virus cRNA promoter sequences as indicated. RNA was extracted at 41 h posttransfection and analyzed by primer extension with vRNA- or cRNA-specific primers. (B) Sample sequence traces of cDNA amplified by cRNA- or vRNA-specific RT-PCR from virus-like CAT RNA described in A. Extracted RNA was TAP treated and circularized prior to RT-PCR amplification across the junction and cloning of the amplicon. The arrows indicate the mutated 3′-terminal residue (1U→A complement) of the template cRNA (left) and the corrected wild-type 5′-terminal residue (5′A) of the replicated vRNA (right).

FIG. 6.

Internally synthesized pppApG derived from the cRNA promoter can realign to a vRNA template for elongation in vitro. (A) Autoradiograph showing pppApG synthesized internally on the cRNA promoter and realigned onto 3′-terminal residues of the vRNA promoter for extension into a trinucleotide by partially purified influenza A/Turkey/England polymerase. Either 1 mM CTP or 1 mM UTP was added to ATP and radiolabeled GTP as indicated for extension to the third nucleotide on either a vRNA or a cRNA promoter. The synthesized di- or trinucleotides are shown on the left. The solid circle on the right marks an unknown band. (B) Characterization of bands A to H by treatment with T₂ RNase and PEI-cellulose TLC. The positions of pppA*p and pp*pGp are shown. *p, ³²P.

FIG. 7.

Different de novo initiation strategies are used by influenza virus RNA polymerase on its cRNA and vRNA promoters for viral RNA replication. (A) Internal initiation and realignment model for the cRNA promoter. (B) Terminal initiation and elongation model for the vRNA promoter. Base-pairing is shown as dotted lines. The phosphodiester bonds formed within nascent di- or trinucleotides are shown as dashes.