Photochemical cross-linking of influenza A polymerase to its virion RNA promoter defines a polymerase binding site at residues 9 to 12 of the promoter

Fodor, Ervin; Seong, Baik Lin; Brownlee, George G.

doi:10.1099/0022-1317-74-7-1327

Cited by 87 publications

(83 citation statements)

References 19 publications

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“…Subunit PB1 has the binding activity of substrate nucleoside 5 H -triphosphates (Asano & Ishihama 1997;Asano et al 1995) and template RNA (Fodor et al 1993;Li et al 1998) and is involved in RNA polymerization (Nakagawa et al 1995;Kobayashi et al 1996). Both PB2 and PA associate with PB1 , thereby forming the ternary complex of functional RNA polymerase (Honda et al 1990;Kobayashi et al 1992).…”

Section: Discussionmentioning

confidence: 99%

Two separate sequences of PB2 subunit constitute the RNA cap‐binding site of influenza virus RNA polymerase

1999

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Background: In¯uenza virus RNA polymerase with the subunit composition of PB1-PB2-PA is a unique multifunctional enzyme with the activities of both synthesis and cleavage of RNA, and is involved in both transcription and replication of the RNA genome. Transcription is initiated by using capped RNA fragments, which are generated after cleavage of host cell mRNA by the RNA polymeraseassociated capped RNA endonuclease. To identify the RNA cap 1-binding site on the RNA polymerase, viral ribonucleoprotein (RNP) cores were subjected to UV-crosslinking with RNA which was labelled with 32 P only at the cap-1 structure.

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Section: Discussionmentioning

confidence: 99%

Two separate sequences of PB2 subunit constitute the RNA cap‐binding site of influenza virus RNA polymerase

1999

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“…Three nucleotides near the 3Ј end of the (Ϫ)-strand RNA were found to be required for synthesis, although accurate initiation was not demonstrated. Subsequently, these nucleotides were shown to be directly contacted by the influenza RdRp (29). However, additional data demonstrated the need for sequences located at the 5Ј end of the influenza RNA which form a partially double-stranded structure recognized by the RdRp (30,31).…”

Section: Discussionmentioning

confidence: 99%

Sequence-specific recognition of a subgenomic RNA promoter by a viral RNA polymerase

Siegel

Adkins

Kao

1997

Proc. Natl. Acad. Sci. U.S.A.

109

129

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RNA templates of 33 nucleotides containing the brome mosaic virus (BMV) core subgenomic promoter were used to determine the promoter elements recognized by the BMV RNA-dependent RNA polymerase (RdRp) to initiate RNA synthesis. Nucleotides at positions ؊17, ؊14, ؊13, and ؊11 relative to the subgenomic initiation site must be maintained for interaction with the RdRp. Changes to every other nucleotide at these four positions allow predictions for the base-specific functional groups required for RdRp recognition. RdRp contact of the nucleotide at position ؊17 was suggested with a template competition assay. Comparison of the BMV subgenomic promoter to those from other plant and animal alphaviruses shows a remarkable degree of conservation of the nucleotides required for BMV subgenomic RNA synthesis. We show that the RdRp of the plant-infecting BMV is capable of accurately, albeit inefficiently, initiating RNA synthesis from the subgenomic promoter of the animalinfecting Semliki Forest virus. The sequence-specific recognition of RNA by the BMV RdRp is analogous to the recognition of DNA promoters by DNA-dependent RNA polymerases.Viral RNA replication, a process fundamental to pathogenicity, requires specific recognition of RNA features by proteins. RNA-dependent RNA polymerase (RdRp) is a complex composed of viral and cellular proteins that directs viral RNA synthesis from infecting RNA templates (1). Many viral RdRp proteins have been sequenced and analyzed (2); however, a comprehensive mechanism describing RNA synthesis is lacking. Consequently, general knowledge of RdRps is significantly less than that of other RNA and DNA polymerases.To investigate the mechanism of RNA-directed RNA synthesis, we study brome mosaic virus (BMV), type member of the bromovirus group of plant viruses in the alphavirus-like superfamily of (ϩ)-strand RNA viruses (3). The BMV genome is comprised of three RNAs designated 1, 2, and 3, and a subgenomic RNA4 which is initiated from (Ϫ)-strand RNA3. Enriched BMV RdRp preparations from infected barley can, in a highly specific manner, synthesize (Ϫ)-strand RNA from (ϩ)-strand templates and subgenomic (ϩ)-strand products from (Ϫ)-strand templates (4-6).We have developed a system to study BMV subgenomic RNA initiation from minimal templates, designated proscripts, because they contain the promoter and template for (ϩ)-strand RNA synthesis. We have shown that the 20 nt 3Ј of the subgenomic initiation nucleotide, recognized as the subgenomic core promoter (7,8), are sufficient to direct (ϩ)-strand RNA synthesis (6), permitting the design of proscripts focusing only on the core promoter.In this paper, we have used a functional assay to determine that nucleotides Ϫ17, Ϫ14, Ϫ13, and Ϫ11 relative to the subgenomic initiation site are required for RNA synthesis from the subgenomic core promoter. Moreover, at least one of these nucleotides, Ϫ17, was found likely to be contacted by the BMV RdRp. The resolution achieved in this study allows us to predict the base moieties contacted by RdRp and dem...

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“…The 5' arm of the postulated cRNA panhandle was investigated for polymerase binding using the photochemical cross-linking protocol used previously to investigate the vRNA panhandle (Fodor et al, 1993(Fodor et al, , 1994. Radiolabelled 14 nucleotide-long RNAs based on the Y-terminal sequence of cRNA, including transversion mutations at each position, were incubated with nuclease-treated RNP at 30 °C for 30min.…”

Section: Cross-linking To the C R N A 5' Aimmentioning

confidence: 99%

“…The ability of added 3' end sequences alone to promote transcription was also observed when polymerase prepared by micrococcal nuclease treatment of viral cores was used (Seong & Brownlee, 1992a, b). Similar polymerase preparations were used to identify specific binding sites in the conserved 3' terminus of vRNA (Fodor et al, 1993). Also using the nuclease-treated polymerase derived from influenza virus, it was found that added 3' templates carrying certain mutations, which reduced transcription drastically, could have that activity 'rescued' if an RNA corresponding to the 5' arm of the panhandle with mutations designed to restore base-pairing with the mutant 3' arm was added.…”

Section: Introductionmentioning

confidence: 99%

In vitro transcription and polymerase binding studies of the termini of influenza A virus cRNA: evidence for a cRNA panhandle

Pritlove¹,

Fodor²,

Seong³

et al. 1995

Journal of General Virology

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Photochemical cross-linking of influenza A polymerase to its virion RNA promoter defines a polymerase binding site at residues 9 to 12 of the promoter

Cited by 87 publications

References 19 publications

Two separate sequences of PB2 subunit constitute the RNA cap‐binding site of influenza virus RNA polymerase

Two separate sequences of PB2 subunit constitute the RNA cap‐binding site of influenza virus RNA polymerase

Sequence-specific recognition of a subgenomic RNA promoter by a viral RNA polymerase

In vitro transcription and polymerase binding studies of the termini of influenza A virus cRNA: evidence for a cRNA panhandle

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