Structural insight into RNA synthesis by influenza D polymerase.

The influenza virus polymerase uses capped RNA primers to initiate transcription, and a combination of terminal and internal de novo initiations for the two-step replication process by binding the conserved viral genomic RNA (vRNA) or complementary RNA (cRNA) promoter. Here, we determined the apo and promoter-bound influenza D polymerase structures using cryo-electron microscopy and found the polymerase has an evolutionarily conserved stable core structure with inherently flexible peripheral domains. Strikingly, two conformations (mode A and B) of the vRNA promoter were observed where the 3'-vRNA end can bind at two different sites, whereas the cRNA promoter only binds in the mode B conformation. Functional studies confirmed the critical role of the mode B conformation for vRNA synthesis via the intermediate cRNA but not for cRNA production, which is mainly regulated by the mode A conformation. Both conformations participate in the regulation of the transcription process. This work advances our understanding of the regulatory mechanisms for the synthesis of different RNA species by influenza virus polymerase and opens new opportunities for antiviral drug design.

PB2 polymerase subunit of Thogoto virus (Orthomyxoviridae family).

Thogoto virus (THOV) represents a new genus in the family Orthomyxoviridae. The three polymerase subunits PB1, PB2, and PA initiate transcription by a unique cap-stealing mechanism that involves the cleavage of only the m(7)GpppAm structure from cellular hnRNAs. Here, we report the cloning of the longest genomic segment of THOV coding for PB2. It comprises 2,375 nucleotides encoding a single large open reading frame flanked by the conserved terminal regions typical for orthomyxoviruses. The deduced amino acid sequence corresponds to a protein of 769 amino acids, with an estimated Mr of 88,042. Expression of the PB2 cDNA in mammalian cells revealed nuclear accumulation of the recombinant protein, consistent with the replication of THOV in the cell nucleus.

An endonuclease switching mechanism in the virion RNA and cRNA promoters of Thogoto orthomyxovirus.

An in vitro assay was developed to investigate endonuclease activity of Thogoto virus, a tick-borne orthomyxovirus. Endonuclease activity relied on an interaction between the 3' and 5' termini of virion RNA (vRNA) and not those of cRNA. Evidence was obtained that cap structures are cleaved directly from cap donors and that cleavage does not occur after pyrimidines. A 5' hook structure, present in the vRNA promoter but not the cRNA promoter, was introduced into cRNA promoter mutants. These mutants stimulated endonuclease activity, although at levels slightly lower than that of vRNA. The ability of the cRNA promoter to stimulate endonuclease activity when mutated to contain a 5' hook structure indicates that this structure constitutes a switching mechanism for endonuclease activity between the vRNA and cRNA promoters.

The Thogoto orthomyxovirus cRNA promoter functions as a panhandle but does not stimulate cap snatching in vitro.

The cRNA promoter of Thogoto virus, a tick-borne orthomyxovirus, was investigated using an in vitro polymerase assay based on purified viral cores and synthetic oligoribonucleotides corresponding to the 3' and 5' ends of cRNA. In vitro polymerase activity relied on an interaction between the 3' and 5' ends of cRNA and was ApG primer-dependent. Mutational analysis of the promoter showed that interstrand base-pairing of residues 11 and 12 of the 3' promoter arm with residues 10 and 11 of the 5' promoter arm, respectively, was essential for polymerase activity. These data provide the first clear evidence for a cRNA panhandle in an orthomyxovirus. No evidence was obtained for the presence of a 5' or 3' hook structure in the cRNA promoter, and transcription could not be primed with rabbit globin mRNA or synthetic cap analogues. This demonstrates that cap snatching activity relies on the presence of the vRNA terminal sequences.

In vivo reconstitution of active Thogoto virus polymerase: assays for the compatibility with other orthomyxovirus core proteins and template RNAs.

Tick-borne Thogoto virus (THOV), the prototype of a new genus in the Orthomyxoviridae family, contains six single-stranded RNA segments of negative polarity. Four of them encode gene products that correspond to the influenza virus PB1, PB2, PA and NP core proteins. Here we describe an in vivo system in which the expression of a THOV model RNA is driven by THOV core proteins synthesized from cloned cDNAs. Our results demonstrated the biological activity of our cloned genes and showed that the three polymerase subunits and the NP are required for gene expression. For comparison, we also used the in vivo reconstituted systems of the influenza A and B viruses. None of the polymerase or NP proteins was active in a heterologous orthomyxovirus core, indicating a high specificity in core assembly and/or function. Interestingly, the THOV polymerase did not recognize the influenza A virus promoter and vice versa.

In vitro polymerase activity of Thogoto virus: evidence for a unique cap-snatching mechanism in a tick-borne orthomyxovirus.

The tick-borne Thogoto virus (THOV) is the type species of a new genus in the family Orthomyxoviridae. Its genome comprises six segments of single-stranded, negative-sense RNA. Each segment possesses conserved regions of semicomplementary nucleotides at the 3' and 5' termini which strongly resemble those of influenza virus. An in vitro polymerase assay based on reconstituted THOV viral cores was developed, and activity was shown to rely on an interaction between the conserved 3'- and 5'-terminal sequences and to be primer dependent. Addition of globin mRNA primed transcription, catalyzing the addition of an extra nucleotide to the transcripts, corresponding to the 5'-terminal m7G cap residue. Priming with various cap analogs suggested that THOV transcription is initiated preferentially with m7GpppAm and involves base pairing. This is the first experimental evidence of endonuclease activity in THOV as part of a unique cap-snatching mechanism.

Striking conformational similarities between the transcription promoters of Thogoto and influenza A viruses: evidence for intrastrand base pairing in the 5' promoter arm.

In the accompanying report, we describe an in vitro polymerase assay based on reconstituted Thogoto virus (THOV) cores which provided evidence of a double-stranded vRNA promoter consisting of both the 3' and 5' sequences of vRNA (M. B. Leahy, J. T. Dessens, and P. A. Nuttall, J. Virol. 71:8347-8351, 1997). This system was used to investigate further the THOV vRNA promoter structure by using short, synthetic vRNA promoters. The results obtained show that interstrand base pairing between residues 10 and 11 of the 3' promoter arm with residues 11 and 12 of the 5' promoter arm, respectively, is important for promoter activity. In addition, intrastrand base pairing between residues 2 and 3 with residues 9 and 8 of the 5' promoter arm, respectively, was shown to be involved in promoter activity, while no evidence of intrastrand base pairing between residues 2 and 9 of the 3' promoter arm was obtained. These observations are consistent with a hook-like structure in the 5' promoter arm of the THOV promoter. The THOV cores were able to transcribe an influenza A virus (FLUA) vRNA-like promoter, as well as hybrid THOV-FLUA promoters. Hence, the THOV and FLUA vRNA promoters appear to be both structurally and functionally similar.

The fourth genus in the Orthomyxoviridae: sequence analyses of two Thogoto virus polymerase proteins and comparison with influenza viruses.

The tick-borne Thogoto virus (THOV) is the type species of a newly recognized fourth genus, Thogotovirus, in the family Orthomyxoviridae. Because of the distant relationship of THOV with the influenza viruses, determination of its genomic information can potentially be used to identify important domains in influenza virus proteins. We have determined the complete nucleotide sequence of the second longest RNA segment of THOV. The molecule comprises 2212 nucleotides with a single large open reading frame encoding a protein of 710 amino acids, estimated Mr 81,284. The protein shares 77% amino acid similarity with the PB1-like protein of Dhori virus, a related tick-borne virus, and 50-53% with the PB1 polymerase proteins of influenza virus A, B and C. All the motifs characteristic of RNA-dependent polymerases were identified including the SSDD motif common to all RNA-dependent RNA polymerases, indicating that the THOV protein is functionally analogous to the influenza virus PB1 proteins and involved in chain elongation. We also report the corrected sequence of the third longest RNA segment of THOV, encoding a protein which shares 44-47% amino acid similarity with the PA-like polymerase proteins of influenza virus A, B and C. The biological significance of conserved domains in these orthomyxovirid proteins is discussed.