Hairpin loop structure in the 3' arm of the influenza A virus virion RNA promoter is required for endonuclease activity.

Previous studies have shown that the 5' arm of the influenza A virus virion RNA promoter requires a hairpin loop structure for efficient endonuclease activity of influenza virus RNA polymerase, an activity that is required for the cap-snatching activity of primers from host pre-mRNA. Here we examine whether a hairpin loop is also required in the 3' arm of the viral RNA promoter. We study point mutations at each nucleotide position (1 to 12) within the 3' arm of the promoter as well as complementary "rescue" mutations which restored base pairing in the stem of a potential hairpin loop. Our results suggest that endonuclease activity is absolutely dependent on the presence of a 3' hairpin loop structure. This is the first direct evidence for RNA secondary structure within the 3' arm being required for a specific stage, i.e., endonuclease cleavage, in the influenza virus replicative cycle.

Mutagenic analysis of the 5' arm of the influenza A virus virion RNA promoter defines the sequence requirements for endonuclease activity.

Short synthetic influenza virus-like RNAs derived from influenza virus promoter sequences were examined for their ability to stimulate the endonuclease activity of recombinant influenza virus polymerase complexes in vitro, an activity that is required for the cap-snatching activity of primers from host pre-mRNA. An extensive set of point mutants of the 5' arm of the influenza A virus viral RNA (vRNA) was constructed to determine the cis-acting elements which influenced endonuclease activity. Activity was found to be dependent on three features of the conserved vRNA termini: (i) the presence of the 5' hairpin loop structure, (ii) the identity of residues at positions 5 and 10 bases from the 5' terminus, and (iii) the presence of base pair interactions between the 5' and 3' segment ends. Further experiments discounted a role for the vRNA U track in endonuclease activation. This study represents the first mutagenic analysis of the influenza virus promoter with regard to endonuclease activity.

Rescue of synthetic RNAs into thogoto and influenza A virus particles using core proteins purified from Thogoto virus.

The ribonucleoprotein (RNP) complexes of Thogoto virus (THOV), a tick-borne orthomyxovirus, have been purified from detergent-lysed virions. The purified RNPs were then disrupted by centrifugation through a CsCl-glycerol gradient to obtain fractions highly enriched in nucleoprotein (NP) and virtually devoid of viral genomic RNA. When these NP-enriched fractions were incubated with a synthetic THOV-like RNA, and the mixtures were transfected into THOV-infected cells, the synthetic RNA was expressed and packaged into THOV particles. Similarly, hybrid mixtures containing purified THOV NP and influenza A virus synthetic RNAs (either a model CAT RNA or a gene encoding the viral neuraminidase), were prepared and transfected into influenza A virus-infected cells. The synthetic CAT RNA, was shown to be expressed and packaged into virus particles, and the neuraminidase gene was rescued into influenza virions. These data are discussed in terms of the similarities observed between THOV and influenza A virus and the potential application of the THOV purified proteins for rescuing synthetic genes into infectious viruses.

A hairpin loop at the 5' end of influenza A virus virion RNA is required for synthesis of poly(A)+ mRNA in vitro.

We present evidence, based on extensive mutagenesis, that a hairpin loop at the 5' end of influenza A virus virion RNA (vRNA) is required for the synthesis of polyadenylated mRNA from model vRNA templates in vitro. The hairpin loop, which we term the vRNA 5' hook, contains a stem of 2 bp formed by the second and third residues pairing with the ninth and eighth residues, respectively, and a 4-nucleotide loop composed of the intervening residues 4 to 7. Disruption of the base pairs of the vRNA 5' hook by introducing point mutations prevented polyadenylation, except in two mutants where a G-U base pair reformed. The polyadenylation activity of point mutants could be rescued by constructing double mutants with reformed base pairs in the stem of the vRNA 5' hook. These results suggest that base pairing rather than a particular nucleotide sequence was critical. We also show that mutation of the analogous region in the 3' arm of vRNA did not interfere with the synthesis of polyadenylated mRNA, suggesting that a hook structure in the 3' arm is not required for transcription of polyadenylated mRNA in vitro.

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 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.

Quantitative measurement of faecal blood loss: comparison of radioisotopic and chemical analyses.

Blood loss in faeces was assessed by three different methods in five patients with recurrent iron deficiency. In short term (12 day) studies chemical analysis of complete stool collections for haemderived porphyrins (HemoQuant) gave results closely correlated with those obtained by measuring stool loss of 51Cr-labelled red blood cells. Whole body counting for 59Fe was relatively insensitive to small blood losses but allowed losses to be followed up over longer periods. Chemical analysis of faecal porphyrins thus provides a satisfactory alternative to radioisotopic techniques in short term quantitation of faecal blood loss, while longer term whole body counting of 59Fe may still be appropriate in a few patients for the detection and quantification of intermittent blood losses.