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Journal of Virology, August 2001, p. 7042-7049, Vol. 75, No. 15
Sir William Dunn School of Pathology,
University of Oxford, Oxford, United Kingdom
Received 13 February 2001/Accepted 25 April 2001
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.
Influenza A virus is a segmented,
negative-sense RNA virus. The virion RNA of influenza virus serves
several functions. During transcription it acts as the template and as
a cofactor in endonuclease cleavage of host pre-mRNA (17,
31), leading to the synthesis of viral mRNA which is capped at
its 5' end and polyadenylated at its 3' end (18). Virion
RNA (vRNA) also acts as the template for full-length cRNA, which in
turn serves as a template for the synthesis of new vRNA molecules.
Influenza virus RNA synthesis takes place within the nucleus of
infected cells, consistent with the requirement for host cell pre-mRNAs
and for splicing (26), suggesting that influenza virus
transcription may be coupled to host cell RNA polymerase II
transcription (14). Inhibitors selective for either
influenza virus endonuclease or RNA polymerase activities suggest that
the active site for endonuclease cleavage is separate from the
transcription active site (50, 51).
The influenza virus RNA polymerase is a heterotrimer formed by the PB1,
PB2, and PA subunits (25, 26). All three subunits are
required for transcription and replication, although the role of PA is
poorly understood (37, 44, 45). In the virion the polymerase proteins are associated with vRNA and the nucleoprotein to
form ribonucleoprotein complexes. It is likely that base pairing between the conserved 3' and 5' termini of the influenza virus vRNA
leads to the formation of an RNA panhandle structure; however, it is
probable that the polymerase itself helps to stabilize the interactions
between the 5' and 3' termini (7, 19, 20, 23, 31, 33, 35).
PB1 interacts with both PB2 and PA (20, 22, 52) and is
also involved in binding vRNA (11, 12, 15, 31). PB1 also binds cRNA, although the binding sites are different from those for
vRNA (16). PB1 contains amino acid motifs present in other RNA-dependent RNA polymerases (38) and is the subunit
responsible for polymerization (24). Although it has been
reported that the endonuclease site residues in PB2 (32),
recent evidence favors its location being in PB1 (31a).
PB2 is the cap-binding protein. Cap-binding regions have been mapped,
and capped RNA can be cross-linked to PB2 (3, 4, 21, 31a, 36, 46,
53). Defective PB2 mutants do not cleave cap structures from
mRNA (54) and there are potential sequence similarities
between PB2 and known cap-binding proteins (8). In
addition, capped RNA-directed RNA synthesis in vitro is inhibited by
antisera against PB2 (5). The extreme N-terminal region of
PB2 interacts with PB1 (21).
The cap-binding activity of the polymerase is stimulated by the
presence of the 5'-terminal nucleotides of the influenza virus vRNA
(6, 31). However, although both vRNA and cRNA bind the polymerase and are efficiently transcribed, only vRNA activates endonuclease cleavage (6). This is presumably because of
subtle differences in the promoters (30) causing separate
regions of PB1 to bind to vRNA and cRNA (16).
Several models have been proposed for the secondary structure of the
influenza virus vRNA promoter in what is a somewhat controversial field
(2, 9-13, 26, 34, 41). It is now accepted that both the
vRNA 5' and 3' ends are required for replication and transcription (6, 12, 13, 17, 41, 49). Chemical probing methods indicate that the promoter structure is made up of a panhandle structure with an internal bulge (2). However, other work
suggests that the 10 5'-terminal vRNA nucleotides and 9 3' nucleotides do not base pair with each other (9, 12). This observation led to the proposal of the "RNA fork" model, based on in vitro transcription assays, in which nucleotides within the 5' promoter arm base pair with cognate nucleotides on the 3' promoter arm, leaving
the terminal nucleotides unpaired (12, 13). The corkscrew model, based on in vivo reporter gene experiments, extends this model
further by proposing local secondary structures, or hairpin loops,
within the "single-stranded" 3'- and 5'-terminal nucleotides (Fig.
1) (9). In all cases the
base pairs within the promoter, rather than the identity of the
nucleotides, were found to be important for polymerase activity.
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.15.7042-7049.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Hairpin Loop Structure in the 3' Arm of the
Influenza A Virus Virion RNA Promoter Is Required for
Endonuclease Activity
and
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
View larger version (11K):
[in a new window]
FIG. 1.
Promoter structure of the 49-nt-long model influenza
virus vRNA used in this study drawn in a corkscrew-type conformation
(9).
In vitro transcription and polyadenylation of orthomyxovirus vRNA templates are dependent on a 5' hairpin loop (27, 43), but it is known that the formation of a 3' hairpin loop is not necessary for replication and transcription primed with a dinucleotide primer (13, 29, 42, 43). Previously, the role of the vRNA 5' promoter sequence on endonuclease activity was studied in isolation from those sequences that regulate transcription (30). Here we extend that study and now report the mutagenic analysis of the 3'-terminal nucleotides of the influenza A virus vRNA with regard to endonuclease activity. We show that a 3' hairpin loop is required, thus formally assigning a role for this structure and reconciling the perceived contradictions between existing in vivo and in vitro data (9, 10, 12, 13, 39, 43).
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MATERIALS AND METHODS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Construction of influenza virus-like model RNAs from a plasmid. The methods used were essentially the same as those published previously (30) and are given here only in brief. Plasmid pBXB49 (30) contains 49 nucleotides (nt) of influenza virus vRNA sequence derived from the 5' and 3' ends of segment 8 of influenza virus A/PR/8/34. Point mutants of pBXB49 were made by standard methods. Influenza virus vRNA-like RNA synthesized by T7 RNA polymerase runoff transcription reactions was worked up and quantitated as before except that unincorporated ribonucleoside triphosphates were removed by a QIAquick nucleotide removal kit.
Preparation of recombinant influenza A virus polymerase/endonuclease and the synthesis of the 67-nt-long cap donor. Recombinant vaccinia virus vectors which express influenza A virus PB1, PB2, or PA protein were used to infect HeLa cells, and nuclear extracts were prepared as described before (30, 48). 32P-labeled cap zero donors (67 nt long) were prepared by a slight modification of a method described previously (17, 30) using QIAquick nucleotide removal columns both for the workup of the transcription reaction and for removing unincorporated [32P]GTP.
Capped RNA endonuclease assays. The endonuclease assay was similar to one described before (30) except that usually 1 µl of recombinant polymerase was used in a 5-µl reaction which typically contained 20 to 30 nM capped 32P-, end-labeled 67-nt-long pGEM-7Zf(+)-derived RNA probe and an excess (about 1 pmol) of wild-type or mutant 49-mer model RNA. The reaction was usually incubated at 30°C for 20 min. Products were analyzed by 15% polyacrylamide gel electrophoresis in 7 M urea and quantified by phosphorimage analysis. The yield of substrate and product was measured, and endonuclease activity was expressed as the percentage of substrate cleaved.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Systematic study of endonuclease activity using vRNA with point mutations in the 3' promoter arm. Flick et al. (9) suggested that a corkscrew model of the vRNA promoter (Fig. 1) was a valid secondary structure model based on in vivo reporter gene assays. In contrast, no evidence for the presence of the 3' hairpin loop component of the corkscrew was obtained in studies of in vitro transcription (13) or in studies of the cis-acting requirements for polyadenylation (43). Here we tested whether the 3' hairpin loop of the corkscrew is needed for endonuclease activity by studying endonuclease cleavage in isolation from transcription and replication. We synthesized 49-nt-long influenza virus-like model RNAs (Fig. 1) by T7 transcription of plasmid pBXB49 which were added to the endonuclease reactions in excess. Point mutants of pBXB49 were then used to study the cis-acting vRNA requirements for endonuclease activity. This methodology was similar to that used previously (30) to study the 5' arm of the vRNA promoter.
Initially, a complete set of point mutants was constructed at each nucleotide, from nt 1 to 12 of the 3' influenza virus vRNA promoter arm, with each nucleotide being mutated to two alternative nucleotides. These mutants were used to determine which nucleotides or possible secondary structures within the 3' arm of the influenza virus vRNA have a role in endonuclease function using recombinant influenza virus polymerase prepared by vaccinia virus expression vectors for the PB1, PB2, and PA proteins. Endonuclease assays (see Materials and Methods) were conducted using a 32P-labeled 67-nt-long cap donor (see Materials and Methods) with each of the mutant constructs, and the percent cleavage activity, which gives rise to a 12-nt-long cleavage product, was expressed relative to the control wild-type construct. A representative autoradiograph is shown in Fig. 2. The results of three independent sets of reactions with each virion RNA construct, quantified by phosphorimage analysis, are summarized in Fig. 3.
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U] and 8 [CU]) in the
presence of RNA with mutations in positions 1 to 3 and 8 to 12, suggesting that these nucleotides were essential for endonuclease
activity per se or that they were involved in base pairs with other
residues, thus forming essential secondary structures.
Hairpin loop in the 3' promoter arm is required for endonuclease
activity.
Nucleotide substitutions at positions 2, 3, 8, and 9 reduced endonuclease activity to the background level of detection
(Fig. 2 and 3). To test whether this effect was due to the specific identity of these nucleotides or to a possible hairpin loop structure formed by the base pairing of nt 2 and 3 with 9 and 8, respectively, a
series of rescue mutations was constructed (Fig.
4). The results of endonuclease assays
(averages of three or more experiments) are summarized in Fig.
5. It was found that a mutation from C to
G at position 2 (Fig. 4B), which reduced endonuclease activity to
background levels, could be rescued to 36% of the wild-type activity
(Fig. 5, average of three experiments) by making the complementary
mutation (GC) at position 9 (Fig. 4F). Likewise, a mutation from C
to U at position 2 (Fig. 4C), which reduced endonuclease activity to
approximately 9% of the wild-type activity, could be rescued to 54%
of the wild-type activity (average of three experiments) by making the
complementary mutation (GA) at position 9 (Fig. 4G).
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G) at position 8 (Fig. 4L). Similarly,
when nt 3 was changed from G to A (Fig. 4I), endonuclease activity was
reduced to background levels, but on addition of a complementary
mutation at position 8 (CU) (Fig. 4M), activity was again increased
to 51% that of the wild type. In agreement with these results, the
point mutants at nt 8 and 9 which disrupt base pairs are in all cases
inactive (Fig. 3 and 4). When nt 8 was mutated from C to U, an activity
of 16% of the wild-type activity was observed, which was higher than
the background, although still significantly lower than that of the
rescue mutants (Fig. 4K and 5). These results indicate that a 3'
hairpin loop structure involving base pairs between nt 2 and 9 and nt 3 and 8 is required for endonuclease activity. It is interesting to note
that when the CU mutation was made at position 2 or 8 endonuclease activity was higher than for other mutations to the stem of the possible hairpin loop (Fig. 5), possibly due to the formation of a G-U
wobble pair (see Discussion).
To further test the hypothesis that a 3' hairpin loop, rather than
specific nucleotides within the influenza virus vRNA 3' promoter arm,
is required for endonuclease activity, double mutants which would
disrupt both base pairs in the stem of the loop were constructed. When
a CG mutation was made at position 2 and a GC mutation was made
at position 3, endonuclease activity was reduced to background levels
(Fig. 5). Likewise, when a CU mutation was made at position 2 and a
GA mutation was made at position 3, endonuclease activity was
abrogated. Again, when a CG mutation at position 8 was combined with
a GC mutation at position 9 or a CU mutation at position 8 was
combined with a GA mutation at position 9, endonuclease activity was
reduced to background levels (Fig. 5). When rescue mutations were made
which allowed the formation of base pairs between nt 2 and 9 and nt 3 and 8 (Fig. 4R and S), endonuclease activity was rescued to 19 and
23%, respectively. In both cases this was significantly higher than background levels, although still significantly lower than any of the
single base pair rescue mutants (Fig. 5).
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The RNA polymerase complex of influenza virus, composed of the PB1, PB2, and PA subunits, carries out all of the essential replicative functions of the virus (26). Not only is the polymerase required for the synthesis of mRNA, vRNA, and cRNA, but it also possesses an endonuclease activity which is strictly dependent on the presence of the vRNA promoter whereby the complex cleaves cap structures together with 9 to 15 or so heterologous nucleotides from host mRNA.
A thorough mutagenic analysis of the 5' arm of the vRNA promoter had previously shown that a 5' hairpin loop formed by base pairing between nt 2 and 9 and nt 3 and 8, respectively, of the 5' promoter arm was essential for cleavage activity (30). These same positions had also been found to be important in previous in vitro and in vivo studies of transcription and polyadenylation (9, 12, 39, 43). In the present study, mutation of nt 2, 3, 8, and 9 of the 3' promoter arm destroyed endonuclease activity (Fig. 2 and 3), suggesting that the 3' arm of the influenza virus vRNA promoter may possess a similar hairpin loop structure to the 5' arm (Fig. 1).
To determine whether the 3' hairpin loop was required for endonuclease
activity, mutations were made at 3' positions 2 and 3, thus disrupting
the stem of the hairpin loop (Fig. 4). Mutations which would destroy a
putative 3' hairpin loop were also made at positions 8 and 9. In all
cases the residues were changed to at least two alternative nucleotides
(usually a transition and a transversion) to rule out possible
sequence-specific effects. All mutants which destroyed possible base
pairing within the stem of a putative hairpin loop reduced endonuclease
activity markedly, usually to background levels of detection.
Complementary mutations made at positions 9 and 8 reformed the base
pairs with nt 2 and 3, respectively (Fig. 5). These complementary
mutations partially rescued endonuclease activity in all cases,
indicating the importance of a 3' hairpin loop in endonuclease
activity. However, the fact that the rescue was not 100%, with typical
values lying between 35 and 55%, indicated that there were some
sequence-specific effects. Interestingly, when a CU mutation was
made at position 2 or position 8, residual endonuclease activity was
higher than when other mutations were made to the stem of the possible
hairpin loop. The fact that these wobble base pairs were only weakly
active argues that two standard Watson-Crick base pairs are required to
ensure sufficient stability (Fig. 4C and K). In agreement with previous
studies, the mutation of 3' nt 10, 11, and 12, which are involved in
base pairs with the 5' promoter arm, destroyed endonuclease activity (Fig. 3) (30). It is possible that insertions or deletions
within the hairpin loop or other mutations beyond the scope of this
paper may affect endonuclease activity. However, the data presented here show that the 3' hairpin loop appears to be the major determinant of endonuclease activity.
Binding of recombinant influenza virus polymerase, devoid of influenza virus vRNA, to a capped RNA is dependent on the addition of the 5' arm of the influenza virus vRNA (6). However, the cap structure is not cleaved from the mRNA unless the 3' arm of the vRNA is also present (6, 17, 31). The 5' component of the influenza virus vRNA promoter was not mutated in the present study and so cap binding should not have been affected. Therefore, the step that was abrogated by the mutants which disrupted the 3' hairpin loop was probably the actual cleavage of the capped substrate. Loss of activity could have occurred either because the polymerase complex failed to bind the 3' promoter arm lacking the hairpin loop structure or because when bound it did not cause the presumed allosteric changes required to activate the endonuclease.
Although the results reported here support the RNA corkscrew promoter
model (Fig. 1), the secondary structure seen is likely to be transient.
The process of transcription and the accompanying conformational
changes that take place within both the vRNA and the PB1, PB2, and PA
subunits are complex and not well understood. Our current understanding
of the role of the 3' hairpin loop and the ordered events leading to
transcription is outlined schematically in Fig.
6. First the PB1 polymerase subunit binds
the 5' hairpin loop of the influenza virus vRNA promoter (Fig. 6A)
(6, 31). This brings about unspecified allosteric changes,
activating a cap-binding site on the PB2 subunit and a vRNA 3' binding
site on PB1 (31). As the PB1 subunit binds the vRNA 3'
promoter arm, base pairs form between the 5' and 3' promoter arms, thus
forming the RNA corkscrew conformation (Fig. 6B). In addition, there is compelling evidence for interactions of the vRNA 3' and 5' termini with
distinct regions of PB1 that are separated by 300 amino acids in the
primary sequence (31). Binding of the 3' hairpin loop stimulates endonuclease cleavage, possibly on the PB1 subunit, leading
to the cleavage of host pre-mRNA (6, 31). Finally, the
capped RNA is used to prime transcription of the vRNA and elongation
ensues (Fig. 6C). It is obvious that either coincident with or after
endonuclease cleavage, melting of the 3' hairpin loop of vRNA must
occur to allow transcription to initiate. During elongation the RNA
polymerase remains bound to the 5' promoter arm, ultimately leading to
reiterative incorporation of A nucleotides directed by a track of
uridine nucleotides, thus forming a poly(A) tail of mRNA (39, 40,
42, 43).
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It is known that influenza virus vRNA activates the polymerase complex, allowing it to cleave mRNA, but influenza virus cRNA, which is superficially similar in sequence, does not (6); however, to date no mechanism has been proposed to explain this. A related orthomyxovirus, Thogoto virus (THOV), appears to have many similarities to influenza virus with respect to transcription and replication and also cleaves host cell pre-mRNA for use as primers (1, 27, 47, 55). In contrast to the present study, however, in the THOV vRNA promoter only the 5' hairpin loop and not a 3' hairpin loop was required for endonuclease activity (28). As the THOV cRNA molecule is a complement of the vRNA, it follows that the cRNA lacks a 5' hairpin loop. Based on these observations an endonuclease switching mechanism was proposed whereby vRNA molecules containing 5' hairpin loops stimulated endonuclease activity and cRNA molecules lacking the 5' hairpin loop structure did not (29). Hairpin loops have now been shown to be present and necessary for endonuclease activity in both the 3' and 5' promoter arms of the influenza virus vRNA. Because hairpin loops are present in both influenza virus vRNA termini, it follows that they are present in cRNA molecules. Therefore, it seems likely that influenza virus distinguishes between vRNA and cRNA with regard to endonuclease cleavage by a different method from that of THOV.
In conclusion, our results show that in contrast to transcription and polyadenylation, which are also catalyzed by the influenza virus RNA-dependent RNA polymerase, a hairpin loop in the 3' arm of the influenza A virus vRNA promoter is stringently required for endonuclease activity studied in vitro. This resolves the apparent contradiction between reporter gene experiments (9) and work carried out in vitro (12, 40, 43). In vivo work has previously stressed the significance of a so-called corkscrew structure involving both 5' and 3' hairpin loops, whereas ApG primed transcription, polyadenylation, and polymerase binding function in vitro without the need for such a structure in the 3' arm. Here we have shown that it is probable that the crucial step in reporter gene work (9) requiring a hairpin loop in the 3' promoter arm is the activation of endonuclease cleavage.
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ACKNOWLEDGMENTS |
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M. B. L. was supported by the MRC (program grant G9523972 to G.G.B.).
We thank Peter Palese and Jane Sharps for plasmids and Alice Taylor for DNA sequencing.
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FOOTNOTES |
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* Corresponding author. Mailing address: Chemical Pathology Unit, Sir William Dunn School of Pathology, University of Oxford, South Parks Rd., Oxford OX1 3RE, United Kingdom. Phone: 44 1865 275559. Fax: 44 1865 275556. E-mail: George.Brownlee{at}path.ox.ac.uk.
Present address: School of Biological Sciences, Biochemistry
Division, University of Manchester, Manchester, United Kingdom.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Virology, August 2001, p. 7042-7049, Vol. 75, No. 15
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.15.7042-7049.2001
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