Unité de Génétique Moléculaire des Virus Respiratoires, URA 1966 CNRS, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15, France1
Author for correspondence: Sylvie van der Werf. Fax +33 1 40 61 32 41. e-mail svdwerf{at}pasteur.fr
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Abstract |
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Introduction |
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There are some interesting differences between influenza A and C viral genomic RNAs (vRNAs). First, based on the few existing sequence data available for type C RNA segments (Clern-van Haaster & Meier-Ewert, 1984 ), 11 nucleotides are conserved at the 3'- and 5'-end of the vRNA on each of the seven segments of influenza C virus, whereas 12 and 13 nucleotides are conserved, respectively, at the 3'- and 5'-end of influenza A virus segments (Robertson, 1979
). Despite the high degree of nucleotide conservation, natural variations have been documented at nucleotide 5 from the 3'-end (Desselberger et al., 1980
) for influenza C virus RNAs and at nucleotide 4 in the conserved 3'-end region of influenza A virus RNAs (Lee & Seong, 1998a
). Second, the base-paired duplex region of the type C vRNA is usually shorter than that of type A vRNA (45 base pairs in influenza C virus and 48 base pairs in influenza A virus). Third, the uridine (U) stretch corresponding to the polyadenylation signal (Poon et al., 1999
) usually consists of 57 U residues in influenza A virus, whereas in influenza C virus the number of residues is 45 and no stretches of 67 U are found.
Influenza virus RNAs are complexed with the nucleoprotein (NP) and associated with the polymerase complex, which consists of three P proteins (PB1, PB2 and PA for influenza A virus or PB1, PB2 and P3 for influenza C virus), to form the viral ribonucleoproteins (RNPs). From biochemical and genetic data, it has been established that the PB1 subunit displays RNA-dependent RNA polymerase activity and that the PB2 subunit binds to the 5'-cap structure and is involved in the cap-snatching mechanism by which viral mRNA synthesis is initiated, whereas the role of the PA/P3 subunit is still unclear. The NP and P proteins have been shown to be the minimum set of viral proteins that are required for the transcription and replication of vRNA for all three types of influenza viruses (Crescenzo-Chaigne et al., 1999 ; Jambrina et al., 1997
; Mena et al., 1996
; Pleschka et al., 1996
).
Most of our knowledge about the transcription and replication processes of the influenza virus genome has been derived from studies carried out on type A viruses (Lamb & Krug, 1996 ). In vivo studies showed that the conserved 5'- and 3'-end sequences were sufficient for the expression, replication and packaging of genome segments (Luytjes et al., 1989
), although nonconserved sequences were found to modulate the efficiency of transcription and replication (Bergmann & Muster, 1996
; Zheng et al., 1996
). The nucleotide sequence requirements within the conserved termini controlling influenza virus transcription and replication have been studied extensively by making use of either in vitro transcription assays (Fodor et al., 1995
; Piccone et al., 1993
) or in vivo reconstitution of functional RNPs with vRNA-like model templates containing a chloramphenicol acetyltransferase (CAT) reporter gene (Neumann & Hobom, 1995
; Pleschka et al., 1996
). Although initial in vitro studies suggested that the promoter for transcription was entirely contained within the conserved 3'-end sequences, it was later recognized that interaction between the 3'- and 5'-end sequences is required for transcription initiation (Hagen et al., 1994
). While the vRNA polymerase strongly binds the vRNA 5'-end (Tiley et al., 1994
), it subsequently interacts with the vRNA 3'-end (Li et al., 1998
).
Our previous work (Crescenzo-Chaigne et al., 1999 ) showed that the influenza A and C virus RNA templates were transcribed and replicated with the same efficiency with the influenza A virus polymerase complex, but that the type A RNA template was amplified by the influenza C virus polymerase with a dramatically reduced efficiency. On the basis of this data, we analysed, in this study, the nucleotides involved in the type-specific interaction of the type A and C polymerase complexes with the viral promoters. By making use of the genetic system described by Pleschka et al. (1996)
for the reconstitution of functional RNPs and by comparing the ability of the polymerase complex of the influenza A and C viruses to transcribe and replicate either wild-type or mutated type A and C RNA templates, we found that nucleotide 5 at the 3'-end and, to a lesser extent, nucleotide 6' as well as the 3':8' base pair in the stem of the hairpin loop at the 5'-end of the vRNA are important determinants of type-specificity (prime notation is used throughout to distinguish 5'-end residues from 3'-end residues; Fodor et al., 1994
). Furthermore, our results suggest that sequence requirements for the type C polymerase are more stringent that those required for the type A polymerase.
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Methods |
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Plasmids for the expression of wild-type and mutated type A and C model RNA templates.
The pA/PRCAT and pC/PRCAT plasmids, which direct the expression of model RNA templates derived from the nonstructural (NS) segments of A/WSN/33 and C/JHB/1/66 viruses, respectively, were described previously (Crescenzo-Chaigne et al., 1999 ). They each contain the CAT gene sequences in an anti-sense orientation flanked by the cDNA sequences corresponding to the 5'- and 3'-end of the NS gene segments inserted at the BbsI site of vector plasmid pPR, which is flanked by the human Pol I promoter and hepatitis delta virus ribozyme sequences, such that exact 5'- and 3'-termini of the model RNA templates are ensured.
Mutations at nucleotides corresponding to the 5'- and/or 3'-end of types A or C model RNA templates were generated by PCR amplification of the CAT gene and NS noncoding sequences in the presence of primers containing appropriate mutations and using either pA/PRCAT or pC/PRCAT, respectively, as a template. The exact sequences of the primers can be obtained from the authors upon request. For PCR amplification, from 10 to 50 ng of plasmid was used as the starting template in 100 µl of 2 mM TrisHCl, pH 7·5, 10 mM KCl, 0·1 mM DTT, 0·01 mM EDTA and 1·5 mM MgCl2 in the presence of 2·5 U Expand High Fidelity polymerase (Roche), 0·25 mM dNTPs and 1 µM of oligonucleotide primers. Amplification was for 25 cycles, each consisting of 30 s at 94 °C, 30 s at 45 or 50 °C and 2 min at 72 °C. The amplified products were purified from low-melting-point agarose gels by the QIAquick gel extraction kit (Qiagen) and inserted at the BbsI sites of pPR treated with Klenow enzyme. Positive clones were purified using a QIAfilter plasmid midi kit (Qiagen) and subsequently subjected to phenolchloroform extraction and ethanol precipitation.
Sequencing.
The presence of mutations was assessed by sequence determination using the Big Dye terminator sequencing kit (Perkin Elmer), according to the suppliers instructions, and analysis on an ABI prism 377 automatic sequencer (Perkin Elmer).
Transfections and CAT assays.
COS-1 cells were grown in Dulbeccos modified Eagles medium containing 10% foetal calf serum. Subconfluent monolayers of COS-1 cells (3x105 in 35 mm dishes) were transfected by using 10 µl FuGENE 6 (Roche) with 1 µg of either pA/PRCAT or pC/PRCAT together with pHMG-PB1 (1 µg), pHMG-PB2 (1 µg), pHMG-PA/P3 (1 µg) and pHMG-NP (4 µg) plasmids. After 48 h of incubation at 37 °C, cells were harvested for lysis and reporter gene assays, as described previously (Crescenzo-Chaigne et al., 1999 ). CAT gene expression was determined using the CAT ELISA kit (Roche), which allowed detection of 0·05 ng/ml CAT.
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Results |
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In the presence of the type A polymerase complex, the levels of CAT for the type A RNA template harbouring single A6'U6' or U5
C5 mutations were found to be reduced approximately 1·3- to 2-fold, with respect to the type A wild-type RNA template (Fig. 2
, lanes 2, 3; P<0·05 and P<0·02 in the Student t-test). When both mutations were present, no further reduction of CAT levels was detected, which is consistent with the CAT level that is observed with the type C wild-type RNA template (Fig. 2
, lanes 4, 5). However, in the presence of the type C polymerase complex, the introduction of either of the A6'
U6' or U5
C5 mutations in the type A RNA template resulted in a 4- to 5-fold increase of CAT levels, with respect to the type A wild-type RNA template (Fig. 2
, lanes 2, 3; P<0·01 and P<0·005 in the Student t-test). When both mutations were present, the level of CAT was found to be increased by up to 7-fold, reaching a level that corresponds to about half of the activity of that measured for the type C wild-type RNA template in the presence of the type C polymerase (Fig. 2
, lanes 4, 5). Thus, mutation A6'
U6' or U5
C5 seemed to affect the recognition of the type A model RNA template by the type A polymerase complex to a lesser extent than recognition of the same template by the type C polymerase complex.
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As shown in Fig. 3, for each of the mutated type C RNA templates, similar or slightly higher levels of CAT activity were obtained with the type C polymerase complex (Fig. 3
, lanes 24). However, introduction of the C5
U5 mutation either alone or in combination with the U6'
A6' mutation resulted reproducibly in an increase of the level of CAT activity in the presence of the type A polymerase complex and reached a level comparable to that achieved with the cognate type A wild-type RNA template (Fig. 3
, lanes 35). Thus, whereas mutations C5
U5 and U6'
A6' did not seem to affect recognition of the type C RNA template by the homotypic type C polymerase complex, the C5
U5 mutation appeared to contribute, to some extent, to the recognition of the heterotypic type C RNA template by the type A polymerase complex, although not statistically significant in this case (P<0·1 in the Student t-test).
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Discussion |
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The conserved sequences of the extremities of the RNA segments of influenza A, B and C viruses (Stoeckle et al., 1987 ) are all compatible with the formation of a corkscrew structure on both the vRNA and the cRNA, which is not the case for Thogoto virus, a related orthomyxovirus, for which the sequence at the 5'-end of the cRNA is not compatible with the formation of a hairpin loop (Leahy et al., 1998
). Differences between the influenza A, B and C virus promoters are localized in both the proximal and the distal elements. Limited sequence requirements (i.e. position 10:11') were identified within the distal promoter element of influenza A virus, although base pairing as well as the length of the distal element, which differs between all three types of influenza virus, were found to be critical for the efficient transcription and replication of vRNA (Flick et al., 1996
; Fodor et al., 1994
, 1995
, 1998
; Kim et al., 1997
; Lee & Seong, 1998b
; Tiley et al., 1994
). Within the proximal element of the promoter, differences between influenza A, B and C viruses are located at the tip of the tetraloop structures of the 3'- and 5'-end of the vRNA (nucleotides 5 and 6') as well as at the second base pair (nucleotides 3':8') of the stem of the hairpin loop structure at the 5'-end. Site-directed mutagenesis of the influenza A virus promoter has shown that nucleotides 5 and 6' can be substituted without significantly affecting transcription and replication of the vRNA (Flick et al., 1996
; Fodor et al., 1994
; Kimura et al., 1993
; Poon et al., 1998
). It has, therefore, been suggested that these residues are not involved in binding the type A polymerase complex (Fodor et al., 1994
). Indeed, as shown here, a substantial activity was retained when nucleotide 6' was substituted. However, when a U5
C5 mutation was introduced at the 3'-end of the type A RNA, a 2-fold reduction of CAT levels was observed. This was in agreement with the data reported by Neumann & Hobom (1995)
, but in contrast to data reported by Flick & Hobom (1999)
, within the context of an up-promoter, which harbours additional substitutions at positions 3 and 8 at the 3'-end. This observation suggests that nucleotide 5 may contribute, to some extent, to the efficient recognition of the type A promoter by the type A polymerase complex. Consistent with this interpretation was the fact that the introduction of the reciprocal mutation, i.e. type A-like, C5
U5,within the type C promoter resulted in a reproducible, albeit not statistically significant (P<0·1), increase of the efficiency with which the type C RNA was transcribed and replicated by the type A polymerase complex. Similarly, we have shown here that substitution of nucleotides 5 and/or 6' within the type C vRNA promoter did not significantly affect the efficiency of transcription and replication of type C RNA by the type C polymerase complex. However, when type C-like nucleotides were introduced at these positions within the type A promoter, the result was a substantial increase of the efficiency of transcription and replication of type A RNA by the type C polymerase complex. Thus, subtle differences that may not show up when using optimal promoter sequences may be revealed within the context of a less active heterotypic promoter sequence. Overall our observations suggest that for both influenza A and C viruses, nucleotide 5 contributes to specific recognition of the promoter by the polymerase complex and, furthermore, that nucleotide 6' may also participate, to some extent, in the efficient recognition of the promoter.
Analysis of the 3':8' base pair of the stem of the 5' hairpin loop structure allowed us to confirm the importance of this structure for recognition of the type A wild-type promoter by the type A polymerase, as shown previously in vitro (Pritlove et al., 1999 )or in vivo in the context of the up-promoter (Flick & Hobom, 1999
; Flick et al., 1996
). In addition, short-range interactions within the 5'-arm of the vRNA were also found to be essential for recognition of the promoter by the type C polymerase complex. In contrast to the type A polymerase, for which a weakly interacting base pair seemed to be preferred both in the context of either the type A or the type C promoter, a strongly interacting base pair, with an additional preference for C:G over G:C at positions 3':8' was required, particularly in the context of the type A vRNA, for optimal transcription and replication by the type C polymerase. However, influenza B virus RNA, which harbours a type A-like U3':A8' base pair as the only difference with type C vRNA within the proximal promoter element, is transcribed and replicated by the type C polymerase as efficiently as the homotypic type C RNA (Crescenzo-Chaigne et al., 1999
). In this case, contribution of the distal promoter element, the length of which is extended in the case of type B (9 bp) as compared to either the type C (5 bp) or the type A (6 bp) vRNA, remains to be determined. It is of interest to note in this context that the type B promoter is the only one which is efficiently recognized by all three types of influenza virus polymerase complexes (Crescenzo-Chaigne et al., 1999
) and also the only heterologous orthomyxovirus promoter which can be used, to some extent, by the Thogoto virus polymerase (Weber et al., 1998
). Comparison of the amino acid sequences of the type A and C polymerase proteins, which share 2739% identity, did not reveal any obvious clues concerning the specificity of their recognition of the vRNAs (Yamashita et al., 1989
).
Overall, our results show that, as for type A, a hairpin loop structure at the 5'-end of the vRNA is required for efficient recognition of the viral promoter in the case of influenza C virus. Whether a similar hairpin loop structure at the 3'-end of the vRNA and/or at the 5'-end of the cRNA is also required at some stage of replication of the type C RNA, as suggested for the type A RNA (Flick & Hobom, 1999 ), remains to be determined. Experiments are in progress to address this question. Furthermore, our results emphasize the importance of nucleotides 5 and, to a lesser extent, 6', for type-specific recognition of the promoter sequence by either the type A or the type C polymerase complexes. A sequence-specific contribution of the 3':8' base pair was also found, in agreement with other reports. More stringent sequence requirements within the proximal promoter element seem to prevail for the type C compared with the type A polymerase, which may account for the fact that the type C promoter could be used quite efficiently by the type A polymerase, but not in reverse (Crescenzo-Chaigne et al., 1999
). Whether or not similar sequence requirements would be found within the proximal promoter element for the type B polymerase remains to be determined. Thus, it may be suggested that residues 5 and 6' of the tetraloop structures at the 3'- and 5'-end of the vRNA are involved in binding of the polymerase complex. The experimental approach used here did not, however, allow us to determine if specific interactions are required both at the level of initiation of transcription and of replication. The involvement of residues of the tetraloop structures in type-specific interactions is further suggested by the overall conservation of the nucleotide sequence of the extremities of the vRNA segments for each of the three influenza virus types. Although sequence data for the extremities of the vRNA segments are limited for type A and even more so for type B or type C influenza virus isolates, such conservation contrasts with the substantial exchangeability of the promoter nucleotide sequence determined experimentally. By making use of reverse genetics, an influenza A virus mutant with a U5
C5 substitution at the 3'-end of NA-coding segment 6 could be rescued as infectious virus (Bergmann & Muster, 1995
). Interestingly, for this virus the overall level of segment 6 vRNA was found to be reduced 3-fold as compared to wild-type virus, in agreement with the 2-fold reduction of the level of transcription and replication observed here. However, a similar U5
C5 mutant could not be rescued as infectious virus when present in the context of a promoter harbouring a C rather than a U at position 4 at the 3'-end (Lee & Seong, 1998a
). Furthermore, among all possible substitutions at position 4, the only position of the conserved 3'-end of type A vRNA in which a unique and natural U or C variation is observed, the only mutant that could be rescued as viable virus was the C4
U4 mutant (Lee & Seong, 1998a
). It was thus postulated that the residue at position 4 of the promoter is involved in segment-specific temporal regulation of transcription and replication of the vRNA. The U4 virus exhibited a higher infectivity than the C4 virus, which may account for the fact that the U5
C5 virus could be rescued in the context of the U4 virus but not in that of the C4 virus. Attempts to rescue mutants with substitutions of the U3':A8' base pair should help to determine to what extent the stem of the hairpin loop might also be involved in sequence-specific interactions with the polymerase complex.
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Acknowledgments |
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References |
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Received 25 September 2000;
accepted 5 January 2001.