Sir William Dunn School of Pathology, University of Oxford, Chemical Pathology Unit, South Parks Road, Oxford OX1 3RE, UK
Correspondence
George Brownlee
George.Brownlee{at}path.ox.ac.uk
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ABSTRACT |
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Published ahead of print on 29 November 2002 as DOI 10.1099/vir.0.18795-0.
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INTRODUCTION |
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The terminal 13 and 12 nt of the 5' and 3' ends, respectively, are conserved in all eight vRNA gene segments and are known to form the vRNA promoter (reviewed by Fodor & Brownlee, 2002). The vRNA promoter is a partially duplex structure formed by base-pairing between nt 11'13' of the 5' end with nt 1012 of the 3' end, which is conserved in all eight gene segments. Adjacent to the conserved base pairs are the segment-specific base pairs that extend the duplex by an additional 1 to 5 bp, depending on the segment (Desselberger et al., 1980
; Robertson, 1979
; Skehel & Hay, 1978
). Following the observation that a stemloop structure in both the 5' and the 3' termini is important for promoter function, a corkscrew conformation has been proposed (Brownlee & Sharps, 2002
; Flick et al., 1996
; Leahy et al., 2001
).
Base pairing of nt 11'13' of the 5' end of the vRNA promotor with nt 1012 of the 3' end of the vRNA promotor was found to be critical for efficient virus replication in MDBK cells (Fodor et al., 1998). However, it was possible to generate influenza viruses in which the conserved base pairs in the duplex region of the NA gene were replaced by alternative base pairs. The D2 mutation (GC
UA at nt positions 12'11) was found to attenuate the virus significantly. Furthermore, it significantly reduced the NA mRNA and protein levels in virus-infected cells but did not dramatically affect replication (Fodor et al., 1998
). The NA D2 virus was also found to be attenuated severely in mice and could be used to elicit protective immunity against a wild-type infection (Solorzano et al., 2000
).
The development of plasmid-based techniques for the rescue of influenza viruses (Fodor et al., 1999; Neumann et al., 1999
) has enabled promoter studies to be extended to other gene segments. In the present study, we test the hypothesis that alternative base pairs in the conserved influenza virus vRNA promoter cause attenuation when introduced into either segment 3 (PA gene) or segment 8 (NS1 and NEP-coding gene).
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METHODS |
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Virus rescue.
Rescued viruses were generated using a 12 plasmid rescue system, essentially as described originally (Fodor et al., 1999) but with some modifications which have also been described elsewhere (Fodor et al., 2002
).
Sequencing the mutated genes of transfectant viruses.
TRIzol reagent (Invitrogen) was used according to the manufacturer's instructions to isolate total RNA from MDBK cells infected at an m.o.i. of 1. vRNA was sequenced by standard methods. Details and primers are available from the authors on request. Segment 3 (PA gene) was sequenced fully in the PA-recombinant viruses. Segment 8 (NS1- and NEP-encoding genes) was sequenced fully in the NS-recombinant viruses. No mutations, other than the desired promoter mutations, were detected.
Growth curves.
Briefly, as described previously (Fodor et al., 1998), near-confluent MDBK cells in 3·5 cm diameter dishes were infected with transfectant viruses at an m.o.i. of 0·001 and at various time-points after infection, virus particles in the medium were titrated by plaque assay in MDBK cells.
RNA primer extension.
Primer-extension analysis was performed as described previously (Fodor et al., 1998, 2002
) with some modifications. MDBK cells were infected typically at an m.o.i. of 2 and RNA was isolated from virus- and mock-infected cells using TRIzol reagent. RNA was harvested at hourly intervals between 7 and 12 h post-infection (p.i.). RNA was reverse-transcribed with Superscript reverse transcriptase (Invitrogen) in the presence of two primers that had been 32P-labelled at their 5' ends with T4 polynucleotide kinase (Roche) and [
-32P]ATP (Amersham). Increasing the primer concentration twofold did not increase the yield of transcription products, indicating that the primers were in excess. When studying the levels of vRNA in NS D2 and PA D2 virus-infected cells, the primers 5'-GGGAACAATTAGGTCAGAAGT-3', complementary to nt 695715 of the NS vRNA, and 5'-TTCTTATCGTTCAGGCTCTT-3', complementary to nt 20212040 of the PA vRNA, were used in the same reaction. The two primers used in the reactions to study levels of mRNA and cRNA in NS D2 virus-infected cells were the primer targeting NS1 mRNA and NS cRNA, 5'-TGCAACTCTTTTGCGGACAT-3', corresponding to nt 7695 of the NS gene, and the internal control primer, targeting PB2 vRNA, 5'-GAGATATGGACCAGCATTA-3', complementary to nt 21332151 of the PB2 vRNA gene. The two primers used in the reaction to study levels of mRNA and cRNA in PA D2 virus-infected cells were the primer targeting positive-sense PA RNAs, 5'-TGAGTGCATATTGCTGCAAAT-3', corresponding to nt 126146 of the PA vRNA, and the internal control primer, targeting PB2 vRNA (described above). Reverse transcription reactions were incubated at 50 °C for 90 min, analysed on 6 % denaturing polyacrylamide gels and quantified by phosphorimager analysis. To compare the levels of vRNA, mRNA and cRNA in NS D2 or PA D2 virus-infected cells with wild-type virus-infected cells, the product of the experimental primer was normalized to the internal control. The product of mutant virus-infected cells was expressed as a percentage of wild-type.
Western blot analysis of proteins in virus-infected cells.
MDBK cells were infected with NS D2, PA D2 or wild-type viruses, as described for primer-extension analysis. At 12 or 24 h p.i., cells were harvested and resuspended in SDS-PAGE loading buffer. The cells were boiled and analysed by SDS-PAGE on 12 % polyacrylamide gels. To ensure an equal amount of protein was loaded for each sample, the amount of protein in each lysate was quantified using a Bradford assay (Pierce). Membranes (Amersham Pharmacia) were probed with either a polyclonal NS1 antibody (kindly donated by P. Palese, Mount Sinai School of Medicine, New York, USA) or a monoclonal PA antibody, PA-2 (kindly donated by A. Portela, Agencia Española del Medicamento, Madrid, Spain) (Barcena et al., 1994; Ochoa et al., 1995
). Bound antibodies were detected by standard methods.
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RESULTS |
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DISCUSSION |
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As observed for the NA-transfectant viruses, the D2 mutation, which replaces a GC base pair with a UA base pair at positions 12' and 11 of the vRNA promoter, caused the greatest degree of attenuation of the three mutations tested in both the NS and the PA genes (Fig. 2). Interestingly, the severity of attenuation varied in the two genes tested. The NS D2 virus was most affected, reaching maximum titres 3 log lower than wild-type. The PA D2 virus was least affected, eventually producing titres similar to wild-type but taking up to 12 h longer to reach this maximum titre. The reason for these differences is unknown. One possibility is that the D2 mutation is having a different effect on the different genes because adjacent cis-acting elements, perhaps non-coding regions, modulate promoter activity (Bergmann & Muster, 1996
; Zheng et al., 1996
). Another possibility is that the D2 mutation has the same effect on both genes but that the virus can tolerate reduced levels of PA protein more readily than reduced levels of NS1 or NEP proteins. To distinguish between these possibilities, we analysed the effect of the D2 mutation on, firstly, levels of vRNA, mRNA and cRNA and, secondly, on levels of NS1 and PA protein in virus-infected cells.
A comparison of the levels of vRNA, mRNA and cRNA in NA D2 (Fodor et al., 1998) with PA D2 and NS D2 viruses (in the present study) highlights both similarities and differences. In all three genes tested, the D2 mutation had a greater effect on transcription than replication, decreasing mRNA levels more than vRNA levels (Table 1
). cRNA levels were apparently not affected in cells infected with any of the three D2-mutated viruses. One possibility that could explain why the decrease in vRNA levels did not result in significantly decreased cRNA levels is that the total amount of vRNA in virus-infected cells is in excess to the amount of vRNA required to act as a template for cRNA synthesis. Therefore, the modest decrease in vRNA levels observed in the D2-mutated virus-infected cells may not have been sufficient to significantly affect cRNA synthesis. It should be noted, however, that we cannot exclude the possibility that the D2 mutation caused a small reduction in cRNA levels below our detection limits. Whilst the general effect of the D2 mutation on vRNA, mRNA and cRNA levels was the same in each of the three genes, the magnitude of the effect showed some variation among the different genes. The mRNA was reduced to the same level in NS D2 and PA D2 virus-infected cells but was reduced to a greater extent in NA D2 virus-infected cells (Table 1
). The magnitude of the effect of the D2 mutation on vRNA production also differed. In this study, we show that the vRNA levels in NS D2 and PA D2 virus-infected cells are reduced, whereas it was reported previously that the vRNA levels in NA D2 virus-infected cells were not affected (Table 1
). It is possible that the NA D2 virus may also produce slightly less vRNA than wild-type, as, when it was characterized previously (Fodor et al., 1998
), a small reduction may not have been considered significant. It should also be noted that, whilst the vRNA levels in NS D2 and PA D2 virus-infected cells are statistically different from one another (70 and 41%, respectively; Table 1
), the difference is still small.
A possible explanation for the variations observed in the effect of the D2 mutation in the different genes (Table 1) is that transcription and replication are influenced by nucleotides outside the conserved vRNA promoter. The non-conserved, non-coding nucleotides that are adjacent to the conserved termini differ in both length and sequence between the genes and have been shown to affect both transcription and replication (Bergmann & Muster, 1996
; Zheng et al., 1996
). One possible mechanism by which they could affect transcription is by controlling the efficiency of polyadenylation. It has been reported previously that the D2 mutation interferes with polyadenylation of mRNA (Fodor et al., 1998
). Differences in the non-conserved nucleotides in the NA, NS and PA genes may modulate any effect on polyadenylation caused by the D2 mutation and therefore, result in differing overall polyadenylation efficiencies. We attempted to characterize the effect of the D2 mutation on the polyadenylation of the viruses presented in this study but were unsuccessful due to very low titres of the NS D2 virus.
To test if the observed decrease in mRNA levels (Table 1) resulted in a decrease in protein levels, we performed Western blots on protein samples from virus-infected cells (Fig. 7
). As expected, both NS1 protein in NS D2 (Fig. 7A
) and PA protein in PA D2 (Fig. 7B
) virus-infected cells were reduced dramatically. The amount of NS1 protein in NS D2 virus-infected cells was estimated at between 8 and 16 times lower than wild-type (Fig. 7C
). Although some caution should be taken in directly comparing values obtained from two very different experimental techniques, this suggests that NS1 protein levels are reduced to a greater extent than may be directly expected by the decrease in mRNA (Table 1
). Whilst we cannot rule out an effect of the D2 mutation on translation (see below), the difference in NS1 protein and mRNA levels may be linked to the properties of the NS1 protein. The NS1 protein is known to play a key role in the inhibition of host gene expression (reviewed by Fodor & Brownlee, 2002
). NS1 interferes with splicing (Fortes et al., 1994
; Qiu et al., 1995
), 3'-end processing (Nemeroff et al., 1998
; Shimizu et al., 1999
) and nuclear export of cellular mRNAs (Chen & Krug, 2000
; Chen et al., 1999
; Qian et al., 1994
; Qiu & Krug, 1994
). In addition, NS1 enhances the translation of viral mRNAs (de la Luna et al., 1995
; Enami et al., 1994
). Specifically, it has been proposed to recruit the eukaryotic translation initiation factor 4GI to viral mRNAs (Aragon et al., 2000
). Consequently, the initial decrease in NS1 protein levels that would result directly from the reduction in mRNA may be enough to prevent an efficient inhibition of host gene expression. Competition for translation of viral transcripts with host transcripts would result in decreased translation of all viral proteins. Western blot analysis supports this hypothesis, as not only are the NS1 protein levels dramatically reduced in NS D2 virus-infected cells but PA protein levels are also reduced (Fig. 7B
, compare lanes 2 and 4).
It should be noted that the D2 mutation creates an alternative AUG translation initiation codon (complement of UAC; Fig. 1B) in the corresponding mRNA. Whilst we cannot exclude the possibility that this will affect translation of NS1 and NEP in NS D2 virus-infected cells, and PA in PA D2 virus-infected cells, previous work on the NA D2 virus suggests that this would be unlikely since the NA D1/2 virus, which lacked this AUG codon yet incorporated the D2 mutation, was shown to have very similar properties to the NA D2 virus (Fodor et al., 1998
). As in the case of the NA D2 virus-infected cells (Fodor et al., 1998
), if translation is initiated from the D2 AUG codon in NS D2 or the D2 AUG codon in PA D2 virus-infected cells, a stop codon is reached soon after (5 and 2 aa residues, respectively) translation initiation and therefore, only a short peptide would be produced. Therefore, we have not isolated NS D1/2 or PA D1/2 viruses as the results in Fodor et al. (1998)
suggest this to be unnecessary.
Interestingly, the PA D2 virus is able to achieve virus titres similar to wild-type but the maximum titre of the NS D2 virus is 3 log lower (Fig. 2). Both viruses have been shown to have similar phenotypes, i.e. decreased vRNA, mRNA and protein levels. We propose that, under these conditions, influenza virus growth can better tolerate a reduction in PA protein levels than NS1 and/or NEP. Western blot analysis of cells infected with the PA D2 virus illustrated that the reduced PA protein levels did not result in a decrease of NS1 protein detected in virus-infected cells (Fig. 7A
, lane 3). These results suggest that, under these conditions, the PA protein may be in excess. This is the first time that an influenza virus has been reported that is able to replicate with reduced levels of one of its polymerase proteins.
Another reason why influenza virus may not be able to tolerate a reduction in NS1 protein levels in MDBK cells is that NS1 is needed to protect the virus against the IFN-I antiviral response, as NS1 is known to act as an IFN-I antagonist (reviewed by García-Sastre, 2001). It has been demonstrated that NS1-deficient influenza viruses replicate efficiently in cells that have deficiencies in the IFN-I response, such as Vero cells, but are attenuated in cell lines with normal IFN-I systems (Egorov et al., 1998
; García-Sastre et al., 1998
). Our preliminary results (data not shown), which suggest the NS D2 virus is less attenuated in Vero cells than MDBK cells, support this hypothesis. These results also suggest that the attenuation of the NS D2 virus is mainly due to the reduction in NS1 protein and not NEP. Further support for this hypothesis is that an influenza virus mutant that produces very little NEP replicates normally (Elton et al., 2001
; Smith & Inglis, 1985
).
In conclusion, we have shown that the base pair mutations in the vRNA promoter cause a similar but probably not identical effect in the different genes tested. Therefore, our results are consistent with the hypothesis that the control of transcription and replication is not solely influenced by the minimal vRNA promoter (residues 113 of the 5' end and 112 of the 3' end). This is the first time the effects of such vRNA promoter mutations have been compared in different genes. Specifically, we have demonstrated that the ability of the D2 promoter mutation to attenuate virus growth is not specific to the NA gene but attenuates influenza virus when present in any of the genes tested and therefore, may be considered to be a general response. Therefore, we propose that the introduction of alternative base pairs into the duplex region of the conserved influenza virus vRNA promoter of any of the eight influenza gene segments could be used in the production of live-attenuated vaccine strains.
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ACKNOWLEDGEMENTS |
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Received 23 August 2002;
accepted 15 November 2002.