1 School of Animal and Microbial Sciences, University of Reading, Whiteknights, PO Box 228, Reading RG6 6AJ; 2 Medicine Research Centre, GlaxoSmithKline, Stevenage SG1 2NY, UK
Received 18 August 2004; returned 22 September 2004; revised 5 November 2004; accepted 8 November 2004
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Abstract |
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Results and conclusions: With the exception of a virus containing the E116G mutation, mutant viruses were attenuated to different levels in comparison with wild-type virus. This attenuation was a result of altered NA activity or stability depending on the introduced mutation. Mutant viruses displayed increased resistance to zanamivir, oseltamivir and peramivir, with certain viruses displaying cross-resistance to all three drugs.
Keywords: reverse genetics , antivirals , zanamivir , oseltamivir , peramivir
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Introduction |
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Neuraminidase (NA) inhibitors (NAIs) are a class of anti-influenza drugs used for both prophylaxis and treatment of influenza virus infections. The drugs are highly potent sialic acid (SA) analogues that selectively target the NA enzyme of both influenza A and B viruses. They interact with NA with a higher affinity than SA in a slow-binding manner, thereby preventing the cleavage of SA molecules from host cell receptors required for viral release. This results in aggregation of progeny virions at the cell surface and prevents viral spread. Two such drugs, zanamivir and oseltamivir (Figure 1), display high potency and specificity both in vitro and in vivo 38 and are effective prophylactics.911 Both drugs were introduced into clinical practice in various parts of the world during 19992002. A third NAI, peramivir (Figure 1), is not yet licensed. The anti-influenza virus activity of peramivir was compared with both zanamivir and oseltamivir in vitro12 and in vivo.13 Results suggest that all three drugs share similar potency and specificity for all subtypes of influenza A and B virus NA.
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NAI resistance can also be conferred by mutations in the NA protein. Changes at four residues, depending on the drug and its interaction with the NA active site, have been described. Two of these residues are framework residues, E119 and H274. The other two are functional residues: R152, which binds directly to sialic acid via the formation of a hydrogen bond to the acetamide group;23
and R292, a residue that forms part of a catalytic triad of arginine residues responsible for binding to, and changing the conformational shape of, sialic acid.24
Residue E119 of influenza A virus N1 and N2 NA subtypes interacts with the guanidinyl group of zanamivir (Figure 1). Mutations of this residue to alanine, aspartic acid or glycine confer resistance.18,23,25
Similarly, in the influenza B virus NA protein, the E116G mutation confers resistance.18,26
It has been reported that these mutations may lower NA activity by decreasing the NA protein stability.2730
There have also been reports of zanamivir-resistant mutant influenza A viruses containing an ArgLys mutation at residue R292 of the N2 NA subtype.30,31
However, this R292K mutation is more commonly associated with oseltamivir resistance and is the mutation most frequently observed in resistant N2 subtype mutant viruses isolated from patients,21,32
although this mutation is not found in oseltamivir-resistant N1 subtype viruses. The R292K mutation prevents the reorientation of E276, required to accommodate the large hydrophobic pentyl ether group of oseltamivir (Figure 1). 33
The H274Y mutation also prevents this reorientation event. Therefore, resistance to oseltamivir has also been associated with this mutation, but interestingly only in the N1 subtype viruses.33,34
A third mutation, E119V, is associated with oseltamivir resistance.35,36
Gubareva et al.12
showed that resistance to peramivir is also associated with the R292K (influenza A virus N2 subtype) and H274Y (influenza A virus N1 subtype) mutations, as this drug also contains a bulky hydrophobic group that requires reorientation of the NA active site (Figure 1). Baum et al.22
recently reported the generation of a peramivir-resistant influenza B virus which harboured an H274Y change in NA and additional changes in HA. This mutant showed cross-resistance to oseltamivir, but retained sensitivity to zanamivir similar to the A N2 R292K and A N1 H274Y mutants. Gubareva et al.12
found that an influenza B virus containing an R152K mutation, from the only in vivo selected zanamivir-resistant virus, was resistant to all three drugs. This residue forms a hydrogen bond to the acetamide group of sialic acid.23
The slight increase in flexibility of the lysine side chain may cause a loss of interaction between this residue and the drugs.
There is no evidence of zanamivir resistance in viruses isolated from normal healthy patients after treatment with the drug. The only case of in vivo zanamivir resistance is that of an 18-month-old immunocompromised child, who acquired an influenza B virus infection and failed to respond to ribavirin treatment.20 The child was subsequently treated with zanamivir and after 12 days of treatment a virus containing an R152K NA mutation was isolated. This virus also contained a mutation in the HA protein, T198I, which had appeared prior to the NA mutation. In contrast, resistance to oseltamivir occurs in 1%4% of adults and 4%8% of the paediatric population.36 These viruses only contain mutations in the NA protein, suggesting that mutations in HA are not important for in vivo drug resistance.37
Previous work on NAI resistance has largely centred on influenza A viruses,12,17,19,21,28,30,31,35 with only limited work on influenza B virus resistance.18,20,22,27,38 This study describes the generation of recombinant influenza B viruses containing NA mutations alone, to study their effects on the NAI susceptibility of influenza B virus in the absence of any compensating HA mutations. This was achieved using an influenza B virus reverse genetics system. Comparison of recombinant viruses with a stable genetic background and in the absence of accompanying HA changes enables growth defects and NA activity decreases to be attributed to the NA mutation alone. Mutant NA proteins have been compared in terms of their enzymatic activity, stability and susceptibility to NAIs.
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Materials and methods |
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293-T cells and Madin-Darby canine kidney (MDCK) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) supplemented with 10% fetal calf serum, 1% glutamine (200 mM), 2% non-essential amino acids and 1% penicillinstreptomycin (5000 IU/mL; 5000 µL) at 37°C and 5% CO2. All viruses were amplified in serum-free DMEM at 34°C in the presence of trypsin 2.5 µg/mL. Viruses for NA-STAR NA activity assays were grown in serum-free DMEM lacking phenol red (Life Technologies).
Plasmids and rescue transfections
The pPRNA rescue plasmid38
was mutated by site-directed mutagenesis (Stratagene) using specific primers. Mutant plasmids were substituted into the influenza B virus rescue system in place of the pPRNA plasmid. Influenza B virus rescue was performed as described previously.38
Briefly 0.5 µg of pPRPB1, pPRPB2, pPRPA, pPRHA, pPRNP, pPRNA, pPRM, pPRNS, pCIPB1, pCIPB2, pCIPA and 1 µg of pCINP were transfected into 80% confluent 293-T cells on a 12-well plate and incubated at 34°C. Sixteen hours after transfection, the 293-T cells were co-cultured with MDCK cells. Each transfection was split into two wells of a six-well plate and incubated at 34°C for 8 h. Serum-free DMEM containing trypsin 2.5 µg/mL was added to each well and 1 mU/mL of Vibrio cholerae NA (Roche) was added to one of each duplicate sample. Cells were incubated at 34°C until a cytopathic effect was observed 6872 h post-transfection. All viruses were passaged twice in MDCK cells before use in subsequent experiments.
Flow cytometry
Two wells of 90% confluent Vero cells were infected with each virus at a multiplicity of infection (MOI) of 1 and incubated at 34°C with 5% CO2 for 16 h. Cells were harvested in 1 mL of PBS/EDTA and pooled. Serum-free DMEM (2 mL per well) was added and samples centrifuged at 500 g (2000 rpm) for 3 min. Samples were split in two, with one half being used in the NA-STAR NA activity assay. Cells were resuspended in 500 µL of the anti-FLAG monoclonal antibody at a 1/100 dilution in PBS/0.5% BSA/0.02% sodium azide (PBN) and incubated at room temperature for 1 h on a tube rotator. Cells were washed three times in PBN followed by centrifugation at 500 g for 3 min. Samples were resuspended in 500 µL of anti-mouse FITC-conjugated secondary antibody at a 1/100 dilution in PBN and incubated at room temperature for 1 h on a tube rotator. Cells were washed three times, followed by centrifugation at 500 g for 3 min. Samples were fixed in 500 µL of CellFix (Becton Dickinson) and added to 1 mL of FACSflow (Becton Dickinson). The samples were then subjected to flow cytometry in a Becton Dickinson FACScan and results analysed on WinMDI 2.8 software.
NA-STAR neuraminidase activity assay
NA-STAR assays were performed based on the protocol of Buxton et al.39 Briefly, Vero cells were infected with each virus at an MOI of 1 and incubated at 34°C with 5% CO2 for 16 h. Cells were harvested in 1 mL of PBS/EDTA and resuspended in PBN; 2x106 cells were used in the NA-STAR assay. In a 96-well microtitre plate, serial two-fold dilutions of each sample were prepared in enzyme buffer (325 mM MES/10 mM CaCl2, 1:1) and 40 µL of each dilution was transferred to a white Packard Optiplate in duplicate rows. Five microlitres of 100 µM NA-STAR substrate mix (Applied Biosystems, Foster City, CA, USA) was added and the plate was incubated at 37°C for 15 min. Fifty-five microlitres of Light Emission Accelerator II (Applied Biosystems) solution was added to each well and the plate was read on the Victor2 1420 multilabel counter (Wallac, Perkin-Elmer, Boston, MA, USA).
For the IC50 assay, dilutions of each virus in enzyme buffer that gave a signal-to-noise ratio of no more than 40/1 were made and 40 µL was added to white Packard Optiplates in duplicate rows. Serial four-fold dilutions of zanamivir (GG167) (GlaxoSmithKline), oseltamivir (GS4071) (Roche) or peramivir (RWJ-270201) (Biocryst Pharmaceuticals Inc., Brimingham, AL, USA) were prepared in H2O (drug concentrations therefore were in the range 0.550.000028 µM) and 10 µL of each dilution was added to all wells. Plates were incubated at room temperature for 30 min. Five microlitres of NA-STAR substrate (100 µM) was added, followed by incubation at 37°C for 15 min. Fifty-five microlitres of Light Emission Accelerator II solution was added and the plate was read on the Victor2 1420 multilabel counter. The results were analysed in Excel using the NA-STAR IC50 assay template (GlaxoSmithKline).
Multi-cycle virus growth curves
Multi-step growth curve infections were performed in six-well plates of confluent MDCK cells. Cells were infected with virus at an MOI of 0.001 and incubated at 34°C for 1 h. Cells were washed in PBS, and 1.5 mL of serum-free DMEM containing trypsin 2.5 µg/mL was added. Supernatant samples were harvested every 12 h until 72 h post-infection and infectivity was determined by plaque assay.
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Results and discussion |
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The recently established influenza B virus plasmid-based reverse genetics system38 was used to introduce specific mutations into the B/Beijing/1/87 virus NA gene. The pPRNA rescue plasmid, encoding the entire B/Beijing/1/87 virus segment six vRNA, was mutated by site-directed mutagenesis to introduce the following amino acid changes in NA: E116D, E116A, E116V, E116G, R149K and R291K (residues E119, R152, R292, respectively, in N2 virus numbering). Silent non-coding mutations were introduced into each of the plasmids to facilitate detection of possible wild-type revertant viruses. Each plasmid was used in the influenza B virus rescue system in place of the pPRNA plasmid. As it was expected that these mutations would reduce the activity of the NA enzyme, exogenous V. cholerae NA was included in the rescue. In fact, NAE116D was the only virus that required bacterial NA for its recovery, although on subsequent passage exogenous NA was not required. The plaque size of mutant NAE116D was, however, smaller compared with the wild-type virus and the other mutants, indicating that NA activity was significantly altered in this mutant.
It is possible to introduce a FLAG epitope into the stalk region of the influenza A virus NA protein.40 Viruses were also rescued in which a FLAG epitope was inserted into the stalk region of rescued B/Beijing/1/87 virus NA for subsequent flow cytometry analysis of infected cell surface NA expression. The FLAG epitope replaced codons 4249 of the B/Beijing/1/87 virus NA. It also resulted in a premature stop codon in the NB open reading frame (ORF), resulting in a 47-amino-acid NB protein, a truncation of 53 amino acids. However, this is unlikely to be detrimental to replication in vitro since viruses completely lacking the NB ORF are viable in tissue culture (D. Jackson & W. Barclay, unpublished data).41 Viruses containing the FLAG epitope, in conjunction with each of the single point mutations in NA, were rescued. The NA and HA genes of all 14 recovered viruses were fully sequenced, to confirm the presence of the desired mutations and the lack of non-specific or compensatory changes.
Determination of enzymatic NA activity of mutant NA proteins
Vero cells were infected at 34°C with each of the seven mutant viruses containing the FLAG epitope, and NA expression and activity were measured 16 h post-infection by flow cytometry and NA-STAR assays39 (Table 1). The results show that five of the six NA mutant viruses displayed similar cell surface NA expression levels to the NA-FLAG wild-type virus, with the exception being the E116D mutant, which displayed an increase in NA expression. This was subsequently proven to be due to a higher titre of virus stock, resulting in a greater MOI being used in this experiment. The E116A and E116V mutations resulted in increased activity compared with wild-type NA. The E116D mutation resulted in a decrease in NA activity, despite the fact that the glutamic acid to aspartic acid mutation is a relatively conservative change. Accordingly, this was the only mutant virus that required exogenous bacterial NA for its recovery. The R291K mutation resulted in the largest reduction in NA activity to only 6% of the NA-FLAG wild-type virus.
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Mutations within the NA protein at positions that confer resistance to NAIs can affect the stability of the protein, which is reflected by a decrease in enzyme activity following incubation of mutant NA at higher temperatures.2830,42 The NA activity of influenza B/Beijing/1/87 mutants was measured by NA-STAR assay after incubation at one of three temperatures, 4°C, 37°C or 50°C for 2 h. No NA activity was measurable for any sample at 50°C, presumably as a result of denaturation of the protein. The decrease in NA activity following incubation at 37°C compared with 4°C was determined (Table 1). The data show that the wild-type protein is the most stable of all assayed proteins. It is interesting to note that the E116V NA enzyme is relatively stable, even though a glutamic acid to valine mutation encompasses both a reduction in the size of the side chain as well as a difference in charge. The effects of the E116G mutation on NA protein stability in the context of both influenza A and B viruses have previously been reported.2730 The results here confirm previous findings that the reduction in relative NA activity of this mutant is not due to a decrease in NA activity but to a less stable protein. The R291K mutation resulted in a significant decrease in protein stability, whereas in the context of influenza A virus NA this mutation does not affect protein stability to the same extent.30 The stability of the NAR149K NA enzyme was only 3.9% that of wild-type NA, therefore despite a high relative activity (82%), this virus is likely to be extremely attenuated in vivo. There are clear discrepancies for some mutants between the relative NA activity when expressed by single cycle infection of Vero cells at 34°C and relative reduction in NA activity after additional incubation at 37°C. These differences in protein stability may contribute to replication efficiency in multiple cycle infections in cell culture or in the patient.
Effects of NA point mutations on the growth characteristics of mutant viruses
Analysis of multi-cycle replication of these viruses showed that, with the exception of a virus containing the E116G mutation, all mutants displayed varying levels of attenuation in tissue culture (Figure 2). Similar data were obtained using the set of recombinant viruses carrying the FLAG tag (data not shown).
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Residue R149 is highly conserved among all known NA proteins and is predicted to be important in the binding of sialic acid. Lentz et al.44
demonstrated that an NA protein (N2 subtype) containing an ArgLys mutation at this position lost all NA activity. The NAR149K virus was significantly attenuated in comparison to the Brec virus (Figure 2). This may be due to the low stability of the enzyme, as it displayed 82% relative NA activity compared with that of the Brec virus NA protein. This result suggests that NA protein stability is a major factor in determining virus viability. The B/Memphis/12/97 virus,20
which contained the R149K mutation, also contained a compensating HA mutation, which may have accounted for the viability of the virus.
Replication of the NAR291K virus was significantly attenuated compared with wild-type virus, and did not attain its peak titre until 60 h after infection, 12 h later than all the other viruses. This may be due to the reduction in NA activity to only 6% of wild-type levels. The recovery of titre at the later time points was not due to the R291K mutation reverting to wild-type sequence: both segment four and six vRNA from 60 and 72 h time point supernatant samples were analysed for sequence changes. It was previously shown by Gubareva et al.30 that an influenza A virus (N2 subtype) containing this mutation grew to titres similar to wild-type virus but displayed a reduction in NA activity. The results in this study support this finding. However, the reduction in NA activity and stability of B/Beijing/1/87 R291K virus is greater than previously observed for influenza A viruses.
Although the attenuation of any individual mutant virus can be accounted for by a comparison of NA protein stability and activity with that of wild-type, it is not always possible to correlate relative fitness of mutant viruses with the NA enzyme data. For example, it is unclear why the activity and stability values of the NAE116D virus NA protein were greater than those of the NAR291K virus NA protein, whereas the NAR291K virus grew to far higher titres. This suggests that other factors might be involved in determining the fitness of NA mutant viruses.
Determination of IC50 values for mutant viruses in the presence of NAIs
The effects of NA mutations on susceptibility to NAIs were determined by measuring the IC50 of each recombinant virus in the presence of each of the three different NAIs, zanamivir, oseltamivir and peramivir (Figure 1; Table 2). The IC50 value of each mutant virus was expressed as fold-increase in IC50 compared with Brec wild-type virus. All mutant viruses displayed increased IC50 results for each drug when compared with wild-type virus. Results suggest that the NAI resistance of the NA mutant viruses is similar to that previously observed for mutant viruses and is probably determined by a reduction in interaction between each drug and the NA active site.
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R149 is a functional residue that directly interacts with sialic acid, forming a hydrogen bond to the acetamide group (reviewed in ref. 23) (Figure 3). As all three NAIs contain the acetamide group, NA altered at this residue might be expected to display global resistance to all the drugs. Indeed, this prediction was borne out in a survey by Gubareva et al.12 Our data suggest that in the context of the B/Beijing/1/87 NA the R149K mutation results in a high level of resistance to oseltamivir and peramivir, but only moderate resistance to zanamivir. The reasons why the NAR149K mutant virus shows only moderate resistance to zanamavir are not clear; however, similar results were also obtained by McKimm-Breschkin et al.48
Figure 3 shows that in the B/Beijing/1/87 NA, residue R291 may interact with the glycerol side group of zanamivir and computational analysis predicts that this may be due to the formation of a hydrogen bond, which was previously suggested by Burmeister et al.46 to support binding of R291 to sialic acid. In order for oseltamivir or peramivir to bind in the influenza A virus NA active site, a conformational change has to take place to accommodate the bulky hydrophobic group of these two drugs. The reorientation of residue E276 allows it to form a salt link with R224, which thereby creates the hydrophobic pocket required to accommodate the large hydrophobic group of the drugs (reviewed in ref. 23). However, it is not known whether this reorientation event occurs in the influenza B virus NA active site, as the region surrounding E276 is naturally hydrophobic; therefore, this event would be energetically less favourable.12 A peramivir-resistant influenza B virus with an H274Y change in NA has been reported22 and this mutant showed cross-resistance to oseltamivir, but retained sensitivity to zanamivir, similar to the A N2 R292K and A N1 H274Y mutants. An R292K mutation in the influenza A virus NA active site prevents the reorientation event, thereby preventing the binding of oseltamivir or peramivir, leading to drug resistance. Influenza A viruses (N2 subtype) containing an R292K mutation have been isolated during treatment with oseltamivir35 and in vitro in the presence of oseltamivir,21 zanamivir30 and peramivir13 (reviewed in ref. 23). These viruses tend to display high-level resistance to oseltamivir and peramivir but low-level resistance to zanamivir. The data in Table 2 show that the characteristics of influenza B virus NA containing alterations at this residue are similar, which makes it likely that the reorientation event may indeed occur in the influenza B virus NA active site. It can be concluded that some of the mutations studied here rendered influenza B virus resistant to more than one NAI. However, mutations E119V and R292K, found in oseltamivir-treated patients infected with influenza A N2 viruses, did not cause resistance to zanamivir in influenza B viruses, offering alternative treatment options.
The structurally designed NA inhibitors closely resemble the natural NA substrate (SA) and data obtained so far indicate that resistance requires changes in the highly conserved active site of NA. Our data showed that all mutants displayed significant alteration in the relative enzymatic NA activity and stability of NA, which seem to be tolerated for replication in tissue culture, but might significantly affect replication in man. Accordingly, it has been shown previously that mutants isolated from oseltamivir-treated patients could be propagated in tissue culture, but reverted immediately in infected animals.49 The development of resistance during an acute, self-limiting viral infection will be different from a chronic viral infection with a limited number of viral replication cycles and opportunities to generate resistance. Future monitoring of NAI treatment will show whether resistant mutants are able to spread from patient to patient, are maintained among circulating viruses and can indeed cause a problem for NAI treatment.
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Footnotes |
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Acknowledgements |
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References |
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