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Compensatory mutations contribute to the appearance of the oseltamivir resistance substitution H274Y in the neuraminidase (NA) gene of H1N1 influenza viruses. Here, we describe a high-throughput screening method utilizing error-prone PCR and next-generation sequencing to comprehensively screen NA genes for H274Y compensatory mutations. We found four mutations that can either fully (R194G, E214D) or partially (L250P, F239Y) compensate for the fitness deficiency of the H274Y mutant. The compensatory effect of E214D is applicable in both seasonal influenza virus strain A/New Caledonia/20/1999 and 2009 pandemic swine influenza virus strain A/California/04/2009. The technique described here has the potential to profile a gene at the single-nucleotide level to comprehend the dynamics of mutation space and fitness and thus offers prediction power for emerging mutant species.
Influenza A viruses, including the well-known 1918 Spanish influenza virus, cause mortality and significant societal cost globally every year (1). Recently, the appearance of swine-origin H1N1 influenza A virus also drew the world's attention (2). Drugs have been developed to offer protection against influenza virus infection (3, 4). However, rapid viral mutations pose a challenge for drug efficacy (5–9). There is a need to develop methods for prediction of the emerging influenza A virus mutants.
Oseltamivir (Tamiflu; Hoffmann-La Roche) was discovered in the late 1990s and has been purchased by more than 70 governments at a cost of multiple billions of dollars (10, 11). Oseltamivir biochemically targets neuraminidase (NA), the viral integral membrane protein, which functions in the releasing of viral particles from the host cells (12, 13). In 2007 to 2008, influenza A virus carrying residue 274 substituted from histidine to tyrosine (H274Y [N2 numbering]), which confers oseltamivir resistance and had long been thought to compromise viral fitness, spread throughout the world (14, 15). The presence of H274Y mutations not only raises a public health concern but also highlights the role of permissive mutations in viral evolution (16).
To understand the dynamic nature of the influenza A mutations, phylogenetic analysis or in vitro selection has been commonly employed (16–22). However, the phylogenetic approach does not allow the evaluation of all possible mutations and has a prediction power restricted by dependency on preexisting clinical isolates. In vitro selection, on the other hand, has limited sensitivity due to its dependence on stochastic spontaneous mutation. In this report, we describe a new experimental approach which couples saturation mutagenesis with next-generation sequencing to study the evolution of the viral NA protein in the oseltamivir-resistant influenza A virus. Our results reveal that the replication fitness of influenza A/WSN/33 (H1N1) virus can be fully restored by the substitution R194G (numbering based on WSN sequence) or E214D (N1 numbering) or partially restored by L250P (N1 numbering) or F239Y (N1 numbering). This high-throughput screening approach is also sensitive to mutations with a minor beneficial effect and allows single-nucleotide profiling of the entire gene or genome. One representative compensatory mutation identified by this method, E214D, is further characterized and suggested to have an important role in the emergence of swine-origin 2009 H1N1 influenza virus.
The C227 cell line, stably expressing dominant-negative IRF-3, is derived from human embryonic kidney (293T) cells (23). The H1N1 strain A/WSN/33 (WSN) eight-plasmid reverse-genetics system was kindly provided by Hoffmann et al. (24). The A/New Caledonia/20/1999 (NC99) NA segment and hemagglutinin (HA) segment, all eight segments of A/Texas/36/1991 (TX91), and all eight segments of A/California/04/2009 (swine-origin influenza virus) were kind gifts from Jesse Bloom at the University of Washington. A 4-μg volume of WSN NA plasmid containing H274Y was used as a template for 28 PCR cycles with error-prone polymerase Mutazyme II (Stratagene) to create the insertion library. Bsa1 restriction enzyme sites were added to the PCR primers (forward, 5′-GTGTGTGGTCTCAGGGAGCGA-3′; reverse, 5′-GTGTGTGGTCTCGTATTAGTA-3′) and cloned into BsmB1-digested pHW2000 vector (24). Transformation was conducted with electrocompetent MegaX DH10B T1R cells (Invitrogen). Around 800,000 colonies were collected to generate the NA plasmid library. A total of 10 clones were submitted for Sanger sequencing (Laragen) to approximate the mutation frequency. N-terminal Flag-tagged NA was created by cloning the WSN NA gene into pcDNA 5/TO (Invitrogen). Individual mutant constructs were created by site-directed mutagenesis (Agilent).
Neuraminidase cDNA was PCR amplified into two nonoverlapping fragments with a multiplex identifier (MID) and the constant region for sequencing flanking the primer according to the primer design instructions for the 454 sequencing system (Integrated DNA Technologies). Briefly, each PCR fragment was approximately 700 bp long, and a bidirectional read was used during the sequencing process. 454 FLX sequencing was performed at UCLA GenoSeq Core. Sequence alignment was done through BLAST 2.2.24. A custom perl script was employed for downstream analysis. After setting the Phred quality score cutoff at 20, the mean Phred quality score was 32.46, which corresponds to a 0.06% base-calling error rate. Assuming the sequencing error rates for each of the 3 changes (e.g., A to T, C, or G) are evenly distributed, the base-calling error rate for each exact change is 0.06%/3 = 0.02%. Using the Poisson distribution model (λ = 0.0002 × total number of base calls at a particular position), a minimum read cutoff with a 5% false-discovery rate can be set for each position.
To produce WSN virus, C227 cells were transfected with Lipofectamine 2000 (Invitrogen) using the neuraminidase mutant plasmid library plus the remaining 7 plasmids of the influenza virus reverse-genetics system. Media were replaced at 24 and 48 h posttransfection. Supernatant containing infectious viral particles was harvested at 72 h posttransfection and filtered with a 0.45-μm-pore-size mixed cellulose ester (MCE) filter. 293T-MDCK coculture cells were transfected to produce NC99 (HA segment and NA segment from NC99 and other 6 segments from TX91) or pdmNA-WSN virus (NA segment from A/California/04/2009 and all of the other 7 segments from WSN). At 24 h posttransfection, cell growth media were replaced with viral growth media (Dulbecco's modified Eagle's medium [DMEM] supplemented with 0.3% bovine serum albumin [BSA], 0.01% serum, 2 μg/ml tosylsulfonyl phenylalanyl chloromethyl ketone [TPCK]-trypsin, and penicillin-streptomycin). Supernatant containing infectious viral particles was harvested at 60 h posttransfection and filtered with a 0.45-μm-pore-size MCE filter.
For infection or selection experiments, A549 cells were used for WSN and MDCK cells were used for NC99 and pdmNA-WSN virus for infection and titering. Briefly, three phosphate-buffered saline (PBS) washes were conducted at 2 h postinfection, and virus in the supernatant was harvested at 24 h postinfection. Titers were determined as described in reference 25. Oseltamivir was a kind gift from Hoffmann-La Roche Pharmaceuticals.
Equal amounts of NA plasmids were transfected into 293T cells using Lipofectamine 2000 (Invitrogen). At 24 h posttransfection, the media were removed and the cells were collected by resuspension in isotonic buffer (15 mM morpholinepropanesulfonic acid [MOPS], 145 mM sodium chloride, 2.7 mM potassium chloride, 4.0 mM calcium chloride, and 2% heat-inactivated fetal bovine serum). NA activity was assayed using the fluorogenic substrate methylumbelliferyl-α-d-N-acetylneuraminic acid (MUNANA) (purchased from Sigma) following a previously described protocol (http://www.nisn.org/documents/A.Hurt_Protocol_for_NA_fluorescence.pdf). The fluorescence readout was subtracted from background and normalized to the cell concentration via cell counting. For the total cell lysate NA activity assay, the same protocol was used except that the cells were lysed in TEB (50 mM Tris [pH 7.5], 150 mM NaCl, 1.0% Nonidet P-40, 10% glycerol, 10 μg/ml aprotinin, 10 μg/ml pepstatin, 0.5 mM phenylmethylsulfonyl fluoride).
293T cells on a 12-well plate were transfected with equal amounts of Flag-tag NA expression plasmid using Lipofectamine 2000 (Invitrogen). At 24 h posttransfection, cells were lysed by TEB and equal volumes of total cell lysate were heated with SDS loading buffer for 5 min and loaded onto a 10% polyacrylamide gel. Milk (5%) was used for blocking. Mouse anti-Flag antibody (Sigma) and sheep horseradish peroxidase-conjugated anti-mouse immunoglobulin G (GE Healthcare) were used as primary and secondary antibodies. For the loading control, mouse anti-beta actin antibody (Sigma) was used as the primary antibody.
293T cells on a 6-well plate were transfected with equal amounts of Flag-tag NA expression plasmid using Lipofectamine 2000 (Invitrogen). At 24 h posttransfection, cells were washed with PBS and incubated at 37°C and 5% CO2 for 1 h with 800 μl DMEM (minus cysteine [Cys]/methionine [Met], 5% fetal bovine serum [FBS]). [35S]Met/Cys Express label (PerkinElmer) was then added (0.1 mCi/well), and the mixture was incubated for 1 h (pulse). Cells were washed with PBS, and the radioactive media were replaced with cell growth media (DMEM with 10% FBS) after the 1-h pulsing. At the indicated time point, cells were washed with PBS and resuspended in PBS. Cell pellets were obtained by centrifugation for 5 min at 3,000 rpm and were stored at −80°C when necessary. Cell pellets from different time points were lysed in parallel on ice for 20 min using TEB (50 mM Tris [pH 7.5], 150 mM NaCl, 1.0% Nonidet P-40, 10% glycerol, 10 μg/ml aprotinin, 10 μg/ml pepstatin, 0.5 mM phenylmethylsulfonyl fluoride). Equal amounts of total protein from different samples were then incubated with TEB and loaded onto an EZview Red Anti-Flag M2 Affinity gel (Sigma) for 2 h. Beads were washed 3 times with TEB. Elution was done in 200 μl SDS loading buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, 0.02% bromophenol blue), and the mixture was heated at 90°C for 5 min. Samples of equal volumes were loaded onto a 10% polyacrylamide gel. The resultant gel was dried at 60°C using an FB-GD-45 gel dryer (Fisher Scientific) and exposed for 3 days. Quantification was done by ImageJ (26).
H274Y mutation was introduced to the NA gene of H1N1 A/WSN/33 (WSN) using site-directed mutagenesis. Random mutagenesis utilizing error-prone polymerase was done on the H274Y NA gene to generate the mutant library with an average mutation frequency of approximately 5.6 per clone. The mutant library was transfected into C227 cells (23), which are subclones of 293T cells stably expressing dominant-negative IRF-3 and are able to increase the viral titer by ~10%, to generate the viral mutant library using the eight-plasmid reverse-genetics system (24). The viral mutant library was then processed through successive rounds of 24-h infections in human lung carcinoma A549 cells at a low multiplicity of infection (MOI) (0.003 to 0.01) to reduce transcomplementation, with increasing oseltamivir concentrations (from 250 nM to 2 μM) throughout selection (Fig. 1A).
We used 454 pyrosequencing to survey the mutant pools at different stages of selection: the DNA plasmid library, the viral library after transfection, selection round 2, and selection round 4. Nucleotides with low-quality base calling (Phred quality score < 20) were filtered out such that a reliability score for each base call of least 99% was achieved. To examine the selection progress at different stages of selection, a preliminary analysis was done to digitally identify beneficial mutations with an occurrence frequency of over 1% and covered with ≥50 reads. Among the mutations identified, 10 satisfied the cutoff in the post-round 2 selection pool and 6 remained in the post-round 4 selection pool (Fig. 1B and Table 1). Therefore, we utilized the data from the post-round 2 selection pool for the rest of the study to achieve higher sensitivity. R194G, which was previously shown to be able to compensate for the H274Y fitness deficiency in WSN (16), appeared to be a positive candidate in both the post-round 2 and post-round 4 selection pools, thus supporting the validity of our approach.
To detect weakly beneficial mutations and also permit gene profiling at a single-nucleotide resolution level, the enrichment ratio of post-round 2 occurrence frequency to posttransfection occurrence frequency was computed for each mutation. To limit statistical errors, only mutations that had a <5% false-discovery rate in the posttransfection pool were considered. We reasoned that a mutant had to acquire a relative growth advantage to maintain its occurrence frequency and avoid being outcompeted. As a result, in addition to a strict cutoff (ratio > 5), we also set a less stringent cutoff (ratio > 1). A total of 9 mutations satisfied the strict cutoff (ratio > 5), and an additional 13 mutations satisfied the less stringent cutoff (ratio > 1) (Fig. 1D and Table 2). A lot of mutations disappeared (ratio = 0) or had a decrease of at least 10-fold in occurrence frequency (ratio < 0.1) after 2 rounds of selection (Fig. 1C and andD).D). These mutations most likely occurred at codons encoding essential residues for viral growth. Utilization of the recently developed sequencing platform with higher throughput would improve the sensitivity.
It was surprising to see E214G (ratio = 1.2) in the candidate list (Table 2). Mutation E214G, which spread globally in circulating influenza virus strains (27), was reported to have a neutral effect on virus harboring the H274Y NA mutation (16). However, our data in combination with the clinical prevalence of E214G suggest that the mutation may provide a weakly beneficial effect for the H274Y influenza virus. The beneficial effect of L250Q (ratio = 1.3) described in our data is also supported by its fixed presence in all swine-origin 2009 H1N1 clinical isolates (data not shown).
Compensatory mutant candidates with a mutation frequency > 1% and at least 50 reads of coverage in the post-round 2 selection pool were reconstructed using site-directed mutagenesis. R194G and E214D fully restored and F239Y and L250P partially restored the growth deficiency of the H274Y mutant while not affecting the replication kinetics in the wild-type (WT) background (Fig. 2A to toC).C). (Note that the E214G and L250Q mutations mentioned above have amino acid substitutions different from those of E214D and L250P at the same positions). Since our random mutagenesis method does not restrict the number of mutations per viral mutant, it is possible that false-positive candidates could be considered bystander mutations that happen to appear in the fittest genome. It is also possible that the level of the beneficial effect of some candidates may be below the sensitivity of our validation experiment. Previously identified compensatory mutations, V234M and R222Q (16), did not appear in our screen, likely due to the strain differences. While seasonal influenza virus strains (A/New Caledonia/20/1999 and A/Brisbane/59/2007) were used in the previous studies to study the compensatory effect of V234M and R222Q (16, 28), we employed a mouse-adapted WSN strain in this study. Indeed, V234M and N222Q (WSN carries an asparagine [N] instead of an arginine [R] in this position) do not restore the growth deficiency of H274Y in strain A/WSN/33. A similar observation was reported in a swine-origin 2009 H1N1 strain, A/California/4/2009, using a neuraminidase functional assay (29). Among the validated compensatory mutations, all but L250P are distant from H274Y (Fig. 2D). A close spatial arrangement (side chains separated by 4.4 Å) suggested that V234M, a proposed permissive mutation (16), and F239Y might exert their compensatory effect through similar molecular mechanisms (Fig. 2D).
To measure the NA activity of different mutants, a fluorometric enzyme activity assay based on the substrate 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (MUNANA) was used (30). Unlike R194G, E214D does not fully restore the NA activity to the wild-type (WT) level but is still capable of restoring the viral fitness to that of the WT. In addition, the compensatory effect of F239Y and L250P in viral replication was not directly correlated with the cell surface NA activity (Fig. 2A and andBB and Fig. 3A). Therefore, we suspect that either the WT has an excess of cell surface NA activity for viral replication or that the cell surface NA enzyme activity is not the sole determinant of viral neuraminidase activity for effective replication. Control experiments utilizing the N-terminal Flag-tagged WSN NA showed that the differences of the functional neuraminidase activity in cell surface and total protein lysates are not due to the differences in protein expression level or stability (Fig. 3B to toEE).
F239Y and L250P offer a compensatory effect for H274Y viral replication in vitro but have a low prevalence in patient samples (Table 3). This finding raised the possibility of their emergence in circulating strains and the need of monitoring these two amino acid residues in clinical isolates. Whereas R194G has been described in the past (16, 31), we turned our attention to E214D, which is present in only 1.3% of seasonal H1N1 strains but is fixed in the swine-origin 2009 H1N1 influenza virus (Table 3). Interestingly, it was reported that H274Y did not significantly decrease the fitness of swine-origin influenza virus (32–34). We hypothesized that the difference between seasonal influenza virus and swine-origin influenza virus at position 214 allows the swine-origin influenza virus to carry H274Y without significantly compromising viral growth.
Consistent with our hypothesis, in pdmNA-WSN (NA segment from A/California/04/2009 and all of the other 7 segments from WSN), the D214E-H274Y double mutant has a replication deficiency whereas H274Y or D214E alone does not (Fig. 4A). In addition, the D214E-H274Y double mutant has a cell surface NA activity less than 5% of that of WT, which correlates with its fitness deficiency (Fig. 4A and andC).C). We also found that E214D is able to compensate for the deficiency of H274Y in NC99 (a seasonal influenza virus model in which the HA and NA segments are from A/New Caledonia/20/1999 and the other 6 segments are from A/Texas/36/1991) (Fig. 4B) and also to rescue the cell surface NA activity to around 80% of the wild-type level (Fig. 4D).
NA H274Y mutation has been identified as the major mutation that confers H1N1 influenza A virus oseltamivir resistance (14). While H274Y has been thought to damage viral fitness, clinical isolations in recent years suggest that permissive mutations exist such that the drop in the fitness effect from H274Y can be minimized (16, 28). Phylogenetic analysis which relies on existing clinical isolations is often used to discover beneficial mutations (16–20, 22). However, the searching space is often limited by the defined number of clinical isolates being sequenced. In vitro selection or serial passaging could be employed for discovering beneficial mutations in virus (21), but it is limited by the stochasticity and frequency of spontaneous mutation. In addition, the selection process restricts the sensitivity to mutants that offer a minor beneficial effect, due to the highly competitive environment of selection. The high-throughput screening method presented in this study provides an alternative approach to screen for beneficial mutations in an unbiased fashion.
A few recent studies have demonstrated the concept of high-throughput surveillance of a mutant library such as functional mapping for the human WW domain using a phage display system (35) and genome functional element screening using random transposon insertion (36, 37). This is the first time, to the best of our knowledge, that this approach has been applied with single-nucleotide resolution to a whole gene. By surveying the mutant pool in early rounds of selection with 454 sequencing, we were able to avoid an out-competing effect exerted by the optimum mutation and discover suboptimum mutations, such as F239Y and L250P. By increasing the scale of the sequencing sampling with newer sequencing technologies, which would provide greater sequencing depth, it is possible to build a single-nucleotide profile of the whole genome and predict the dynamics of viral mutation space under different conditions. The technique employed in this study is widely applicable to different fields where reverse-genetics systems exist such as human immunodeficiency virus (38), hepatitis C virus (39), etc. It also has the potential to be adapted to screen for mutations in cellular genes.
The screen performed here identified a novel mutation, E214D, which can fully restore the H274Y viral replication efficiency. In contrast to the seasonal H1N1 strain, aspartic acid at position 214 is present in every clinical isolate of the swine-origin 2009 H1N1 strain. It could provide an explanation for the phenomenon that H274Y imposed a less deleterious effect on swine-origin influenza virus than on seasonal influenza virus (32–34). The novel compensatory mutations presented in this study enhance the forecasting power for the emergence of oseltamivir-resistant influenza virus. Influenza virus harboring a compensatory mutation is likely to acquire the oseltamivir-resistant mutation NA H274Y under conditions of oseltamivir treatment and to be less responsive to the treatment without any compromise in fitness. Therefore, to prevent future spread and minimize the social cost, precautions should be taken before treating an influenza virus-infected patient with oseltamivir when most isolates in an area are acquiring the compensatory mutations.
In summary, we present a new approach allowing the identification of beneficial mutations in a comprehensive fashion with a high sensitivity for detection of mutations with suboptimal potency. Besides identifying a novel compensatory mutation, E214D, which can fully restore the viral replication fitness of the H274Y mutant, our data also revealed mutations that could offer a smaller degree of beneficial effect, such as F239Y and L250P. The results of this study not only demonstrate the usage of high-throughput functional profiling to study a gene at single-nucleotide resolution but also broaden the understanding of NA protein evolution of oseltamivir-resistant influenza virus by offering a tool for quasispecies dynamic prediction.
We thank G. Hobom and Y. Liang for the 8-plasmid reverse-genetics system for WSN and J. D. Bloom for the reverse-genetics system for TX91, NC99, and swine-origin influenza virus and insightful discussions, E. Botz for proofreading the manuscript, and Hoffmann-La Roche Pharmaceuticals for oseltamivir carboxylate.
This work was supported by the National Institutes of Health (reference R01-EB-009764), a UCLA Molecular Biology Whitcome Pre-Doctoral Training Grant, Oppenheimer Endowment Awards, and Clinical Translational Seed Grants, UCLA Center for AIDS Research, and UCLA Jonsson Comprehensive Cancer Center.
N.C.W. performed the majority of experiments and all the data analysis, S.D. and H.W. performed the 454 sequencing run, A.P.Y. assisted with experimental design, L.Q.A. assisted with the structural study and provided intellectual support, T.-T.W. provided intellectual and experimental support, A.P.Y. and R.S. supervised the project, and N.C.W, A.P.Y., and R.S. wrote the text.
Published ahead of print 14 November 2012