A Computational-Experimental Approach Identifies Mutations That Enhance Surface Expression of an Oseltamivir-Resistant Influenza Neuraminidase

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1 A Computational-Expeimental Appoach Identifies Mutations That Enhance Suface Expession of an Oseltamivi-Resistant Influenza Neuaminidase Jesse D. Bloom, Jagannath S. Nayak, David Baltimoe* Division of Biology, Califonia Institute of Technology, Pasadena, Califonia, United States of Ameica Abstact The His274?Ty (H274Y) oseltamivi (Tamiflu) esistance mutation causes a substantial decease in the total levels of suface-expessed neuaminidase potein and activity in ealy isolates of human seasonal H1N1 influenza, and in the swineoigin pandemic H1N1. In seasonal H1N1, H274Y only became widespead afte the occuence of seconday mutations that counteacted this decease. H274Y is cuently ae in pandemic H1N1, and it emains unclea whethe seconday mutations exist that might similaly counteact the deceased neuaminidase suface expession associated with this esistance mutation in pandemic H1N1. Hee we investigate the possibility of pedicting such seconday mutations. We fist test the ability of seveal computational appoaches to etospectively identify the seconday mutations that enhanced levels of suface-expessed neuaminidase potein and activity in seasonal H1N1 shotly befoe the emegence of oseltamivi esistance. We then use the most successful computational appoach to pedict a set of candidate seconday mutations to the pandemic H1N1 neuaminidase. We expeimentally sceen these mutations, and find that seveal of them do indeed patially counteact the decease in neuaminidase suface expession caused by H274Y. Two of the seconday mutations togethe estoe suface-expessed neuaminidase activity to wildtype levels, and also eliminate the vey slight decease in vial gowth in tissue-cultue caused by H274Y. Ou wok theefoe demonstates a combined computational-expeimental appoach fo identifying mutations that enhance neuaminidase suface expession, and descibes seveal specific mutations with the potential to be of elevance to the spead of oseltamivi esistance in pandemic H1N1. Citation: Bloom JD, Nayak JS, Baltimoe D (2011) A Computational-Expeimental Appoach Identifies Mutations That Enhance Suface Expession of an Oseltamivi-Resistant Influenza Neuaminidase. PLoS ONE 6(7): e doi: /jounal.pone Edito: Ron A. M. Fouchie, Easmus Medical Cente, Nethelands Received May 5, 2011; Accepted June 16, 2011; Published July 20, 2011 Copyight: ß 2011 Bloom et al. This is an open-access aticle distibuted unde the tems of the Ceative Commons Attibution License, which pemits unesticted use, distibution, and epoduction in any medium, povided the oiginal autho and souce ae cedited. Funding: JDB was funded by a Beckman Institute postdoctoal fellowship at the Califonia Institute of Technology. JSN was funded by an Amgen undegaduate summe eseach fellowship. The fundes had no ole in study design, data collection and analysis, decision to publish, o pepaation of the manuscipt. Competing Inteests: The authos have declaed that no competing inteests exist. * baltimo@caltech.edu Intoduction In molecula evolution, multiple mutations ae often equied to confe an advantageous phenotypic change. Fequently, one mutation diectly causes a beneficial functional alteation (such as a shift in substate specificity o dug esistance), but is deleteious to potein-level popeties such as folding, stability, o expession. A seconday mutation bolstes the potein-level popeties damaged by the functional mutation, but by itself may confe no majo adaptive benefit. Both mutations ae needed to yield a potein that possesses the beneficial functional alteation and the equisite potein-level popeties. Examples of this phenomenon may include the evolution of antibiotic esistance [1,2], vial immune escape [3], steoid-ecepto specificity [4], cytochome P450 enzymatic activity [5,6], HIV co-ecepto usage [7], and influenza antivial esistance [8]. When the functional mutation occus fist and is followed by a seconday mutation that epais potein-level popeties, the seconday mutation is typically efeed to as compensatoy. Howeve, if an initial occuence of a seconday mutation enables the potein to toleate the subsequent functional mutation, the seconday mutation is efeed to as pemissive [4]. It is often impossible to detemine which of these two scenaios actually occued, but in some cases it appeas that evolution poceeded via pemissive mutations [4,8]. This fact aises the tantalizing pospect that it may be possible to pedict seconday mutations that could foeshadow futue evolutionay change. In this pape, we exploe the possibility of identifying mutations of possible elevance fo the evolution of esistance to the neuaminidase-inhibito oseltamivi (Tamiflu) in the 2009 swine-oigin pandemic H1N1 influenza. Resistance to oseltamivi is confeed on N1 influenza neuaminidases by the His274?Ty mutation (H274Y, N2 numbeing), which causes a subtle stuctual change in the potein s active site that weakens the binding of oseltamivi [9]. Although H274Y could occasionally be identified in human seasonal H1N1 isolates fom people taking oseltamivi [10], it was thought that this mutation was unlikely to spead appeciably. The eason fo this view was that H274Y damatically attenuated a vaiety of seasonal H1N1 stains in tissue cultue and animal models, including A/WSN/1933 [11], A/Texas/36/1991 [12], A/New Caledonia/20/1999 [13], and A/Mississippi/3/2001 [14]. This attenuation coincided with a potein-level defect caused by H274Y that deceased the amount of neuaminidase expessed on the cell suface [8]. But by 2007, H274Y no longe detectably attenuated seasonal H1N1 isolates [14 16], and viuses caying that mutation began to spead globally, going to nea fixation in PLoS ONE 1 July 2011 Volume 6 Issue 7 e22201

2 the season [17 19]. This spead of esistance was peceded by seconday mutations that counteacted the decease in neuaminidase suface expession caused by H274Y [8]. In the sping of 2009, human seasonal H1N1 was displaced by a new pandemic swine-oigin H1N1 stain that continues to ciculate globally [20,21]. Cuently, only about 1% of tested pandemic H1N1 isolates have caied H274Y [22,23]. Most of these esistant isolates have been fom immunocompomised patients o individuals taking oseltamivi, with only a few epoted cases of H274Y vius being tansmitted in healthy unteated adults [22,23]. At the potein level, H274Y causes the same defect in neuaminidase suface expession obseved in ealy seasonal H1N1. Specifically, H274Y causes a substantial decease in the total potein and activity expessed on the suface of cells tansfected with plasmids encoding pandemic H1N1 neuaminidase [8], while pandemic H1N1 viuses with H274Y possess between fou and 10-fold less total neuaminidase activity [24 26]. Howeve, as discussed immediately below, it emains unclea whethe this decease meaningfully attenuates vial fitness. A numbe of expeimental studies have compaed the gowth o tansmission of matched isolates of wildtype and H274Y pandemic H1N1. In MDCK-deived cell lines, H274Y vius gew slightly but detectably wose than wildtype in five of eight cases [24,27 30]; in the othe thee cases, thee was no discenible diffeence [25,29,30]. H274Y vius gew slightly moe pooly than its wildtype countepat in diffeentiated human aiway epithelium cells [25]. Upon diect inoculation of high doses into feets o mice, both wildtype and H274Y viuses eplicated efficiently and caused disease in all studies [27 31]. Similaly, in all studies, both wildtype and H274Y viuses tansmitted by diect contact with 100% efficiency between co-caged feets [29,31] o guinea pigs [29]. Pehaps the most biologically elevant expeimental measue of vial fitness is aibone tansmission in feets o guinea pigs. In two of five compaisons, both wildtype and H274Y vius tansmitted apidly to all exposed animals in the expeimental conditions used [29,30]. But in the thee compaisons without complete apid tansmission, the H274Y vius eithe tansmitted makedly moe slowly [30] o completely failed to infect some of the exposed animals [27,29]. The authos of these studies diffe about whethe thei esults imply attenuation by H274Y clealy, pandemic H1N1 is not seveely cippled by the mutation as was ealy seasonal H1N1. This diffeence in the extent of attenuation caused by educed neuaminidase levels could be due to as yet undefined diffeences elsewhee in the vial genome, such as in hemagglutinin ecepto avidity [32]. Howeve, fom an evolutionay pespective, a eduction of vial fitness by even a few pecent would likely pevent the spead of H274Y in pandemic H1N1, since only a small faction of infected individuals use oseltamivi [33]. We theefoe consideed it wothwhile to investigate whethe we could identify seconday mutations that counteact the deceased neuaminidase suface expession caused by H274Y in pandemic H1N1. We began by testing the ability of seveal computational appoaches to etospectively identify seconday mutations that incease the total suface-expessed neuaminidase activity in seasonal H1N1. We find that the PIPS computational appoach [34] is the most capable of coectly identifying seconday mutations in this etospective test. We then use this computational appoach to pedict 12 candidate seconday mutations to pandemic H1N1. We expeimentally sceen these mutants, and show that seveal of them do indeed incease the total sufaceexpessed potein and activity of H274Y pandemic H1N1 neuaminidase. Combining two of these seconday mutations with H274Y estoes suface-expessed activity to appoximately wildtype levels, and also escues the modest attenutation that H274Y causes fo vial gowth in tissue cultue. Ou wok theefoe identifies seveal seconday mutations that have the potential to be of elevance fo the evolution of oseltamivi esistance in pandemic H1N1. Results Retospective testing of computational appoaches fo identifying impotant seconday mutations in seasonal H1N1 The goal of ou study is to pedict seconday mutations that enhance the suface-expessed activity and potein levels fo H274Y pandemic H1N1 neuaminidase. Thee ae vaious computational appoaches that conceivably could be applied towads this goal. We theefoe began by testing the ability of seveal computational appoaches to etospectively identify impotant seconday mutations fom the evolution of seasonal H1N1 neuaminidase. The A/New Caledonia/20/1999 seasonal H1N1 stain is attenuated by H274Y [13], while the A/Bisbane/59/2007 stain is not attenuated by this mutation [14] and is an immediate ancesto of the lineage of oseltamivi-esistant viuses that went to fixation beginning in We pefomed assays to measue both the total suface-expessed neuaminidase activity and potein levels in mammalian cells tansfected with plasmids encoding wildtype and H274Y neuaminidase poteins fom these two stains. As descibed peviously [8], H274Y caused an appoximately two-fold decease in suface-expessed neuaminidase potein and activity fo the 1999 stain (Figue 1). In compaison, the wildtype 2007 neuaminidase was expessed on the cell suface at ove 1.5-fold highe levels than its 1999 countepat, and the elative magnitude of the decease caused by H274Y was substantially smalle (Figue 1). A total of 12 amino acid mutations sepaate the neuaminidases fom these stains (H45N, V48I, K78E, E214G, R222Q, V234M, G249K, T287I, K329E, D344N, G354D, and D382N; N1 numbeing). Two of these mutations (R222Q and V234M) have been shown expeimentally to be sufficient to alleviate the attenuation of vial gowth in tissue cultue caused by H274Y in the backgound of the 1999 neuaminidase [8]. A thid mutation (D344N) has been suggested to enhance neuaminidase substate affinity [15,35,36]. We pogessively added these mutations to the 1999 neuaminidase in the ode that they appeaed in natual sequences (V234M, then R222Q, then D344N). When all thee mutations wee added to the 1999 neuaminidase, it exhibited simila levels of total suface-expessed potein and activity to the 2007 neuaminidase, both in the pesence and absence of H274Y (Figue 1). Of the emaining mutations, thee (V48I, E214G, and D382N) have been tested peviously [8]. In the backgound of an H274Y seasonal H1N1 neuaminidase, none of these mutations caused a substantial change in suface-expessed neuaminidase potein o activity. Since the divegence in suface-expessed potein and activity between 1999 and 2007 is explained by the thee mutations R222Q, V234M, and D344N, fo the pupose of the etospective testing in this section, we placed these thee mutations in one goup. We then placed all of the emaining mutations in anothe goup although we stess that some of these emaining mutations have not been explicitly tested fo thei effect on neuaminidase suface-expessed activity. We next sought to test whethe computational appoaches could identify the thee known enhancing mutations fom the complete set of mutations that sepaated the 1999 and 2007 stains. We PLoS ONE 2 July 2011 Volume 6 Issue 7 e22201

3 Figue 1. The thee seconday mutations V234M, R222Q, and D344N lagely explain the diffeences in total suface-expessed activity and potein between 1999 and 2007 seasonal H1N1 neuaminidases. Shown ae wildtype (WT) and indicated mutants of the A/ New Caledonia/20/1999 neuaminidase, in addition to WT and H274Y neuaminidases fom the A/Bisbane/59/2007 (BR07) stain. All neuaminidases contain C-teminal epitope tags, except fo the untagged WT and H274Y A/New Caledonia/20/1999 vaiants. Fo the measuements, 293T cells wee tansfected with plasmids encoding the neuaminidase poteins. Afte 20 hous, the cells wee assayed fo the total suface-expessed neuaminidase activity (top panel) o potein using an antibody against the epitope tag (bottom panel). Bas show the mean and standad eo fo at least six eplicates. doi: /jounal.pone g001 easoned that a computational appoach that could coectly identify these thee mutations might also be able to pedict new mutations that enhance the suface expession of pandemic H1N1 neuaminidase. Because seveal of the candidate computational appoaches utilize stuctual data, we esticted the analysis to the mutations that occued in the cystallized [37] ectodomain of the neuaminidase (this excludes mutations H45N, V48I, and K78E). Ou test theefoe consisted of assessing the ability of the computational appoaches to distinguish R222Q, V234M, and D344N fom the emaining six ectodomain mutations (E214G, G249K, T287I, K329E, G354D, and D382N) that occued duing the divegence of the 1999 and 2007 stains. We tested fou diffeent computational appoaches. CUPSAT is a compute pogam that combines stuctual infomation with statistically deived potentials to pedict the changes in potein stability associated with amino acid mutations [38]. FoldX is a compute pogam that uses a full atomic desciption of a potein s stuctue to pedict mutational effects on potein stability [39]. The consensus appoach assumes that the individual contibution of a mutation has a diect logaithmic (Boltzmann-like) elationship to its fequency in a sequence alignment of homologous poteins, such that the consensus esidue is always assumed to be the most favoable [40 42]. Finally, PIPS is a method that we developed to infe mutational effects based on an analysis of potein phylogenies, and which has been shown to be able to pedict seconday mutations that alleviate tempeatesensitive defects in influenza hemagglutinin [34]. The impoved implementation of the PIPS appoach used hee is descibed in detail in the Mateials and Methods section, as ae the datasets used fo the CUPSAT, FoldX, and consensus pedictions. Figue 2 shows the ability of each of the fou computational appoaches to distinguish R222Q, V234M, and D344N fom the othe six mutations. Neithe CUPSAT no FoldX showed any efficacy. Both of these methods placed the pedicted effects of the nine actual ectodomain mutations nea the cente of the distibution fo all possible neuaminidase mutations, and failed to sepaate R222Q, V234M, and D344N fom the othe six mutations. The consensus appoach did identify the nine actual ectodomain mutations as being among the most pefeable of all possible mutations, although this is a somewhat tautological esult since by constuction the appoach pefes mutations that ae pevalent in natual sequences. Howeve, the consensus appoach PLoS ONE 3 July 2011 Volume 6 Issue 7 e22201

4 failed to sepaate R222Q, V234M, and D344N fom the othe six mutations. The PIPS appoach was clealy the most successful. It classified the nine actual ectodomain mutations as being moe pefeable than most of the distibution of all possible mutations, and was futhe able to pase R222Q, V234M, and D344N as the most favoable of these nine mutations. We took this esult as evidence that PIPS is the most pomising appoach fo pedicting mutations that enhance neuaminidase suface-expessed potein o activity. Pediction of mutations that counteact the neuaminidase defect associated with H274Y in pandemic H1N1 We next used the PIPS computational appoach to pedict the top 12 candidates fo enhancing neuaminidase suface expession fom the entie set of possible mutations to the ectodomain of the pandemic H1N1 A/Califonia/4/2009 neuaminidase. These pedictions ae shown in Table 1. Plasmids wee constucted encoding epitope-tagged H274Y neuaminidases with each of these seconday mutations. Among the seconday mutations discussed above as enhancing the suface-expessed activity of seasonal H1N1 neuaminidase, D344N is aleady pesent in the pandemic H1N1 neuaminidase. The identities of esidues 222 Table 1. Top twelve PIPS pedicted neuaminidase mutations to pandemic H1N1. mutation N369K T289M V166A S366K P126N N386E V83M I389S G454N V106I R257K N221K PIPS pediction Top pedicted mutations to A/Califonia/4/2009 neuaminidase, excluding mutations not in cystallized ectodomain and only consideing the top pediction at each site. Mutations named in N1 numbeing scheme. doi: /jounal.pone t001 Figue 2. PIPS is the most effective computational appoach fo etospectively identifying the seconday mutations that inceased seasonal H1N1 neuaminidase suface expession and activity. The histogams show the distibution of pedicted effects fo all possible single amino-acid mutations to the A/New Caledonia/20/1999 neuaminidase, fo each of the fou computational appoaches (CUPSAT, FOLDX, the consensus appoach, and PIPS). The A/Bisbane/59/2007 stain contains nine mutations in the cystallized ectodomain potion of the neuaminidase elative to the A/New Caledonia/20/1999 stain. The thee mutations that wee expeimentally show to enhance neuaminidase suface expession o activity (R222Q, V234M, and D344N) ae indicated with ed squaes, while the othe six mutations ae indicated with geen cicles. The units fo the diffeent pediction methods ae abitay, but in all cases moe negative numbes coespond to mutations that ae pedicted to be moe favoable. Shown ae one-sided P-values fo the hypothesis that the pediction method assigns moe negative values to the known enhancing mutations (ed squaes) than the othe six mutations (geen cicles), as detemined using the Mann-Whitney test. The most successful computational appoach appeas to be PIPS, which coectly places all thee ed squaes to the left of all six geen cicles. doi: /jounal.pone g002 PLoS ONE 4 July 2011 Volume 6 Issue 7 e22201

5 and 234 in pandemic H1N1 ae aspaagine and valine, espectively. We theefoe also constucted plasmids with the seconday mutations N222Q and V234M. Each of these seconday mutations was tested fo its effect on the total amount of neuaminidase activity and potein expessed on the suface of tansfected cells (Figue 3). H274Y deceases suface-expessed activity and potein to less than half of wildtype levels. Seveal of the seconday mutations patially escued this defect, with the stongest effects being mediated by R257K, T289M, N369K, and V234M (N1 numbeing scheme). Othe seconday mutations had no effect, o even deceased neuaminidase suface expession, indicating that the computational pedictions ae impefect. Nonetheless, we consideed it heatening that combining the computational pedictions with a modest amount of expeimental sceening allowed us to identify seveal mutations of possible elevance. The two seconday mutations with the stongest effects wee R257K and T289M. We constucted plasmids encoding both mutations in the backgound of eithe wildtype o H274Y, and measued the total suface-expessed neuaminidase activity and potein (Figue 4). Combining both R257K and T289M with H274Y escued total suface-expessed activity to appoximately wildtype levels. In the absence of H274Y, these two mutations inceased total suface-expessed activity to levels 50% highe than wildtype. Inteestingly, in both backgounds, the effects of the R257K and T289M on the levels of suface-expessed potein wee substantially lage than those on activity. The potein levels fo the H274Y-R25K-T289M tiple mutant wee twice those of wildtype, while the levels fo the double mutant without H274Y wee five times highe than wildtype. This finding suggests that these seconday mutations eithe decease the pe-potein enzymatic activity, o cause a potion of the potein to each the cell suface in an inactive fom. Howeve, this effect is outweighed by the oveall incease in suface potein levels, such that the seconday mutations still enhance total suface-expessed activity. Seconday mutations eliminate the mild tissue-cultue gowth defect caused by H274Y in pandemic H1N1 To test the effects of the top candidate pemissive mutations on vial gowth, we used evese genetics to geneate pandemic H1N1 viuses caying GFP in the PB1 segment [8]. These viuses deived thei gene segments fom the A/Califonia/4/2009 stain, Figue 3. Seveal of the pedicted seconday mutations patially counteact the decease that H274Y causes in total sufaceexpessed activity and potein fo the pandemic H1N1 neuaminidase. Shown ae wildtype (WT) and indicated mutants of the A/Califonia/ 4/2009 neuaminidase. All neuaminidases contain C-teminal epitope tags, except fo the untagged WT. Fo the measuements, 293T cells wee tansfected with plasmids encoding the neuaminidase poteins. Afte 20 hous, the cells wee assayed fo the total suface-expessed neuaminidase activity (top panel) o potein using an antibody against the epitope tag (bottom panel). Bas show the mean and standad eo fo at least six eplicates. doi: /jounal.pone g003 PLoS ONE 5 July 2011 Volume 6 Issue 7 e22201

6 vaiants gew appeciably. But both the H274Y and H274Y- R257K-T289M vaiants gew as well as they had in the absence of oseltamivi. Theefoe, the seconday mutations do not geatly affect vial esistance to oseltamivi pe se, but may alleviate the slight tissue-cultue gowth defect caused by H274Y. Figue 4. Combining seveal seconday mutations can fully counteact the effect of H274Y on suface-expessed pandemic H1N1 neuaminidase activity. Shown ae wildtype (WT) and indicated mutants of the A/Califonia/4/2009 neuaminidase, all containing C-teminal epitope tags. Fo the measuements, 293T cells wee tansfected with plasmids encoding the neuaminidase poteins. Afte 20 hous, the cells wee assayed fo the total suface-expessed neuaminidase activity (top panel) o potein using an antibody against the epitope tag (bottom panel). Bas show the mean and standad eo fo at least six eplicates. doi: /jounal.pone g004 with the hemagglutinin containing the commonly occuing T197A mutation (which makes the sequence match that fom the vaccine stain A/Califonia/7/2009). We successfully escued viuses with wildtype, H274Y, R257K-T289M, and H274Y- R257K-T289M neuaminidases. We pefomed vial gowth assays in MDCK-SIAT1 cells that constitutively expessed the PB1 potein. As has been obseved in the majoity of pevious studies [24,27 30] with 2009 pandemic H1N1 stains, we found that H274Y caused a slight decease in vial gowth (Figue 5). Ou esults most closely esemble those obtained by [29] with the A/Califonia/4/2009 stain, with the H274Y vaiant gowing to slightly lowe tites at all timepoints, with a maximal diffeence of about 10-fold. Howeve, the H274Y-R257K-T289M and R257K-T289M vaiants gew to tites simila to wildtype (Figue 5), suggesting that these two seconday mutations may escue a slight attenuation in tissue-cultue gowth associated with H274Y. In the pesence of 50 nm oseltamivi, neithe the wildtype no the R257K-T289M Discussion We have investigated the possibility of pedicting seconday mutations that counteact the deceased neuaminidase suface expession associated with the H274Y oseltamivi esistance mutation in pandemic H1N1. We began with a etospective test to find the most effective computational appoach fo identifying mutations that enhanced total suface-expessed activity and potein among all of neuaminidase mutations that occued duing the divegence of 1999 and 2007 stains of seasonal H1N1. We then used this computational appoach to pedict 12 new candidate mutations to pandemic H1N1. Thee of these candidates (R257K, T289M, and N369K), as well as one of the seconday mutations fom seasonal H1N1 (V234M), patially escued the defect in suface-expessed neuaminidase activity and potein associated with H274Y in a 2009 pandemic H1N1 stain. Combining the two best candidates (R257K and T289M) with H274Y estoed total suface-expessed activity to wildtype levels. These two mutations also appeaed to escue the slight defect in tissue-cultue gowth associated with H274Y in pandemic H1N1. As discussed in the Intoduction, the question of whethe H274Y meaningfully attenuates pandemic H1N1 is a subject of continuing debate [24 31]. It theefoe emains unclea whethe the fact that H274Y pandemic H1N1 isolates have thus fa been evolutionay dead ends [22,23] is simply a matte of luck, o is because they ae less fit than thei oseltamivi-sensitive countepats. Ou esults cannot esolve this question, which will ultimately be answeed only by continuing to obseve the natual evolution of the vius. Howeve, ou esults do clealy demonstate that a measuable phenotype associated with H274Y in pandemic H1N1 a decease in the total amount of suface-expessed neuaminidase potein and activity has the potential to be counteacted by seconday mutations. Futhemoe, we have identified fou specific mutations (R257K, T289M, N369K, and V234M) with the potential to exet this effect. Note that this is unlikely to epesent an exhaustive list of all mutations that enhance neuaminidase suface expession, since we only expeimentally sceened 14 of the nealy 9,000 possibilities. Nonetheless, these fou mutations may be wothy of monitoing duing suveillance of pandemic H1N1. Regadless of the eventual fate of H274Y in pandemic H1N1, ou findings ae elevant to boade issues in potein evolution. We began this pape by descibing the bugeoning set of examples whee a mutation causes a beneficial phenotypic alteation only when it is paied with a seconday mutation. We futhe noted that these seconday mutations often act in a geneal manne by bolsteing a potein-level popety such as folding, stability, o expession, theeby alleviating defects caused by a vaiety of othe mutations [1,5,43 48]. The potential fo this phenomenon appeas to be pevasive in influenza neuaminidase, as evidenced by the existence of multiple seconday mutations that patially counteact the deceased suface expession caused by H274Y. The exact biophysical mechanism emains unclea, and is an impotant aea fo futhe eseach. Howeve, it is inteesting to note that the mutations ae scatteed about the neuaminidase potein stuctue (Figue 6), and so appea to be geneally pomoting suface expession athe than foming a specific stuctual inteaction with H274Y. PLoS ONE 6 July 2011 Volume 6 Issue 7 e22201

7 Figue 5. Gowth in tissue-cultue of pandemic H1N1 vaiants caying neuaminidase mutations. The plot at left shows gowth in media lacking oseltamivi, while the plot at ight shows gowth in media containing 50 nm oseltamivi. Viuses contain all genes fom the A/ Califonia/4/2009 stain with the T197A mutation to hemagglutinin, with the exception of the PB1 segment which is engineeed to cay GFP. MDCK- SIAT1-CMV-PB1 cells wee infected with the viuses at initial multiplicities of infection of 5 10 {4 infectious paticles pe cell. At the indicated times, vial supenatants wee havested and titeed on fesh cells. Shown ae the mean and standad eo fo fou eplicates. doi: /jounal.pone g005 It is the geneality of this buffeing of potein popeties that povides a basis fo the stategy we used to identify potentially impotant seconday mutations. The PIPS computational appoach is built on the idea that a single additive dimension captues the buffeing effects of mutations on the whole set of evolutionaily constained potein popeties. Clealy this is a sevee appoximation, since mutations can have complex effects on each of these popeties. But the appoximation captues enough of the tuth to be useful, since combining the esulting computational pedictions with a modest amount of expeimental sceening was sufficient to identify seconday mutations that indeed enhanced neuaminidase suface expession. Whethe any of these seconday mutations ae actually found to play a ole in inceasing the pemissiveness of pandemic H1N1 to oseltamivi esistance duing futue natual evolution will of couse be the tuest test of the pactical value of this appoach. Figue 6. Sites of the mutations mapped onto the neuaminidases potein stuctue. Shown in dak geen is one monome fom an N1 neuaminidase cystal stuctue ([37], PDB code 3BEQ]. Residue 274 (N2 numbeing) is shown in ed, and the sites of the seconday mutations (N1 numbeing) ae shown in blue. Oseltamivi (yellow sphees) is modeled in its binding site based on a elated cystal stuctue ([83], PDB code 2HU0). The othe thee monomes of the full neuaminidase tetame ae shown in light geen, based on modeling fom a elated cystal stuctue ([83], PDB code 2HU0). The image was endeed with PyMOL. doi: /jounal.pone g006 Mateials and Methods PIPS computational appoach fo pedicting seconday mutations The PIPS appoach that we used to pedict seconday neuaminidase mutations that might enhance neuaminidase suface expession is an impoved vesion of that descibed in [34]. The appoach is based on the idea that mutations fequently cause changes in potein-level popeties that ae unde evolutionay constaint, such as stability, folding o expession. Peviously [5,34,47,49], we cast the evolutionaily elevant popety solely as potein themodynamic stability, DG f. Howeve, in the couse of wok by ouselves [8] and othes [50,51], it has become inceasingly obvious that themodynamic stability is not always the potein-level popety unde the stongest evolutionay constaint. We will theefoe fomalize a cetain level of biophysical evasiveness by defining a vaiable F, epesenting an appoximate agglomeation of evolutionaily constained popeties such as themodynamic stability, kinetic stability, folding efficiency, esistance to aggegation, intacellula tafficking, etc. In this fomulation, F epesents the best one-dimensional pojection of all of these popeties, to which in pactice mutational effects ae fequently [50,52 56] but not always [45,57] coelated. Descibing each popety individually would be moe biophysically accuate, but would not be mathematically tactable in the appoach that follows. The ultimate justification fo a fomalism based on the biophysically appoximate vaiable F is expeimental validation of some of the esulting pedictions descibed hee and in [34]. Moe negative values of F coespond to bette potein popeties, while moe positive values coespond to wose popeties. We assume that evolution selects to maintain F below some theshold (chosen hee as zeo) to ensue that the potein adopts and maintains its folded confomation. Howeve, as long as F v0, selection is indiffeent to its exact value. When F 0, a potein is nonfunctional. Theefoe, a mutation that wosens potein popeties (inceases F) will not be toleated by a potein that has a maginal value of F (top panel of Figue 7A). But the same mutation is toleated by a potein with a lage magin in F (bottom panel of Figue 7A). This elationship between F and mutational toleance coesponds to the expeimental obsevation that moe stable poteins tend to be moe obust to mutations PLoS ONE 7 July 2011 Volume 6 Issue 7 e22201

8 Figue 7. Rationale fo assuming that the fixation pobability of a mutation depends on its effect on evolutionaily constained potein popeties. (A) Evolution is assumed to select in a theshold manne fo popeties such as folding, stability, o expession (appoximated by the vaiable F). A mutation deleteious to F will not be toleated by a potein that has a maginal value of F (top panel). But the same mutation is toleated by a potein with an exta buffe in F (bottom panel). (B) Most mutations ae deleteious to F, and theefoe have positive DF values. Shown is an example distibution of DF fo all mutations to a potein, taken fom [49]. (C) The time-aveaged pobability distibution of F fo an evolving potein will tend towads values just maginally below the theshold. Shown is an example of this distibution, taken fom [49]. (D) As a consequence, mutations with negative DF values will geneally be toleated, but those with positive DF ae less likely to be toleated. Shown is a plot of the elationship between the pobability fxy that mutating esidue fom y to x will be toleated as a function of the associated DF xy value, as defined in Equation 3. doi: /jounal.pone g007 [5,47,48], and the classic finding that cetain mutations can globally suppess the deleteious effects of many othe mutations by inceasing stability o folding efficiency [43 46]. Each mutation is associated with a DF value, which is the diffeence between the F of the mutated potein and the wildtype one. Most mutations wosen potein popeties, coesponding to an incease in F, o a positive DF value. Figue 7B shows a epesentative distibution of DF values fo all mutations to a potein. The time-aveaged pobability distibution of F fo an evolving potein is detemined by the balance between the selection pessue to maintain F v0 and the opposing pessue of mutations with mostly positive DF values. The exact distibution of F also depends on factos such as mutation ate, population size, and the specific DF values associated with that potein [49,58,59]. Howeve, the distibution will have the geneal featue that most of the time F is just maginally below the selection theshold of zeo. Figue 7C shows a epesentative time-aveaged pobability distibution of F. The foegoing facts lead to an obvious elationship between a mutation s DF value and the pobability that it will be fixed duing neutal evolution. Specifically, let DF xy be the change in F associated with mutating esidue fom y to x. Given the above assumptions, when DF xyƒ0, the mutation will always be selectively neutal, since it will neve push F ove the theshold of zeo. On the othe hand, when DF xyw0, the mutation will only be selectively neutal if the potein possess a sufficient buffe in F, which will be the case when F vdf xy. Given the time-aveaged distibution of F shown in Figue 7C, it is clea that mutations just slightly inceasing F will fequently be neutal, while mutations with vey lage DF xy will only aely be neutal. Let f xy be the pobability that the mutation is selectively neutal. The elationship between fxy and DF xy will have the geneal qualitative fom shown in Figue 7D. We will use this elationship to infe DF xy values fom the mutational histoies contained in potein phylogenies. Fo each esidue, we want to infe the set fdf xwt g ðþ of the DF xwt values fo mutating the esidue fom its wildtype (WT) identity to some othe esidue x. We will assume that the DF xwt values fo all esidues ae independent and additive, an assumption that although obviously impefect is nonetheless likely to fequently be easonable [60 65]. The specification of fdf xwt g ðþ allows fo calculation of abitay DF xy as DF xy ~DF xwt {DF ywt : ð1þ PLoS ONE 8 July 2011 Volume 6 Issue 7 e22201

9 The coesponding deivatives ae LDF xy LDF zwt 8 >< 1, if x~z=y ~ {1, if y~z=x >: 0, othewise: We have descibed fxy as the pobability that the mutation of esidue fom y to x goes to fixation at the neutal expectation. Hee we give an exact functional elationship between fxy and DF xy. We have chosen this functional fom abitaily, fo simple easons of mathematical convenience. Howeve, it captues the key qualitative attibutes discussed above. Specifically, we assume that ð2þ fxy ~ 1 2 { 1 2 tanh b DF xy { 1 2 ln c, ð3þ 1{c whee bw0 is a constant descibing the steepness of the cuve and c gives the value of fxy at DF xy ~0. We use a ange of g~20 and constain {gvdf xwtvg. We set c~0:8, and then choose b~0:265 so that 10 {4 ~ 1 2 { 1 2 tanh b g{ 1 2 ln c. 1{c Equation 3 is plotted in Figue 7D. The coesponding deivatives ae Lf xy LDF xy and so by the chain ule, Lf xy LDF zwt ~2bfxy f xy {1, ð4þ 8 2bfxy f xy {1 if x~z=y >< ~ {2bfxy f xy {1 if y~z=x >: 0 othewise: As in [34], define G as the matix with elements ð5þ 8 < f Gxy ~ xy c xy, if x=y { P fzy c zy, if x~y, ð6þ : z=y whee c xy is the pobability that a andom nucleotide mutation to a codon fo amino acid y changes this codon to be fo amino acid x. We efe to the set of all c xy values as C. Again using the chain ule, LG xy DF zwt 8 c xy 2bfxy f xy {1 if z~x=y {c xy 2bfxy f xy {1 if z~y=x >< ~ {c zy 2bfzy f zy {1 if x~y=z X c wy 2bfwy f wy {1 if x~y~z w=y >: 0 othewise: The pobability that a substitution changes esidue fom y to x afte an elapsed time t is given by element Mxy ðþof t the matix ð7þ M ðþdefined t by M ðþ~exp t ðutg Þ, ð8þ whee u is the pe codon mutation ate. Let D be the diagonal matix with enties equal to the eigenvalues of G, let S be the matix with columns equal to the ight eigenvectos of G, and let S {1 be the invese of S, so that G ~S D S {1 : ð9þ The matix M ðþis t conveniently computed as M ðþ~s t expðutd ÞS {1 : ð10þ The deivatives of M ðþae t given by [66] as whee the elements of V,z ae Vxy,z ~ 8 < : B,z xy LM ðþ t LDF ~S V,z S {1, ð11þ zwt ð exp utd xxþ{exp utd yy D xx {D yy B,z xx ut exp D xx ut if x=y if x~y, ð12þ whee D xx and D yy ae the diagonal elements of D epesenting the eigenvalues of G, and B,z xy ae the elements of the matix B,z defined by B,z ~S {1 LG LDF zwt S : ð13þ Let the pobability p x of finding esidue x at position in the long-time limit be given by element x of the vecto p. The vecto p epesents the stationay solution to Equation 8, and so is the pobability vecto (enties sum to one) that satisfies the eigenvecto equation p ~ ðizg Þp, ð14þ whee I is the identity matix. Given a value of G, the uniqueness of p is guaanteed by the Peon-Fobenius theoems, since IzG is a nonnegative and acyclic stochastic matix. The deivatives of p ae given by [67] as Lp LDF zwt ~{ ðg Þ # LG LDF p, zwt ð15þ whee ðg Þ # is the goup invese of G as descibed in [68]. In pactice, we want to infe fdf xwt g fom a phylogeny built fom a set of potein sequences. Let S~ s k j1ƒkƒn consists of N aligned homologous sequences of length L, with s k denoting the kth sequence. Fo each sequence s k, we know the identity s k of the amino acid at position (whee 1ƒƒL). The set of amino acid identities fo all N poteins at a single site is denoted by S ðþ ~ s k j1ƒkƒn. Let T be the phylogenetic tee giving the elationship among these sequences. The pobability of S given fdf xwt g, the set C of c xy values, the mutation ate u, and the tee PLoS ONE 9 July 2011 Volume 6 Issue 7 e22201

10 T, is the poduct of the pe-site likelihoods, P SjfDF xwt g,c,u,t L ~ P P S ðþ jfdf xwt g ~1 Fo the example tee in Figue 8, ðþ,c,u,t : ð16þ P S ðþ jfdf xwt g ðþ,c,u,t ~ X P A,C,D,E,F,x,y,z,wjfDF xwt g,c,u,t ð17þ : x,y,z,w Using the puning appoach of [69,70], P S ðþ jfdf xwt g ðþ,c,u,t ~ X! X p x Myx ð t 6Þ MAy ð t 1Þ MCy ð t 2Þ ð18þ x y " X Mzx ð t 8Þ MDz ð t 3Þ X #! Mwz ð t 7Þ MEw ð t 4Þ MFw ð t 5Þ : z w The deivatives of Equation 18 can be computed using the ecusive natue of the likelihood calculation. This is most easily seen by intoducing the notation whee L nð q Þ epesents the likelihood that node n has esidue q at position given all the data in the subtee ooted at node n. With this notation, Equation 18 is P S ðþ jfdf xwt g ðþ,c,u,t ~ X x p x L 9 ðxþ, ð19þ LL 9 ðxþ ~ LDF qwt X LMyx LDF y qwt "!# L 6 ðy LL ÞzM 6 ðyþ X yx z and X y LL 8 ðþ z LDF qwt! X Myx ð t 6ÞL 6 ðyþ z ~ LM Dzð t 3Þ LDF qwt LDF qwt LDF qwt z! Mzx ð t 8ÞL 8 ðþ z "!# LMzx L 8 ðþzm z LL 8 ðþ z zx,! X Mwz t y L 7 ðwþ z w LDF qwt " MDz ð t 3Þ X!# ð24þ LMwz ð t 7Þ LDF L 7 ðwþzm wzð t 7Þ LL 7 ðwþ w qwt LDF, qwt whee the deivatives of the Myx ae given by Equation 11. As discussed in [34], a pio pobability distibution can be specified fo each DF xwt value. These pios can intoduce specific biophysical knowledge as might be computed using molecula modeling pogams, o can simply seve a egulaizing ole [71] to avoid ovefitting the DF xwt values. The pios also enfoce the constaint that {gvdf xwtvg. We define the pio pobability distibutions as beta distibutions peaked at a pio estimate DF xwt,prior fo the DF xwt in question, and with the sum of the beta distibution a and b paametes equal to B sum, P DF xwt DF ~ xwt zg a{1 g{df b{1 xwt (23) Bða,bÞð2gÞ azb{1 ð25þ whee the likelihoods ae calculated ecusively down to the tee tips, so that fo example, L 9 ðxþ~ X y! Myx ð t 6ÞL 6 ðyþ X z Mzx ð t 8ÞL 8!, ðþ z ð20þ and L 8 ðþ~m z Dzð t 3Þ X w Mwz ð t 7ÞL 7!: ðwþ ð21þ Using this epesentation, LP S ðþ jfdf xwt g ðþ,c,u,t ~ X x LDF qwt Lp x DF qwt L 9 ðxþzp x LL 9 ðxþ LDF qwt! ð22þ whee the deivatives of the p x values ae given by Equation 15, and the deivatives of the likelihoods ae calculated ecusively, as fo example, Figue 8. An example phylogenetic tee T. This tee shows the sequence data S ðþ fo five sequences at a single site. The amino acid codes at the tips of the banches (A, C, D, E, and F) show the esidue identities fo the five sequences at this site. The vaiables at the intenal nodes (x, y, z, w) ae the amino acid identities at the site fo the ancestal sequences, and must be infeed. The numbes next to the nodes ae unique identifies fo the nodes. The banch lengths (t 1, t 2, ) ae popotional to the time since the divegence of the sequences. doi: /jounal.pone g008 PLoS ONE 10 July 2011 Volume 6 Issue 7 e22201

11 DF xwt, PRIOR zg ðb sum {2Þ whee B is the beta function, a~ 2g z1, and b~b sum {a. Note that DF xwt,prior must satisfy {gvd F xwt, PRIORvg. The deivative of Equation 25 is LP DF xwt LDF ~P DF xwt xwt a{1 b{1 DF { xwtzg g{df xwt : ð26þ The oveall pio pobability of the set of fdf xwt g ðþ of DF xwt values fo esidue is simply the poduct of the pio pobabilities fo the individual DF xwt values, P fdf xwt g ðþ so the deivative is LP fdf xwt g ðþ LDF xwt ~P fdf xwt g ðþ ~ P DF xwt P DF xwt, ð27þ a{1 b{1 { zg DF xwt g{df xwt : ð28þ Equations 16 and 18 povide a method fo computing P SjfDF xwt g,c,u,t. But goal is to infe the fdf xwt g, which is equivalent to computing P fdf xwt gjs. Using Bayes Theoem, P fdf xwt gjs ~ X P C,u,T P fdf xwt g,c,u,t fdf xwt gp fdf xwt g,c,u,t P SjfDF xwt g,c,u,t P SjfDF : ð29þ xwt g,c,u,t Rathe than solving fo all of the unknown vaiables, hee we will take the computational shotcut of using othe methods to assign fixed values to C, u, and T, so that P fdf xwt gjs P fdf xwt ~ g P SjfDF xwt g,c,u,t P fdf xwt g P fdf xwt g P SjfDF : xwt g,c,u,t ð30þ Futhemoe, athe than fully solving the ight-hand side of Equation 30 as might in pinciple be done using Makov-chain Monte Calo methods [72 74], we will simply compute the maximum a posteioi value fdf^ xwt g of fdf xwtg, defined as fdf^ xwt agmax g~ fdf xwt g P fdf xwt g P SjfDF xwt g,c,u,t :ð31þ Above we have povided equations fo all of the deivatives necessay to pefom this maximization using gadient-based techniques. Implementation of the computational appoach in the PIPS pogam A compute pogam that solves Equation 27 to infe fdf xwt g was witten in the Python pogamming language and given the name PIPS (Phylogenetic Infeence of Potein Stability), vesion 1.0. This pogam and the aw data fom the analyses descibed in this pape will be made feely available at bloom/. As input to the PIPS pogam, we used MUSCLE [75] to build a multiple-sequence alignment of all 3,731 unique full-length N1 neuaminidase potein sequences that wee available fo download fom eithe NCBI s Influenza Vius Resouce ([76], ncbi.nlm.nih.gov/genomes/flu/flu.html) o GISAID s EpiFlu Database ([77], as of June 21, The aligned sequences wee then used to build a neighbo-joining phylogenetic tee without a molecula clock, using the PHYLIP package [78]. This tee was used as input to the PIPS pogam. The PIPS pogam was used to compute the fdf xwt g values fo mutations to the neuaminidases fom the seasonal H1N1 stain A/New Caledonia/20/1999 and the pandemic H1N1 stain A/Califonia/4/2009. The pio pobability distibutions in Equation 22 wee set so that all mutations had pio estimates of D F xwt, PRIOR ~5, based on the idea that most mutations will be modeately deleteious to F. The value of B sum in Equation 22 was set to thee. The mutation biases given by C in Equation 6 wee calculated by assuming that each amino acid is equally likely to be encoded by any of its possible codons, and that nucleotide mutations occu with a tansition-to-tansvesion atio of fou. The value of u in Equation 8 was set to 10. The maximization in Equation 31 was pefomed using the conjugate-gadient algoithm. Although this algoithm is deteministic given specific stating values, thee may be local maxima. Theefoe, fo each esidue we pefomed five diffeent maximizations stating fom diffeent andomly chosen DF xwt values, and used the values that gave the highest a posteioi pobability as the final estimates. Running the pogam in this fashion gave the PIPS pedictions shown in Figue 2 fo the specified mutations to the neuaminidase fom A/New Caledonia/20/1999. Fo the mutations to the neuaminidase fom A/Califonia/4/2009, Table 1 lists the 12 mutations with the most negative pedicted DF xwt values, consideing only the best mutation fo each esidue and only esidues found in the ectodomain of the cystal stuctue of a closely elated N1 neuaminidase ([37], PDB code 3BEQ). CUPSAT, FoldX, and consensus pedictions We also used CUPSAT, FoldX, and the consensus appoach to pedict the effects of mutations to the A/New Caledonia/20/1999 (H1N1) neuaminidase, as shown in Figue 2. Text files giving all of these pedictions ae available along with the PIPS pogam and aw data that ae being made available at bloom/. CUPSAT and FoldX both take as thei input a potein s stuctue. We used the cystal stuctue fom PDB code 3BEQ [37], which is of the 1918 H1N1 influenza neuaminidase. This neuaminidase aligns to that of A/New Caledonia/20/1999 with no gaps and 89% potein identity ove the 385 esidues in the cystallized ectodomain. Fo the CUPSAT pedictions, this potein stuctue was submitted to the webseve tu-bs.de/cupsat/custompdb.htm to geneate pedictions fo all single mutations. Fo FoldX, we made the pedictions using the FoldX executable vesion 3.0 beta 4 fo Mac OS X, as downloaded fom The FoldX RepaiPDB function was fist un to efine the PDB stuctue. The pedictions wee then made using the default paametes and the Position- Scan function. Fo the 89% of the esidues in which the A/New Caledonia/20/1999 neuaminidase sequence exactly matched that in the 3BEQ cystal stuctue, the pedicted mutational effects wee simply the pedictions fo that mutation. Fo esidues that diffeed between the two sequences, the pedicted mutational PLoS ONE 11 July 2011 Volume 6 Issue 7 e22201

12 effect was calculated as the pedicted effect of mutating the PDB esidue to the taget amino acid minus the pedicted effect of mutating the PDB esidue to the A/New Caledonia/20/1999 esidue. Fo both CUPSAT and FoldX, highly destabilizing mutations (values geate than the leftmost histogam ba shown in Figue 2) ae counted in this last ba to avoid having to damatically expand the x-axis of the plot in the positive diection. Fo the consensus pedictions, we used the same sequence data set of 3,731 full-length N1 neuaminidases that is descibed above fo the PIPS pogam. The pedicted effect of mutating a esidue fom amino acid x to y was calculated as ln N yz1 N x z1 whee N x and N y ae the numbe of sequences that have amino acids x and y at that position, espectively. The one in the fomula epesents a single pseudocount added to each sequence tally to avoid undefined values fo mutations to esidues that ae not pesent in the natual sequence alignment. Neuaminidase suface expession and activity assays To test the effect of the pedicted pemissive mutations on the levels of suface-expessed neuaminidase activity and potein, we ceated plasmids encoding vaious mutants with C-teminal HA epitope tags. Each neuaminidase potein-coding sequence was diectly fused to the epitope tag (YPYDVPDYA) and inseted into a plasmid (HDM) containing a CMV pomote and 59 EcoRI/39 NotI cloning sites, followed by an intenal ibosome enty site (IRES) expessing the mchey ed fluoescent potein. As was peviously obseved [8], the addition of the C-teminal epitope tag led to at most a slight (less than 10%) decease in the total sufaceexpessed neuaminidase activity elative to an untagged vaiant (Figues 1 and 3), indicating that the tag did not substantially alte the potein o activity levels. Plamids wee constucted fo all of the mutants of the A/New Caledonia/20/1999 neuaminidase shown in Figue 1 and all of the mutants of the A/Califonia/4/ 2009 neuaminidase shown in Figues 3 and 4. In the naming of the mutations, H274Y was named in the N2 numbeing scheme to adhee to histoical convention this is actually esidue 275 in sequential numbeing of the N1 neuaminidase. All of the othe mutations ae named accoding sequential N1 neuaminidase numbeing. Fo the assays, the plasmids wee tansfected into 293T cells in 12-well dishes that had been seeded at unifom densities of cells pe well. At 20 hous post-tansfection, the cells wee collected using a vey bief teatment with EDTA-typsin, and esuspended in an isotonic assay buffe at ph 7.4, consisting of 15 mm MOPS, 145 mm sodium chloide, 2.7 mm potassium chloide, 4.0 mm calcium chloide, and 2% heat-inactivated fetal bovine seum. A faction of these cells (5% of the total numbe collected pe well) wee then assayed fo the total neuaminidase activity expessed on the cell suface using the fluoogenic MUNANA assay. Fo this assay, the cells wee incubated with 0.1 mm MUNANA (Sigma M8639) in a total volume of 150 ml in black 96-well plates at 37 0 C fo 45 minutes. The eactions wee quenched by adding 100 ml of 150 mm sodium hydoxide in 84% ethanol. The fluoescence was ead using a Tecan Safie 2 plate eade (excitation 360 nm, slit width 5 nm; emission 448 nm, slit width 20 nm). The activities wee quantified as the fluoescence above the backgound fom untansfected cells, nomalized by the faction of cells tansfected with the plasmid as detemined by flow cytomety fo mchey fluoescence as descibed below. Each ba fo the activity measuements in Figues 1, 3, 4 epesents the mean and standad eo fo at least six individual measuements. A emaining faction of the cells wee stained with a fluoescently conjugated antibody against the epitope tag (Santa Cuz Biotechnology, HA pobe F-7 Alexa-Fluo 647 conjugate, sc AF647, 1:100 dilution). The stained cells wee analyzed by flow cytomety to detemine the faction of cells expessing the mchey potein (these ae the cells tansfected with the plasmid), and the mean signal fom the antibody staining among these mchey positive cells. The staining signal above backgound was assumed to be popotional to the amount of neuaminidase potein on the cell suface. Each ba fo the stain measuements in Figues 1, 3, 4 epesents the mean and standad eo of at least six individual measuements. Vial gowth assays Revese genetics plasmids fo the A/Califonia/4/2009 H1N1 stain wee constucted by using evese-tansciptase PCR to amplify the genome segments fom total RNA extacted fom vius obtained fom the Biodefense and Emeging Infections Resouce Repositoy (BEI Resouces, catalog numbe NR-13658). The hemagglutinin gene fo A/Califonia/4/2009 was modified by adding the T197A mutation, since this mutation is pesent in the majoity of 2009 pandemic H1N1 isolates including the A/ Califonia/7/2009 vaccine stain, and has been epoted to aid in vius escue by evese genetics [79]. The gene segments wee cloned into the BsmBI sites of the bidiectional RNA polymease I/polymease II cassette plasmid phw2000 [80], which was kindly povided by Robet Webste of St. Jude Childen s Reseach Hospital. Mutations to the neuaminidase wee intoduced by site-diected mutagenesis. Viions caying GFP in the PB1 segment wee escued as descibed in [8]. Biefly, the plasmid phh-pb1flank-egfp encodes a vial RNA with the untanslated egions and 80 teminal coding nucleotides fom each end of the PB1 gene segment fom A/WSN/33 influenza, with potential stat codons mutated. This plasmid and the evese genetics plasmids fo the othe seven influenza segments (PB2, PA, HA, NP, NA, M, and NS) wee co-tansfected into a co-cultue of 293T (ATCC CRL11268) and MDCK-SIAT1 ([81], HPA Cultues ) cells that constitutively expessed the A/WSN/33 PB1 potein unde a CMV pomote (293T-CMV-PB1 and MDCK-SIAT1- CMV-PB1 cells), with the PB1-F2 peptide eliminated by intoduction of a stop codon in the manne descibed by [82]. At 12 hous post-tansfection, the cells wee washed once with PBS and the media changed to influenza gowth media (Opti- MEM I supplemented with 0.3% bovine seum albumin, 0.01% heat-inactivated fetal bovine seum, 100 U/ml penicillin, 100 mg/ml steptomycin, and 100 mg/ml calcium chloide) containing 3 mg/ml TPCK-teated typsin. Afte anothe 60 hous, at which point essentially all cells had tuned geen and wee undegoing visible cytopathic effect, the viuses wee havested by filtation though a 0.45 mm filte. The viuses wee titeed by infecting MDCK-SIAT1-CMV-PB1 cells in influenza gowth media, and then quantifying the pecentage of GFP positive cells at 15 hous post-infection using flow cytomety. Each vius vaiant (wildtype, H274Y, R257K-T289M, and H274Y-R257K-T289M neuaminidase) was escued and titeed in duplicate. Fo the gowth assays, MDCK-SIAT1-CMV-PB1 cells wee seeded in 6-well dishes so that they wee at cells pe well at the time of vial infection. Immediately befoe infection, the medium was changed to 3 ml of influenza gowth media plus 3 mg/ml TPCK-typsin. Some wells also contained 50 nm oseltamivi caboxylate (kindly povided by J. Smith and A. Pein of F. Hoffmann-La Roche), as indicated in Figue 5. Each well was then infected with an amount of vius equal to 300 infectious paticles accoding to the flow cytomety titeing. At the time points indicated in the figues, supenatant was collected and the PLoS ONE 12 July 2011 Volume 6 Issue 7 e22201