Restrictions to the Adaptation of Influenza A Virus H5 Hemagglutinin to the Human Hos

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http://jvi.asm.org/cgi/content/full/78/1/502


Restrictions to the Adaptation of Influenza A Virus H5 Hemagglutinin to the Human Host

Ruth Harvey,¹^,2^, Andrew C. R. Martin,¹ Maria Zambon,² and Wendy S. Barclay¹^* School of Animal and Microbial Sciences, University of Reading, Reading RG6 6AJ,¹ Health Protection Agency, Enteric and Respiratory Virus Laboratory, Colindale, London NW9 5HT, United Kingdom²
Received 30 June 2003/ Accepted 17 September 2003
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Abstract
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The binding specificities of a panel of avian influenza virussubtype H5 hemagglutinin (HA) proteins bearing mutations atkey residues in the receptor binding site were investigated.The results demonstrate that two simultaneous mutations in thereceptor binding site resulted in H5 HA binding in a patternsimilar to that shown by human viruses. Coexpression of theion channel protein, M2, from most avian and human strains testedprotected H5 HA conformation during trafficking, indicatingthat no genetic barrier to the reassortment of the H5 surfaceantigen gene with internal genes of human viruses existed atthis level.
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Influenza A viruses with at least 15 antigenically distincthemagglutinin (HA) proteins and nine different neuraminidasesubtypes can be isolated from avian species (29). Some of themolecular changes which occur when an avian influenza virusadapts to a new host (e.g., humans), such as changes in receptorrecognition and mutations in polymerase protein PB2, have alreadybeen characterized (11, 15, 24, 29), but the genetic restrictionswhich define the species barrier are still not fully understood.The consequence of the adaptation of a new antigenic subtypefor replication and transmission in humans is pandemic influenza,which carries a heavy morbidity and mortality toll.
A major determinant of host range is the affinity of the viralHA protein for the host cell sialic acid (SA) receptor. In thenatural avian host, SA is joined to the sugar chain throughan

2

3 linkage, and viruses isolated frombirds possess HAs with high affinity for this type of sugar.On the other hand, in the human respiratory tract, terminalSA is linked through an

2

6 bond. Virusescirculating in humans have acquired mutations in their HAs whichresult in the loss of affinity for

2

3 SAand the concomitant increase in

2

6 binding(4, 21, 28). This has been particularly well characterized forthe H3 subtype, which crossed from ducks into the human populationin 1967 and 1968 and caused the 1968 (Hong Kong) influenza pandemic.HAs from viruses isolated early in the pandemic differed fromtheir avian progenitors by a change from glutamine to leucineat residue 226 and from glycine to serine at residue 228 inthe HA receptor binding site (RBS) (15). This may representthe minimum change necessary for the H3 subtype to establishitself in the new human host.
In 1997, a highly virulent H5N1 virus spread to live poultrymarkets in Hong Kong. Eighteen people became infected, and sixdeaths resulted (3). The viruses recovered from these individualswere identical in all eight RNA segments to those isolated fromthe chickens at the same time, indicating for the first timethat a virus seemingly unadapted for mammalian replication couldreplicate in humans (1, 25). However, these avian viruses didnot transmit between humans and this may be why a pandemic didnot ensue.
The crystal structure of an H5 HA from A/Duck/Singapore/3/97virus, which is closely related to the HAs of the viruses isolatedin Hong Kong in 1997, such as the primary human isolate A/HK/156/97,has been determined (9). The width of the receptor binding pocketis less than that for the previously studied human H3 HA protein.It is hypothesized that changes at residues 226 and 228 mayallow H5 HA to better interact with the human form of the SAreceptor. We have used cloned H5 HA proteins to test whethersuch mutations indeed result in human-receptor binding characteristics.
The full-length cDNA encoding the H5 HA protein from the humanindex isolate of the outbreak, A/HK/156/97, was amplified byreverse transcription and PCR from viral RNA and cloned intothe expression plasmid pcDNA3 (2). A series of mutations inthe H5 HA cDNA which altered the nucleotides encoding the RBS,specifically at residues 226, 227, and 228, were engineered.At position 226, the mutations changed glutamine to either leucineor valine (the Q226L and Q226V mutants), and at residue 228,glycine was changed to serine (the G228S mutant). Changes atresidues 226 and 228 were also generated in combination to createLSS and VSS mutants. We also generated mutation S227I. Thischange was present in a subset of the H5 viruses isolated fromhumans in Hong Kong (11).
A hemadsorption assay was used to measure red blood cell (RBC)binding to exogenously expressed H5 HA. Following transfectionof Vero cells with 1 ?g of appropriate plasmids and 3?l of Lipofectamine and infection with a recombinant fowlpoxvirus (FPV)-expressing T7 RNA polymerase, cells were treatedwith 5.5 mU of bacterial neuraminidase/ml for 1 h (6). Thistreatment was necessary because the sugar modifications on theHA protein itself are sialylated and, in the absence of viralneuraminidase, the SA will block access to the RBS (19). Itwas also necessary to coexpress the HK156 M2 protein with HK156HA in order to facilitate cell surface transport of the functionalprotein. A 0.2-?g amount of the appropriate M2 expressionplasmid was cotransfected with each of the HA mutants.
We measured the binding of HA to different linkages of SA byanalyzing differential levels of binding to erythrocytes ofdifferent species which vary in the types of SA they display.Horse RBCs exclusively display

2

3 terminallylinked SA, whereas guinea pig RBCs predominantly display SAof the

2

6 linkage and express approximately1/10 of their SA in the

2

3 linkage (13,16; R. Harvey and W. S. Barclay, unpublished data). Accordingly,cells infected with apathogenic H5 avian virus, A/Duck/Singapore/3/97,bound horse RBCs efficiently and also hemadsorbed smaller amountsof guinea pig RBCs, possibly via the

2

3SA which they display. On the other hand, cells infected withthe human H3 subtype strain A/Sydney/5/97 bound only small amountsof horse RBCs but bound larger amounts of guinea pig RBCs dueto specific binding of

2

6 SA (Fig. 1A).Each of the H5 HA mutants was assessed for its ability to hemadsorbhorse or guinea pig RBCs (Fig. 1B). By using fluorescence-activatedcell scanning analysis of cell surface expression, we confirmedthat the mutations did not significantly alter the ability ofthe HA proteins to be transported to the plasma membrane (datanot shown). As expected, HK156 wild-type HA showed a typicalavian-virus-like pattern of efficient binding to

2

3-linkedSA. The S227I or Q226V changes did not affect receptor binding.The Q226L and G228S changes each decreased the binding to horseRBCs without any change in the binding of guinea pig RBCs. TheLSS and VSS double mutants showed a considerable loss in theirability to hemadsorb horse erythrocytes, and in the case ofthe LSS mutations, this was not at the expense of overall SAaffinity since binding to guinea pig RBCs was high. In fact,the LSS mutant behaved very much like the human-adapted H3 HAprotein of A/Sydney/5/97 virus that was used as a control inthis assay (Fig. 1A).
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FIG. 1. Hemadsorption of horse or guinea pig erythrocytes by avian or human influenza A virus HA. (A) Vero cells were infected with either A/Duck/Singapore/97 (D/S) or A/Sydney/5/97 (SYD) at a multiplicity of infection of >1. Twenty-four hours postinfection, a 0.5% suspension of horse or guinea pig erythrocytes was added for 1 h at room temperature. Following washing with phosphate-buffered saline, Vero cells and bound erythrocytes were lysed. The absorbance of the clarified lysate was measured at 540 nm. (B) Vero cells were infected with FPV directing the expression of T7 polymerase and were then transfected with plasmids directing the expression of mutant H5 HA (1 ?g) and HK156 M2 (0.2 ?g) proteins. Forty-eight hours posttransfection, cells were treated with 5.5 mU of bacterial neuraminidase (Vibrio cholerae sialidase; Roche) per ml for 1 h. Then a 0.5% suspension of horse or guinea pig erythrocytes was added, and after 1 h of incubation at room temperature, cells were washed and lysed for absorbance measurement. Cells infected with FPV T7 but untransfected were processed in the same way, and the mean absorbance from these wells was subtracted as background. All transfections were performed in triplicate, and mean absorbance values with standard errors are shown. WT, wild type.
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For Fig. 2, we used MODELLER (22) to model the mutations ontothe structure of the A/Duck/Singapore/3/97 H5 HA protein. Acomparison of panels B to F with the wild-type structure inpanel A shows the increase in the width of the RBS pocket, whichis predicted to allow greater access of the more bulky

2

6SA linkage and concomitantly decrease the stabilizing interactionsof HA with the

2

3 SA-conjugated receptor.The latter effect may be important for the resistance of virusto naturally occurring inhibitors in human sera and respiratorysecretions which display

2

3-linked SA.Ha et al. (10) have recently described the structure of an avianH3 HA that may be a progenitor to human H3 strains. A comparisonwith the human H3 structure shows that there are structurallysignificant differences in the human HA RBS that cause the pocketto open. Moreover, the Q226L and G228S substitutions are alsopredicted to cause some rearrangement of other residues in theRBS. Whether similar rearrangements occur in the H5 HA to accompanythe 226 and 228 mutations we have engineered in this study couldonly be addressed by a crystallization of mutant H5 proteins. <!-- null -->



FIG. 2. Modeling of point mutations into the receptor binding site of H5 HA. (A) Wild type; (B) G228S mutant; (C) Q226L mutant; (D) Q226V mutant; (E) LSS double mutant; (F) VSS double mutant. Native residues 226Q, 227S, and 228G are shown in orange, pink, and yellow, respectively. Mutations to a hydrogen bonding residue (serine) are shown in red, and mutations to a hydrophobic residue (leucine-valine) are shown in blue.
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Matrosovich et al. (15) identified human isolates from earlyin the Asian influenza pandemic of 1957 that still carried theavian-virus-like residues at positions 226 and 228. Other strainsfrom that year contained just one of the two changes associatedwith human viruses, the Q

L mutation at position 226, and alreadyshowed altered receptor binding characteristics. By 1958, only1 year into the pandemic, simultaneous humanizing mutationsat residues 226 and 228 were fixed in the H2 human virus lineage.This example serves to illustrate the proposition that receptorbinding changes are not a prerequisite for early zoonotic eventsbut are strongly selected for during the circulation of newsubtypes of viruses in humans. Applying this proposition tothe H5 situation, it can be hypothesized that if the H5 viruseswhich transmitted to humans in 1997 had continued to circulate,changes at HA residues 226 and 228 might have been selected.The data presented in this study illustrate that the H5 proteincan tolerate these changes and that they do alter the characteristicsof the protein towards those of an HA with higher affinity forhuman receptors. It is likely that the changes occur in a stepwisefashion, as reported for the above-described H2 example, andwe show here that both single changes Q226L and G228S affect

2

3 SA binding. It is possible that themutations we have introduced into H5 HA would not be toleratedin the context of infectious virus. Previously, it has beenshown that changes in the RBS can affect the pH of fusion ofHA (5). To fully establish the viability and characteristicsof viruses carrying these mutations, it would be necessary tointroduce them into recombinant viruses by using reverse-geneticstechniques (7, 17). However, such viruses would require isolationand handling under high containment conditions, which limitseasy experimental confirmation. It has recently been suggested that certain internal gene productsfrom human viruses, such as the matrix protein M1 and/or theion channel protein M2, encoded on RNA segment 7, and certainavian HA subtypes are incompatible (23). Reassortment with HAgenes like the HK/97 H5 HA is even more restricted than forother HAs because of a string of basic amino acids at the HA1/HA2cleavage site which render the HA susceptible to cleavage byubiquitous intracellular proteases, such as furin. Althoughthis susceptibility to cleavage increases the tissue tropismof the virus, it also places the newly synthesized HA at riskfor premature fusogenic conformational change. This possibilityis avoided by viral expression of an ion channel protein, M2,which allows protons to flow out from the late Golgi apparatus,thus maintaining this compartment at a higher pH than usual(18, 27). It was previously shown that the potency of the M2ion channel varies among virus strains and that some reassortantsare not viable due to an incompatibility between the gene productsof segment 4, encoding HA, and segment 7, encoding M2 (8). Inaddition, functional transport of the multibasic H7 RostockHA protein required four times as much of the human A/Udorn/72M2 ion channel as of the homologous Rostock avian M2 protein(27). We therefore asked whether human M2 proteins from strainscirculating in 1997 and later could facilitate transport offunctional multibasic H5 HA to the mammalian cell surface.
We cloned the M2 open reading frames from avian and human viralsources into pcDNA3 and cotransfected equal concentrations (0.2?g) of plasmid DNA with 1 ?g of wild-type HK156H5 HA plasmid. We used M2-specific antisera to check that thelevels of expression of each M2 protein were similar (Fig. 3A and B).Although it is not possible to make a complete comparisonbecause different antibodies were needed to analyze the expressionof human or avian M2 proteins due to differences in their sequences,the results showed that all M2 proteins were expressed welland that within the two sets analyzed, expression levels wereequivalent. Significantly, the two human M2 genes cloned fromH3N2 viruses circulating in 1997 and 1999, A/Sydney/5/97 andA/Panama/99, were as effective in this assay as the homologousM2 ion channel derived from the human index isolate HK156 orthe avian virus M2 gene cloned from A/Duck/Singapore/3/97 virus(Fig. 3C). To confirm that the ion channel activity was indeedresponsible, we repeated each assay in the presence of amantadine,a drug that inhibits the ion channel activity of M2. Followingtransfection of the two HA and M2 plasmids, 5 ?M amantadinewas added and the cells were incubated for a further 48 h beforethe hemadsorption assay was performed. The facilitative effectof M2 on HA transport was inhibited (Fig. 3C). The ability ofhuman M2 protein to support H5 HA transport is significant becauseit indicates that there was no genetic barrier operating atthis level to prevent the generation of an avian-human reassortantvirus containing segment 4 RNA, encoding a novel HA subtype,mixed with a human segment 7 RNA.
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FIG. 3. (A and B) Human and avian virus M2 protein expression. Forty-eight hours following transfection of 0.2 ?g of plasmid DNA, cells were incubated with primary antibodies against M2. A mouse monoclonal antibody (14C2) was used to detect the human M2 proteins (A), and a rabbit polyclonal antiserum raised against a swine influenza virus M2, kindly provided by P. Heinen, was used to detect the avian M2 proteins (B). Fluorescein isothiocyanate-conjugated secondary antibody was used, and the mean fluorescence intensity of each sample was measured by fluorescence-activated cell sorter analysis. (C) Representation of the ability of different M2 proteins to support hemadsorption by H5 HA. Vero cells were transfected with 1 ?g of plasmid directing the expression of HK156 HA and 0.2 ?g of plasmid expressing different avian or human viral M2 proteins and incubated either with (grey bars) or without (black bars) 5 ?M amantadine. Forty-eight hours posttransfection, cells were treated with 5.5 mU of bacterial neuraminidase (V. cholerae sialidase) per ml, and a 0.5% suspension of horse erythrocytes was added. After 1 h of incubation at room temperature, cells were washed and lysed for absorbance measurement at 540 nm. Cells infected with FPV T7 but untransfected were processed in the same way, and the mean absorbance from these wells was subtracted as background. All transfections were performed in triplicate, and mean absorbance values with standard errors are shown. D/S, A/Duck/Singapore/3/97; 156, A/HK/156/97; Pan, A/Panama/99; Syd, A/Sydney/5/97.
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Interestingly, we found that WSN M2 was much less active insupporting the functional transport of H5 HA in the hemadsorptionassay even though it was expressed to the same amount as theother human virus M2 channels. This finding suggests that theion channel activity of this protein is low. The WSN M2 proteinhas an S-to-N change at residue 31, which is associated withresistance to the drug amantadine. However, it should be statedthat amantadine resistance does not necessarily result in theloss of ion channel activity since several strains of avianinfluenza virus with multibasic HA cleavage sites have acquiredmutations which lead to drug resistance (reviewed in reference12). We did not determine the levels of activity of differentM2 proteins. However, bearing in mind that M2 is highly expressedin the infected cells and that we coexpressed M2 from only one-fifththe amount of plasmid DNA used for HA expression in these studies,it is not likely that our results have been affected by theoverexpression of M2. It is possible that by transfecting increasedamounts of the WSN M2 protein, H5 HA transport would be rescued,but how these levels of M2 and HA expression relate to virusinfection is not clear. It is already known that some M2 ionchannels are not active enough to support the transport of multibasicHA in a viral context (8). It might be important to considerthis during the designing of recombinant viruses to be usedas pandemic vaccines: several groups have recently describedthe recovery of viruses containing human internal proteins andavian H5 HA genes (14, 26) with the multibasic site H5 HA removed(because of obvious safety considerations). Since influenzavirus has a known capacity to acquire mutations and insertionsaround this region (20), coupling such engineered HA genes withan incompatible M2 protein may limit the risk of reversion duringvaccine propagation. The hemadsorption assay that we have presentedhere is a functional test for monitoring the ability of exogenousproteins to mediate changes in the pHs of intracellular compartmentsand assist in the transport of viral membrane proteins. As such,it may prove useful for the analysis of potential proton channelactivity bestowed by different M2 proteins as well as by otherviral or host cell proteins. 
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We thank Mike Skinner, Institute for Animal Health, Compton,United Kingdom, for the gift of FPV T7 recombinant virus; R.A. Lamb, Northwestern University, for the gift of 14C2; PaulHeinen, formerly of ID Lelystad and currently of the HealthProtection Agency, for the gift of antiserum to avian M2 proteins;and James White, University of Reading, for assistance withestablishment of the hemadsorption assay.
R.H. was a recipient of a PHLS studentship.
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<!-- null --> * Corresponding author. Mailing address: School of Animal and Microbial Sciences, P.O. Box 228, Whiteknights, University of Reading, Reading RG6 6AJ, United Kingdom. Phone: 44 1189 316368. Fax: 44 1189 316671. E-mail: w.s.barclay@reading.ac.uk<script type="text/javascript"><!-- var u = "w.s.barclay", d = "reading.ac.uk"; document.getElementById("em0").innerHTML = '<a href="mailto:' + u + '@' + d + '">' + u + '@' + d + '<\/a>'//--></script>.
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</sup> Present address: Sir William Dunn School of Pathology, Universityof Oxford, Oxford OX1 3RE, United Kingdom.
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<hr> [SIZE=-1] Journal of Virology, January 2004, p. 502-507, Vol. 78, No. 1
0022-538X/04/$08.00+0 DOI: 10.1128/JVI.78.1.502-507.2004
Copyright ? 2004, American Society for Microbiology. All Rights Reserved. [/SIZE]