Changes in H5N1 influenza virus hemagglutinin receptor binding domain affect systemic spread Hui-Ling Yena, Jerry R. Aldridgea, Adrianus C. M. Boona, Natalia A. Ilyushinaa, Rachelle Salomona, Diane J. Hulse-Posta, Henju Marjukia, John Franksa, David A. Boltza, Dorothy Bushb, Aleksandr S. Lipatova, Richard J. Webbya, Jerold E. Rehgb, and Robert G. Webstera,1 aDivision of Virology, Department of Infectious Diseases and bDepartment of Pathology, St. Jude Children’s Research Hospital, Memphis, TN 38105 Contributed by Robert G. Webster, November 6, 2008 (sent for review August 14, 2008) The HA of influenza virus is a receptor-binding and fusion protein that specimens outside the respiratory tract, including blood, cere- is required to initiate infection. The HA receptor-binding domain brospinal fluid, intestine, feces, placenta, and a fetus (16–20). determines the species of sialyl receptors recognized by influenza The mechanism of systemic spread of H5N1 viruses in mammals viruses. Here, we demonstrate that changes in the HA receptor- has not been established, although detection of virus in blood binding domain alter the ability of the H5N1 virus to spread system- (18, 21) or nerves (22) suggests possible routes. ically in mice. The A/Vietnam/1203/04 (VN1203) and A/Hong Kong/ We and others have found that A/Vietnam/1203/04 (VN1203) 213/03 (HK213) viruses are consistently lethal to domestic chickens virus, whose HA recognizes synthetic ␣2,3-linked sialyl receptor, is but differ in their pathogenicity to mammals. Insertion of the VN1203 highly pathogenic in the ferret and mouse models, whereas A/Hong HA and neuraminidase (NA) genes into recombinant HK213 virus Kong/213/03 (HK213) virus, whose HA recognizes both ␣2,3- expanded its tissue tropism and increased its lethality in mice; con- linked and ␣2,6-linked synthetic sialyl receptors, is less lethal (8, 9, versely, insertion of HK213 HA and NA genes into recombinant 23, 24). Surface glycoproteins have also been reported to play a role VN1203 virus decreased its systemic spread and lethality. The VN1203 in the pathogenicity of H5N1 viruses isolated in Hong Kong in 1997 and HK213 HAs differ by 10 aa, and HK213 HA has shown greater (11, 25), H7N7 virus isolated in the Netherlands (26), and the H1N1 binding affinity for synthetic ␣2,6-linked sialyl receptor. Introduction 1918 pandemic virus (27). We hypothesized that changes in the HA of an S227N change and removal of N-linked glycosylation at residue receptor binding domain might modulate the virulence of H5N1 158 increased the ␣2,6-binding affinity of VN1203 HA. Recombinant viruses in mammals by affecting the spread of virus from the VN1203 virus carrying the S227N change alone or with the residue- respiratory tract to other tissues. 158 glycosylation site removed showed reduced lethality and sys- temic spread in mice but not in domestic chickens. Wild-type VN1203 Results virus exhibited the greatest efficiency in systemic spread after intra- VN1203 HA and Neuraminidase Increase the Virulence of HK213 in muscular inoculation and in infection of mouse bone marrow-derived Mice. The VN1203 virus spreads systemically in inoculated mice and dendritic cells and conventional pulmonary dendritic cells. These ferrets whereas the HK213 virus is detected largely in their respi- results show that VN1203 HA glycoprotein confers pathogenicity by ratory tracts (8, 9, 23, 24). To determine whether the HA and facilitating systemic spread in mice; they also suggest that a minor neuraminidase (NA) of VN1203 contribute to its systemic spread, change in receptor binding domain may modulate the virulence of we first investigated the tissue tropism of HK213 virus carrying H5N1 viruses. VN1203 HA or VN1203 HA and NA. HK213, HK213xVN1203HA, and HK213xVN1203HANA recombinant viruses grew to compara- dendritic cell ͉ H5N1 ͉ pathogenesis ͉ mice bletitersinvitro(Table S1), and one 50% mouse lethal dose (MLD50) in BALB/cJ mice was 4,295, 413.3, and 41.8 pfu, respec- he HA glycoprotein of influenza A virus is a type I transmem- tively. Therefore, the lethality of HK213 virus was increased one log Tbrane protein that binds to sialic acid residues on target-cell by inserting VN1203 virus HA and two logs by inserting VN1203 glycoproteins or glycolipids (1). The receptor-binding domain is HA and NA. Furthermore, Ϸ40% of mice inoculated with located at the HA membrane distal end, where the 130-loop, HK213xVN1203HA or HK213xVN1203HANA virus, but none of the 220-loop, 190-helix, and other conserved residues (Y98, W153, mice inoculated with HK213, developed neurological signs (hind H183, Y195) form the receptor-binding site (1, 2). HA is a limb paralysis or convulsion). The lung titers of HK213 and host-range determinant, because human and avian influenza virus HK213xVN1203HANA virusesdifferedonday1(P ϭ 0.0174, HAs preferentially recognize ␣2,6- and ␣2,3-linked sialyl receptors, Tukey’s test) but were comparable on days 3, 5, and 7 after respectively (3, 4). In addition to the terminal sialic-acid linkages, inoculation (P ϭ 0.0802, 2-way ANOVA) (Fig. 1A). internal linkages, as well as fucosylation, sulfation, and sialylation at HK213xVN1203HA and HK213xVN1203HANA viruses were de- the inner oligosaccharides, also determine receptor-binding affinity tected in brain or blood, suggesting that infection was not contained and specificity (2, 5). Despite their preferential recognition of in the mouse lungs (Fig. 1A). The results suggest that the HA ␣2,3-linked sialyl receptors, highly pathogenic H5N1 avian influ- glycoproteins of VN1203 contribute to viral spread from the enza viruses are able to infect humans and cause severe disease. respiratory tract. Glycan-array profiling has shown differences between seasonal human influenza virus and H5N1 viruses (2, 6), but current techniques are at an early stage to map the glycans in tissues, and Author contributions: H.-L.Y., A.C.M.B., R.J.W., J.E.R., and R.G.W. designed research; H.- it is not known whether differences in glycan recognition affect L.Y., J.R.A., A.C.M.B., N.A.I., H.M., J.F., D.A.B., D.B., A.S.L., and J.E.R. performed research; R.S. and D.J.H.-P. contributed new reagents/analytic tools; H.-L.Y. and J.R.A. analyzed data; influenza virus pathogenicity. and H.-L.Y., J.R.A., and R.G.W. wrote the paper. The highly pathogenic H5N1 viruses are consistently lethal to The authors declare no conflict of interest. domestic chickens but differ in their pathogenicity to small 1To whom correspondence should be addressed at: Department of Infectious Diseases, St. mammals, including mice and ferrets (7–9). Although H5N1 Jude Children’s Research Hospital, 332 N. Lauderdale, Memphis, TN 38105. E-mail: virulence in mammals is known to be a polygenic trait (10–15), [email protected]. H5N1 viruses that are highly lethal to mice or ferrets commonly This article contains supporting information online at www.pnas.org/cgi/content/full/ spread systemically after intranasal inoculation (8, 9). Further- 0811052106/DCSupplemental. more, H5N1 virus or RNA has been isolated from human © 2008 by The National Academy of Sciences of the USA 286–291 ͉ PNAS ͉ January 6, 2009 ͉ vol. 106 ͉ no. 1 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0811052106 Downloaded by guest on September 28, 2021 Table 1. Affinity of the recombinant H5N1 influenza viruses to sialyl substrates Recombinant virus Fetuin p6ЈSL p3ЈSL p3ЈSLN HK213 0.18 Ϯ 0.03 5.78 Ϯ 0.82* 1.05 Ϯ 0.21* ND VN1203 0.14 Ϯ 0.06 Ͼ100 0.23 Ϯ 0.04 1.81 Ϯ 0.22 VN1203 HAS227N 0.26 Ϯ 0.03* Ͼ100 0.13 Ϯ 0.05 6.35 Ϯ 0.34* VN1203 HAS227Nϩ158Gly⌬ 0.11 Ϯ 0.04 19.49 Ϯ 1.21* 0.17 Ϯ 0.03 6.81 Ϯ 0.29* diss ϭ mean Ϯ SE ϫ t␣,n-1 (␮M sialic acid), where t␣ is Student’s coefficient with probability ␣ ϭ 0.95, from four independent experiments. *P Ͻ 0.05. alone is not sufficient to confer preferential binding to p6ЈSL. Our results suggest that the increased binding affinity of HK213 for p6ЈSL was mediated mainly by the S227N change and the absence of N-linked glycosylation at residue 158. Introduced HA Mutations Do Not Affect Viral Growth In Vitro or Membrane Fusion pH. We assessed whether the introduced HA changes affected growth of recombinant VN1203 viruses in vitro. VN1203xHK213HANA virus was generated for comparison. The recombinant viruses grew comparably in Madin-Darby canine kidney (MDCK) cells but formed different plaque sizes (see Table S1). At a multiplicity of infection (MOI) ϭ 0.0001 pfu/cell, multistep growth curves showed comparable titers of VN1203, VN1203- Fig. 1. VN1203 HA contributes to systemic spread in mice. Virus titers in HAS227N, and VN1203-HAS227Nϩ158Gly⌬ viruses in MDCK cells lungs, brains (including olfactory bulbs), and blood of BALB/cJ mice inoculated (Fig. S1). We also evaluated whether the introduced HA mutations intranasally with 100 pfu/50 ␮L of recombinant of HK213 (A) and VN1203 (B) Ϯ would affect membrane fusion activity of the VN1203 recombinant viruses. Each data point represents the mean virus titer SD from 3 mice. *, viruses by determining the pH of syncytium formation in Vero cells. P Ͻ 0.05; , P Ͻ 0.01. ** Vero cells infected with VN1203, VN1203-HAS227N, and VN1203- HAS227Nϩ158Gly⌬ viruses formed syncytia at or below pH 5.8, 5.9, Determinants of HA Affinity for ␣2,6-Linked Sialyl Receptor. We and 5.8, respectively. Syncytium formation was observed at or below assess possible functional differences between the HK213 and pH 5.7 for the VN1203xHK213HANA virus. The results suggest that VN1203 HA glycoproteins. The two HA glycoproteins were the introduced HA mutations did not significantly impair viral shown to differ in their affinity for synthetic ␣2,3-linked 3Ј- fitness in vitro.