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Structure and Receptor Speciflcity of the Hemagglutinin from an H5N1 Influenza Virus


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The hemagglutinin (HA) structure at 2.9 angstrom resolution, from a highly pathogenic Vietnamese H5N1 influenza virus, is more related to the 1918 and other human H1 HAs than to a 1997 duck H5 HA. Glycan microarray analysis of this Viet04 HA reveals an avian α2-3 sialic acid receptor binding preference. Introduction of mutations that can convert H1 serotype HAs to human α2-6 receptor specificity only enhanced or reduced affinity for avian-type receptors. However, mutations that can convert avian H2 and H3 HAs to human receptor specificity, when inserted onto the Viet04 H5 HA framework, permitted binding to a natural human α2-6 glycan, which suggests a path for this H5N1 virus to gain a foothold in the human population.
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DOI: 10.1126/science.1124513
, 404 (2006); 312Science
et al.James Stevens,
Hemagglutinin from an H5N1 Influenza Virus
Structure and Receptor Specificity of the
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15 November 2005; accepted 20 March 2006
Structure and Receptor Specificity of
the Hemagglutinin from an H5N1
Influenza Virus
James Stevens,
Ola Blixt,
Terrence M. Tumpey,
Jeffery K. Taubenberger,
James C. Paulson,
Ian A. Wilson
The hemagglutinin (HA) structure at 2.9 angstrom resolution, from a highly pathogenic Vietnamese
H5N1 influenza virus, is more related to the 1918 and other human H1 HAs than to a 1997 duck
H5 HA. Glycan microarray analysis of this Viet04 HA reveals an avian a2-3 sialic acid receptor
binding preference. Introduction of mutations that can convert H1 serotype HAs to human a2-6
receptor specificity only enhanced or reduced affinity for avian-type receptors. However, mutations
that can convert avian H2 and H3 HAs to human receptor specificity, when inserted onto the Viet04
H5 HA framework, permitted binding to a natural human a2-6 glycan, which suggests a path for
this H5N1 virus to gain a foothold in the human population.
he H5N1 avian influenza virus, com-
monly called Bbird flu,[ is a highly con-
tagious and deadly pathogen in poultry.
Since late 2003, H5N1 has reached epizootic
levels in domestic fowl in a number of Asian
countries, including China, Vietnam, Thailand,
Korea, Indonesia, Japan, and Cambodia, and
has now spread to wild bird populations. More
recently, the H5N1 virus has spread to infect
bird populations across much of Europe and
into Africa. However, its spread to the human
population has so far been limited, with only
191 documented severe infections, but with a
high mortality accounting for 108 deaths in
Indonesia, Vietnam, Thailand, Cambodia, Chi-
na, Iraq, Turkey, Azerbaijan, and Egypt Eas
of 4 April 2006, see the World Health
Organization Web site (1)^. Of these, evi-
dence suggests direct bird-to-human transmis-
sion, although indirect transmission, perhaps
through contaminated water supplies, cannot
be ruled out.
Of the three influenza pandemics of the last
century, the 1957 (H2N2) and 1968 (H3N2)
pandemic viruses were avian-human reassort-
ments in which three and two of the eight avian
gene segments, respectively, were reassorted
into an already circulating, human-adapted virus
(2, 3). The origin of the genes of the 1918 in-
fluenza virus (H1N1), which killed about 50
million people worldwide (4), is unknown. The
extinct pandemic virus from 1918 has recently
been reconstructed in the laboratory and was
found to be highly virulent in mice and chicken
embryos (5, 6). With continued outbreaks of the
H5N1 virus in poultry and wild birds, further
human cases are likely, and the potential for the
emergence of a human-adapted H5 virus, either
by reassortment or mutation, is a threat to pub-
lic health worldwide.
Hemagglutinin (HA), the principal antigen
on the viral surface, is the primary target for
neutralizing antibodies and is responsible for
viral binding to host receptors, enabling entry
into the host cell through endocytosis and sub-
sequent membrane fusion. As such, the HA is
an important target for both drug and vaccine
development. Although 16 avian and mamma-
lian serotypes of HA are known, only three
(H1, H2, and H3) have become adapted to the
human population. HA is a homotrimer; each
monomer is synthesized as a single polypeptide
(HA0) that is cleaved by host proteases into
two subunits (HA1 and HA2). HA binds to
receptors containing glycans with terminal sialic
acids, where their precise linkage determines
species preference. A switch in receptor speci-
ficity from sialic acids connected to galactose in
a2-3 linkages (avian) to a2-6 linkages (human)
is a major obstacle for influenza A viruses to
cross the species barrier and to adapt to a new
host (7, 8).OnH3andH1HAframeworks,as
few as two amino acid mutations can switch
human and avian receptor specificity.
Of the H5N1 viral isolates studied to date,
A/Vietnam/1203/2004 (Viet04) is among the
most pathogenic in mammalian models, such as
ferrets and mice (9, 10). This virus was orig-
inally isolated from a 10-year-old Vietnamese
boy who died from bird flu. Because of the im-
portance of HA in viral pathogenesis and host
response to viral infection, we cloned and
expressed the ectodomain (HA0) of its HA gene
(fig. S1) in a baculovirus expression system,
using the same strategy that led to the crystal
structure of the 1918 influenza virus HA0
(11, 12). Viet04 HA0 was cleaved during protein
production into its activated form (HA1/HA2)
and was crystallized at pH 6.55 (13). Its struc-
ture was determined by molecular replacement
(MR) to 2.95 A
resolution (table S1) (14). In
addition, we have investigated the potential of
this H5 HA to acquire human receptor specificity
by introducing mutations known to effect such a
specificity switch on H1 and H3 frameworks.
Structural overview. The overall fold of the
Viet04 HA trimer (Fig. 1, A and B) is very
similar to other published HAs, as expected,
with a globular head containing the receptor
binding domain (RBD) and vestigial esterase
domain, and a membrane proximal domain
with its distinctive, central a-helical stalk and
HA1/HA2 cleavage site (essential for viral
pathogenicity). Although Viet04 HA and the
only other avian H5 HA structure, Sing97
[A/Duck/Singapore/3/1997; Protein Data Bank
(PDB) entry 1jsm (15)], are closely related in
sequence (HA1, 90%; HA2, 98%), the best mo-
lecular replacement (MR) solutions were sur-
Department of Molecular Biology,
Glycan Array Synthesis
Core-D, Consortium for Functional Glycomics,
Institute for Chemical Biology, The Scripps Research
Institute, 10550 North Torrey Pines Road, La Jolla, CA
92037, USA.
Influenza Branch, Division of Viral and
Rickettsial Diseases, Centers for Disease Control and
Prevention, Atlanta, GA 30333, USA.
Department of
Molecular Pathology, Armed Forces Institute of Pathology,
Rockville, MD 20306, USA.
*To whom correspondence should be addressed. E-mail: (I.A.W .) and (J.S.)
on July 27, 2010 www.sciencemag.orgDownloaded from
prisingly achieved by using the 1918 H1
structure (sequence identity: HA1, 58%; HA2,
85%) as a search model (16). Superimposition
of human, avian, and swine HA structures by
using their HA2 domains (table S2) or individ-
ual domains (table S3) confirms that the Viet04
HA is more closely related to human 1918
H1 HA [root mean square deviation (RMSD)
1.2 A
] than to Sing97 H5 HA (RMSD 1.7 A
For example, an interhelical loop between the
two major helices in HA2 is stabilized by a
hydrogen bond between HA2 Arg
and HA2
, resulting in its having an overall con-
formation much more akin to the 1918 H1 loop
than to that of Sing97 or H3 (Fig. 1C).
The amino acid sequence of Viet04 HA
predicts seven possible glycosylation sites per
monomer, although one is in the cytoplasmic
tail and unlikely to be glycosylated. Interpretabl e
electron density is observed at 16 of the possi-
ble 54 glycosylation sites in the asymmetric
unit (nine monomers), which represents car-
bohydrates at two sites, Asn
and Asn
HA1 (17).
Hemagglutinin is synthesized as a single-
chain precursor (HA0) in the endoplasmic re-
ticulum, where it is assembled as a trimer, and is
then exported to the cell surface via the Golgi
network. On the cell surface, HA0 is cleaved by
specific host proteases, such as tryptase Clara
(18), into HA1 and HA2 (19). For the majority
(20) and the narrow tissue distribution of the
relevant proteolytic enzymes restricts infection
to the lung in mammals. However, for H5 and
H7 subtypes, a polybasic sequence has been
associated with high virulence in birds (21),
because of enhanced cleavage susceptibility by a
broader range of cellular proteases, as seen with
our baculovirus-expressed Viet04 HA (fig. S1)
(22). Consequently, the tissue tropism for H5
viruses in mammals is not restricted to the lungs,
but extends to other organs, including the brain
(10). In the Viet04 structure, the C-terminal HA1
cleavage site region could be interpreted only as
far as Pro
and does not account for the
remaining QRERRRKKR residues before Gly
at the N terminus of HA2 (fig. S3). As in other
HAs, the HA2 N terminus is stabilized within
an electronegative cavity by hydrogen bonds
from its backbone amide groups to Asp
to Ser
of the adjacent HA2 (fig. S3).
From our previous 1918 HA0 structure, we
proposed that a pH-sensitive histidine patch
)(14), together
with the adjacent HA2 Trp
in fusion peptide destabilization and release (Fig.
1A) (11). This structural feature is conserved in
other avian and human H1, H2, and H5
serotypes, as well as in Viet04 HA (fig. S3).
In 1918 HA0, a second patch of four exposed
histidines within the vestigial esterase domain
(Fig. 1A and fig. S4A), together with a nearby
lysine, was also implicated in pathogenicity via
enhanced membrane fusion (11). Of the five
HA1 residues in this basic patch (His
, and His
), only three are
conserved in avian H5 structures (His
and Sing97 HAs have an additional lysine
) and histidine (His
and E). Furthermore, Viet04 has yet another
lysine (Lys
), which renders this patch even
more basic and is found in two strains (1203/
1204) that were isolated from the same patient
(10) (fig. S5). The contribution of this region to
virulence, if any, is as yet unknown, but is
worthy of further investigation.
H5N1 anti genic variation. Phylogenetic
analysis of H5 HA genes from 2004 and 2005
has revealed two distinct lineages, termed
clades1and2(23); Viet04 belongs to the
Indochina peninsula lineage (clade 1). Compar-
ison of their amino acid sequences identified 13
positions of antigenic variation that are mainly
clustered around the receptor-binding site; the
rest are within the vestigial esterase domain (Fig.
2 ) . Escape mutants of H5 HAs (24, 25)canbe
clustered into three epitopes (24), as follows:
site 1, an exposed loop (HA1 140 to 145) that
overlaps with antigenic sites A (26)ofH3(27)
and Ca2 of H1 (28); site 2, HA1 residues 156
and 157, which correspond to antigenic site B
in H3 serotypes; and site 3, HA1 129 to 133,
which is restricted to the Sa site in H1 HAs (28)
and H9 serotypes (29). Thus, natural variation
Fig. 1 . Crystal structure of
Viet04 HA and comparison
with 1918 human H1, duck
H5, and 1968 human H3 HAs.
trimer , represented as a ribbon
diagram. For clarity , each mono-
mer has been colored different-
ly . Carbohydrates observed in
the electron-density maps are
colored orange, and all the
asparagines that make up a
glycosylation site are labeled.
Only Glu
are not labeled, as these are on
the back of the molecule. The
location of the receptor bind-
ing, cleavage, and basic patch
sites are highlighted only on
one monomer . All the figures
were generated and rendered
with the use of MacPymol (66).
(B) Structural comparison of
the Viet04 monomer (olive)
with duck H5 (orange) and
1918 H1 (red) HAs. Structur es
were first superimposed on the
HA2 domain of Viet04 through the following residues: Viet04, Gly
to Pro
1918 H1 (PDB: 1rd8), Gly
to Pro
to Pro
1jsm), Gly
to Pro
. Orientation of the overlay approximates to the blue
monomer in (A). (C) Superimposition of the two long a-helices of HA2 for 1918
H1 (PDB: 1rd8), avian H5 (PDB: 1jsm), human H3 (PDB: 2hmg), and Viet04 reveal that the extended interhelical loop of Viet04 is more similar to the 1918
H1 than to the existing avian H5 structure. The side chain of Phe
is illustrated as an example of the close proximity of the two structures.
on July 27, 2010 www.sciencemag.orgDownloaded from
(yellow in Fig. 2), as well as escape mutants
(blue in Fig. 2, green in both 2004 and 2005
viral isolates), suggests continued evolution of
the virus that impacts decisions on which strain
should be considered for a bird flu vaccine. One
mutation that has alanine at residue 160 replaced
by threonine (A160T), which is present in all
2004–05 strains, introduces a new glycosylation
site at Asn
, consistent with a strategy com-
monly used by influenza viruses to mask and
unmask antigenic sites from the immune sys-
tem (30, 31). This glycosylation likely results in
steric hindrance to antigenic site 2 (around
residues 156 and 157), thus reducing the ability
of the host to mount an effective immune
response to these more recent H5N1 viruses.
Receptor binding domain. The RBD is at
the membrane distal end (HA1) of each HA
monomer (Fig. 1A) and binds to its sialic acid–
containing receptors with very weak (millimolar)
affinity (32). However, influenza virus can in-
crease its avidity to host cells through multivalent
binding via a high density of HA trimers on the
virus surface. Avian viruses bind to sialosides
with an a2-3 linkage in the intestinal tract,
whereas human-adapted viruses are specific for
the a2-6 linkage in the respiratory tract (7),
although H5 viruses have also been reported in
human intestine (33). A switch from a2-3 to
a2-6 receptor specificity is a critical step in the
adaptation of avian viruses to a human host and
appears to be one of the reasons why most avian
influenza viruses, including current avian H5
strains, are not easily transmitted from human to
human after avian-to-human infection.
All HA structures, including Viet04 (Fig. 3A),
have similarly configured RBDs. The binding
site comprises three structural elements, namely
an a-helix (190-helix, HA1 188 to 190) and two
loops (130-loop, HA1 134 to 138, and 220-loop,
HA1 221 to 228) (Fig. 3A). A number of con-
served residues are involved in receptor bind-
ing, including Tyr
1) (19). Superimposition of the RBD structural
elements of Viet04 with Sing97 H5 reveals a
very close relation (RMSD 0.3 A
) (Fig. 3B).
Indeed, all key residues implicated in receptor
specificity [reviewed in (19)] (Table 1) are con-
served between structures, although loop 210 to
221 is displaced È1A
from its equivalent in
Sing97 (Fig. 3B). Otherwise, only two RBD
residues differ between these two H5 HAs
(Viet04, Arg
and Ser
; Dk97, Glu
). Thus, the question arises as to how a
current H5 virus could adapt its HA for binding
to human receptors.
Receptor binding specificity of Viet04 HA.
Our cloning and expression strategy produces
HA with a His-tag at the C terminus, which fa-
cilitates receptor-binding studies using a glycan
microarray (34–37). Glycan binding analyses of
Viet04 HA reveal an avian a2-3 specificity in
which the highest affinity is for glycans with sul-
fate on the 6 position of the N-acetylglucosamine
(GlcNAc) residue at the third position in the glycan
chain (Fig. 4A and table S4) (38, 39). Con-
si d er a b l e binding to only one a2-6–linked sialo-
side was observed (6-sialyllactose, no. 49), but
this glycan is only found in milk and is not a
receptor candidate for influenza (40). We also
expressed and investigated the glycan-binding
properties of A/Duck/Singapore/Q-F119-3/
1997 (Dk97), whose sequence is identical to
that of Sing97, for correlation with its structure
(15). Binding of glycoproteins (nos. 1 to 6) and
sulfated glycans was comparab le to those of
Viet04, but binding to other a2-3 sialosides was
reduced relative to Viet04 (Fig. 4B).
Mutational analysis of the RBD. Previous
studies using whole virus identified a number
of key RBD mutations that were implicated in
avian-human receptor specificity switching in
H1, H2, and H3 serotypes. However, adaptation
of avian H1 and H2/H3 serotypes for human
receptor binding occurs by different mecha-
Fig. 3. Analysis of Viet04 receptor
binding site. (A)TheViet04receptor-
binding domain (RBD) with the side
chains of key residues for receptor binding
labeled. The binding site comprises three
structural elements: an a-helix (190-helix)
and two loops (130-loop and 220-loop).
Residues mutated in this study are labeled
red. (B)OverlayoftheRBDsofViet04
with Sing97 structure (PDB: 1jsm) reveals
a similar RBD. The most divergen t part of
the pocket is the loop made up of residues
210 to 221, in which the Viet04 loop is
displaced È1A
farther away from the
binding pock et compared with the 1997
avian H5. Only two residues, at position
216 and 221, differ in these two RBDs.
Fig. 2. Antigenic varia-
tion in recent H5N1 vi-
ruses mapped onto the
Viet04 structure. (Left)
Side view of the Viet04
structure in which natural
mutations identified by
comparison of 2005 with
2004 isolates (23)are
colored yellow; escape
mutants (24, 25) are blue;
and those that overlap in
both analyses are green.
All of the 2004 and
2005 strains have a new
potential glycosylation
site at position 158 in
the HA1 chain (orange).
The receptor binding site
is highlighted with a red
oval. (Right)Topview
looking down onto the
globular membrane distal
end of the trimer around
the RBD showing that
the mutations mainly
cluster around the RBD.
on July 27, 2010 www.sciencemag.orgDownloaded from
nisms. For H2 and H3, mutation of Gln
in avian strains to Leu
and Ser
human viruses correlates with a shift to human
receptor specificity (41, 42). In H1 serotypes,
the avian Gln
and Gly
framework is main-
tained and a Glu
to Asp
mutation now
appears critical for adaptation to human a2-6 re-
ceptors (43, 44). Indeed , glycan microarray and
cell-based assays revealed that the 1918 HA
could be readily converted from classic a2-6
receptor specificity to classic avian a2-3 spe-
cificity by only two mutations (D190E and
D225G) (35, 45). Here, the reverse experiment
was performed with an avian H1 virus [A/Duck/
Alberta/35/1976 (Dk76)] in which the same two
residues were mutated to the ‘ ‘human’ sequences
(E190D and G225D), which completely con-
verted Dk76 to exclusive a2-6 specificity, similar
to that seen for the South Carolina 1918 virus
(Fi gs. 4C and 5, A to C; and table S4) (11, 46).
However, which mutations are likely to mod-
ulate receptor specificity in the H5 serotype is
not so obvious. Based on sequence similarity,
H5 is in the same clade as H1, H2, and H6
serotypes (47). So, to address that issue, we
analyzed glycan binding of Viet04 HA (Fig. 5
and fig. S6) by generating a panel of mutants
Table 1. Conserved residues within the RBDs of H1 and H5 serotypes that
are implicated in receptor specificity. Accession numbers for each wild-type
HA are listed in supporting online material. Residues mutated in this study
are highlighted in gray . The last two columns give a qualitative assessment of
a2-3/a2-6 binding preferences for each mutant with the glycan array .
Qualitative binding assessments were based on a combination of the signal
strength and the number of glycans bound for a given linkage. The binding
of Viet04 was used as a standard for strong binding to the a2-3 linkage
((((), and the double mutant for Dk76 (E190D,G225D) was used for
strong binding (((()tothea2-6 linkage.
Viral strain
Amino acid position Specificity
98 136 153 183 190 193 194 216 221 222 225 226 227 228 a2-3 a2-6
H1 serotype
A/Duck/Alberta/35/1976 Y T W H E S L E P K G Q A G (( O
A/Duck/Alberta/35/1976 (E190D) Y T W H D S L E P K G Q A G ( O
A/Duck/Alberta/35/1976 (G225D) Y T W H E S L E P K D Q A G O O
A/Duck/Alberta/35/1976 (E190D,G225D) Y T W H D S L E P K D Q A G O (((
H5 serotype
A/Duck/Singapore/Q-F119-3/1997 Y S W H E K L E P K G Q S G (( O
A/Vietnam/1203/2004 Y S W H E K L R S K G Q S G ((( (
A/Vietnam/1203/2004 (E190D) Y S W H D K L R S K G Q S G (( O
A/Vietnam/1203/2004 (G225D) Y S W H E K L R S K D Q S G ((( (
A/Vietnam/1203/2004 (E190D,G225D) Y S W H D K L R S K D Q S G O O
A/Vietnam/1203/2004 (Q226L) Y S W H E K L R S K G L S G ( O
A/Vietnam/1203/2004 (S227N) Y S W H E K L R S K G Q N G ((( (
A/Vietnam/1203/2004 (G228S) Y S W H E K L R S K G Q S S ((( ((*
A/Vietnam/1203/2004 (Q226L,G228S) Y S W H E K L R S K G L S S (( ((*
A/Vietnam/1203/2004 (R216E) Y S W H E K L E S K G Q S G ND ND
A/Vietnam/1203/2004 (S221P) Y S W H E K L R P K G Q S G ((( (
A/Vietnam/1203/2004 (R216E,S221P) Y S W H E K L E P K G Q S G ((( (
*Although Viet04 mutants (G228S and Q226L,G228S) only bound a limited number of a2-6 ligands, they bound strongly to these glycans and were, therefore, assessed as (( for a2-6
specificity. No binding is represented by ‘O’’; ND indicates binding to the array was not determined.
Fig. 4. Glycan microarray analy-
ses of (A) Viet04, (B) Dk97, and
(C) an avian H1, Dk76. The Dk97
HA sequence is identical to that in
the published structure of duck
virus Sing97, so a direct structural
comparison can be made. Binding
to different types of glycans on
the array are highlighted where
orange represents glycoproteins;
yellow, a2-3 ligands; green, a2-6
ligands; blue, a2-8 ligands; and
purple, other ligands such as b-linkages, modified sialic acid analogs or glycolylsialic acid
glycans. Red bars indicate sulfated or additional negatively charged ligands. See table S4
for list and tabulated binding results. Because of continual glycan microarray development,
a number of new ligands were printed between analyzing the Dk76 protein (C) and the
remaining samples reported in this study. Binding to glycans nos. 37 to 44, 56, 58 to 60,
67, and 70 was not determined for Dk76 and its three mutants in Fig. 5.
on July 27, 2010 www.sciencemag.orgDownloaded from
(Fig. 3A and Table 1) in and around the RBD to
explore whether this H5 HA can readily become
adapted to humans through mutations that are
known to change receptor specificity in H1 and
H3 serotypes. Mutations at positions 190 and
225 did not reveal any adaptation of Viet04 to
human receptor analogs (Fig. 5, D to F) (48), in
contrast to H1 Dk76 (Fig. 5, A to C) and 1918
HAs (35). Indeed, the single E190D mutation on
the Viet04 framework reveals markedly reduced
affinity to a2-3 sialosides (Fig. 5D), whereas
the double mutant (E190D,G225D) did not in-
teract at all with the glycan microarray (Fig.
5F) (49, 50). However, sulfated glycans bound
equally well to the single E190D mutant and to
the wild type (Figs. 4A and 5D), which sug-
gests that other residues within the Viet04
RBD, such as Lys
or Lys
(Fig. 3A), may
enhance interaction with charged glycans.
Mutation of residues 226 and 228, which
enable H3 viruses to switch from avian to human
specificity, was also evaluated as a potential
route for H5 viruses to acquire human receptor
specificity. Although a dramatic switch to a clas-
sic a2-6 human receptor binder was not observed
(51), the double mutant (Q226L,G228S) showed
substantially reduced affinity to a2-3 sialo-
sides, as noted for mutants of the H3 A/Hong
Kong/156/1997 virus (52).Butitwasnotable
that significant binding to a natural, branched
a2-6 biantennary glycan (nos. 56 and 57) was
observed for both the double mutant and the
single G228S mutant (Fig. 5H). Although the
glycan composition of lung epithelia have not
been analyzed in detail, the mammalian sia lyl-
transferase that produces a2-6–linked structures
on many human tissues (53, 54) is found in
lung epithelial cells (55–57). Thus, these two
effects could offer advantages for an H5N1
virus to adapt to a human host. Decreased
binding to a2-3–linked glycans would help
circumvent the inhibitory effects of respiratory
mucins (58), whereas increased binding to
biantennary N-linked glycans with a2-6–linked
sialic acids would allow the virus to attach to
the surface of epithelial cells that express this
carbohydrate receptor (55–57). In this regard,
human H1 viruses before 1957 were reported to
bind sialic acid receptors with both a2-3 and
a2-6 linkages; post 1957 viruses were specific
only for a 2-6 linkages (37). These binding
patterns suggest that, once a foothold in a new
host species is made, the virus HA optimizes its
specificity to the new host. It is noteworthy
that, of the HAs tested on the array, the
humanized avian H1 (Dk76) double mutant
(E190D,G225D) (Fig. 5C) an d the hum a n H3
HA (A/Moscow/10/1999) (35) did not bind a2-6
biantennary glycans, in contrast to 1918 South
Carolina H1 HA and human H1, A/Texas/36/
1991 (35). Therefore, the HAs of some viruses
may be able to increa se avidity through
interaction with such bivalent structures on N-
linked glycans, whereas, for others, the geom-
etry of the bivalent structure appears to restrict
binding to linear sequences containing a2-6
Fig. 5. Glycan microarray analysis of mutants of Viet04 and Dk76.
Mutations of an avian H1, Dk76: (A) E190D, (B) G225D, and (C) E190D
and G225D were generated and subjected to glycan microarray analysis.
Both positions were reported to be important for conversion of a2-6
receptor specificity of the human 1918 virus HA to avian a2-3 specificity
(35, 45). These mutations did indeed resul t in exclusive a2-6 specificity for
this avian H1 HA. (D to F) Consequently, Viet04 mutations were generated at
the same positions, but did not result in a switch of receptor specificity ,
except to 6-sialyllactose, although they did result in decreased a2-3
binding, particularly to nonsulfated glycans (compare Fig. 4A). (G to I)
Viet04 was mutated at positions 226 and 228, known to be important for H3
HA a2-6 receptor adaptation. Again, no clear switch in receptor specificity
was observed, although binding to biantennary a2-6 moieties was observed,
as well as reduced a2-3 binding in the double and single (Q226L) mutant.
Graphs are generated as described in the legend for Fig. 4 and labels to the
introduced mutations.
on July 27, 2010 www.sciencemag.orgDownloaded from
linkages. Thus, although human viral HAs have
a prima ry specificity for a2-6 linkages, each
may use a different spectrum of glycan recep-
tors for cell entry.
All key residues within the RBD are con-
served in the majority of H5 strains that
have infected humans (fig. S5). However, two
A/Hong Kong/2003 (HK2003) isolates acquired
a S227N mutation within the binding site, where-
as a double mutation (E216R,P221S) in the
220-loop is observed in all 2003–05 isolates
(fig. S5). The possible effect of these natural
mutations on Viet04 HA binding specificity
(Table 1) was, therefore, assessed. The S227N
mutation had comparable specificity to that of
Viet04, with the exception of increased binding,
particularly for branched a2-3 fucosylated
glycans (nos. 26 to 29) and for 6-sialylated
N-acetylgalactosamine (GalNAc) (no. 20) (fig.
S6A) (59, 60), contrary to previous reports that
HK2003 isolates had increased affinity toward
a2-6 analogs, but decreased affinity toward
a2-3 analogs (39). However, in a previous study
from a 1997 isolate, such changes were also not
observed (52), although Viet04 differs at a num-
ber of other positions around the RBD compared
with the Hong Kong isolates that could ac-
count for this difference (61) (Fig. 3A). Reverse
R216E and S221P mutants were also generated,
as well as the double mutant (R216E,S221P),
but the R216E mutant expressed poorly and
could not be analyzed. However, only the
double mutant is found in natural isolates, sug-
gesting a pressure to select for both mutations,
Whereas Viet04 HA binds to branched fuco-
sylated sialosides (nos. 26 to 29) (Fig. 4A), the
S221P mutation showed weaker binding,
whereas the double mutant abrogated binding
to all branched fucosylated glycans unless sul-
fated (no. 25) (fig. S6, B and C). In the Viet04
HA structure, these residues hydrogen bond to
an adjacent monomer in the trimer (Arg
and Ser
with Asp
bilize the displaced 210 to 229 loop (Fig. 3B),
which, therefore, could possibly enhance bind-
ing to branched fucosylated glycans.
So how might H5 avian HA adapt to human
receptors? Knowledge of genetic changes in
circulating viral isolates (39) by themselves ob-
viously cannot be used to predict the impact on
receptor specificity, let alone predict the effect
of future mutations. Here, we use a completely
recombinant system for structural and functional
analyses that enables such investigation in the
laboratory. Our conclusion is that the mutations
that cause a shift from the avian-type to human-
type specificity on the H1 and H3 frameworks
do not cause an equivalent shift in specificity on
the H5 framework of the Viet04 isolate. How-
ever, the mutations that give rise to a2-6
specificity in H3 HAs do in fact reduce avidity
to a2-3 sialosides and increase specificity for
a2-6–linked biantennary N-linked glycans that
could serve as receptors for the virus on lung
epithelial cells. These combined effects could
allow the Viet04 virus to escape entrapment by
mucins and increase the likelihood of binding to
and infection of susceptible epithelial cells (52).
Thus, such mutations provide one possible
route by which H5 viruses could gain a foot-
hold in the human population, although it is
possible that other, as yet unidentified, muta-
tions may allow the H5N1 virus to effect a
switch in receptor specificity.
This glycan microarray technology can,
therefore, be used to analyze not only existing
viral HAs, but as we show here, to identify
mutations that enable adaptation of the remain-
ing influenza serotypes into the human popula-
tion. Monitoring such changes in the ‘ ‘receptor
binding footprint’ ’ in the field on whole viruses
using the glycan microarray could be invaluable
in the identification of emerging viruses that
could cause new pandemics or epidemics.
References and Notes
1. WHO (
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12. Materials and Methods are available as supporting
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13. Viet04 HA at a concentration of 9 mg/ml was used to
grow crystals in sitting drops with a precipitant solution of
22% polyethylene glycol 2000 and 0.1 M Hepes, pH
6.55 (see also supporting online material).
14. 1918 H1 HA0 (PDB: 1rd8), truncated to remove residues
around the cleavage site, was used as the initial MR
model. The final R
and R
values are 26.9 and
31.9% respectively, at 2.9 A
resolution. The crystal
asymmetric unit contains nine hemagglutinin monomers
(six HA monomers in two noncrystallographic trimers and
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crystallographic trimers) with an estimated solvent
content of 57% based on a Matthews’ coefficient (V
2.9 A
/dalton (fig. S2). For comparison with previous
structures, the Viet04 sequences are numbered as for the
H3 subtype. A, C, E, G, I, K, M, O, and Q refer to the
nine HA1 subunits in the asymmetric unit, and B, D, F, H,
J, L, N, P, and R refer to the nine HA2 subunits; e.g.,
refers to HA1 residue 18 in the A subunit and
refers to HA2 residue 111 in the B subunit of the
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16. Scores from the molecular replacement program PHASER
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(Z score: 37.2; and log-likelihood gain, 3412), as
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log-likelihood gain, 768).
17. Two N-acetyl glucosamines were interpretable at 13 of
these sites (Asn
, Asn
, Asn
, Asn
, Asn
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, Asn
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, Asn
, Asn
, Asn
), but an additional mannose residue could be
interpreted at a further three sites (Asn
, Asn
). The glycans are stabilized at Asn
by a
neighboring residue (Gln
) in the same chain, whereas
at Asn
, an additional mannose was visualized because
of stabilization with Lys
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in a symmetry-related monomer.
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20. Single-letter abbreviations for the amino acid residues
are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G,
Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q,
Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; X, any amino
acid; and Y, Tyr.
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linkage preference, but also for additional features, such
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fucosylation, and sialylation. Of the 265 glycans currently
imprinted on the array, 6 are glycoproteins; 38 have
sialic acids with a2-3 linkages; 16 have a2-6 linkages; 7
have a2-8 linkages; and a further 16 are b-linkages,
modified sialic acid analogs, or glycolylsialic acid glycans.
(See table S4 for the glycans analyzed in this study. Of
the a2-6 sialosides, only natural full-sized N-linked
glycans represented on the array are the biantennary
structures (nos. 56 and 57). The remaining sialosides are
fragments or terminal sequences found on glycoproteins.
For full information on the array, contact the Consortium
for Functional Glycomics (62). Previous binding data using
this technology and cell-based assays with whole viruses
show that N-linked glyc ans close to the receptor-binding site
can affect receptor binding through steric hindrance
(35, 63). Insect cells do not produce complex glycans
containing terminal galactose and/or sialic acids, as seen in
mammalian cells, although high-mannose glycans are
produced (64). However, because of the presence of the
influenza sialidase, complex glycans of influenza HAs usually
terminate only in galactose, and thus the size of the
N-glycans elaborated by insect cells approximate to the size
of the complex N-glycans in mammalian host cells. Thus,
any importance of complex glycans for HA function is still
unknown. Indeed, results for the avian H3 HA (A/Duck/
Ukraine/1/1963), published recently (35), are in agreement
with previous whole viral studies (65). However, indepen-
dent studies are ongoing to develop the array for whole-
virus analyses so that a dir ect comparison can be made.
Such initial experiments are promising, because the strict
a2-3 specificity observed here for Dk76 is also seen with
whole-virus studies (37) and pr eliminary experimen ts with
A/Puerto Rico/8/1934 virus that reveal both a2-3 and a2-6
specificity (34), in agreement with experimen ts from cell-
based assays (37).
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38. Whole-virus studies, including those for Viet04 virus, also
revealed a2-3 specificity with a preference for sulfation
in current H5N1 strains (39). However, this assay used
only seven ligands (one a2-6 and six a2-3), which is
considerably fewer than the 84 sialosides, sialoside analogs,
and glycoproteins analyzed here. In our glycan array ,
on July 27, 2010 www.sciencemag.orgDownloaded from
sulfation on the second galactose was not tolerated (no. 37)
for Viet04, although binding was apparent for sialosides
with Gal in either b1-3 or b1-4 linkage to a GlcNAc or
GalNAc (nos. 21 to 23, 32, 33), as well as to fucosylated
glycans (nos. 26 to 29).
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sulfated and/or negatively charged glycans (Figs. 4C and 5,
A to C). The single E190D mutation reduced binding to most
a2-3 glycans, except to sulfated sialosides (Fig. 5A). These
results suggest mutation at both 190 and 225 positions is
always a requirement for H1 serotypes to adapt to a human
47. R. A. Fouchier et al., J. Virol. 79, 2814 (2005).
48. The E190D mutation (Fig. 5D) reduced overall binding of
a2-3 ligands and glycoproteins, except for the sulfated
and/or negatively charged glycans (nos. 18, 20, 24, 25,
and 38). The G225D mutation (Fig. 5E) appeared to have
little effect on the binding profile, in contrast to avian H1,
where binding was not detected (Fig. 5B). The double
mutant (E190D,G225D) did not bind to any glycan on the
array (see Fig. 5F).
49. S. J. Gamblin et al., Science 303, 1838 (2004).
50. For the human H1 HA from A/Puerto Rico/8/1934, the
longer side chain of Glu
can form hydrogen bonds
to sialic acid of both a2-6 and a2-3 sialosides, whereas
for structures of A/Swine/Iowa/1930, H1 HA bound to
human receptor analogs, the shorter side chain of Asp
can only interact with the GlcNAc to stabilize the a2-6
conformation (49). Binding data, with the 1918 South
(E190D,G225D) (Fig. 5C), show that some sulfated glycans
with a2-6 sialic acid linkages can bind. However, this
situation does not arise for the Viet04 double mutant.
Although the G225D mutation would have been expected to
enhance a2-6 specificity, the additional stabilizing influence
of the E190D mutation toward the GlcNAc may not be
possible because of the neighboring Lys
, which could
inhibit interaction of Asp
with the glycan either by steric
hindrance or by dir ect interaction with Asp
. Experiments
are in progr ess to test this notion.
51. The Q226L mutation eliminated binding to the micro-
array, except for two negatively charged a2-3 glycans
[with either an extra sialic acid on the 6-position of a
GalNAc (no. 20) or 6-sulfation on GlcNAc with a branched
fucose (no. 25)]. The G228S mutation did not have any
significant effect compared with Viet04, except that
sialosides with sulfation on the 6-position of the
galactose, with or without branched fucosylation on
the GlcNAc (nos. 12, 37) were tolerated. Stronger binding
was observed for fucosylated glycans (nos. 26 to 29),
and reduced binding was observed for sialosides with
b1-3 linkages between the galactose and GlcNAc/GalNAc
(nos. 21 to 23) (Fig. 5H). In addition to 6-sialyllactose
(no. 49), as seen for Viet04, binding was observed for
a2-6 biantennary structures (nos. 56 and 57). The
double mutant (Q226L,G228S) showed reduced binding
to a2-3 sialosides. Only sulfated and long-chain
glycans were tolerated (nos. 16, 20, 24, 25, 35),
but binding to a2-6 biantennary structures (nos. 56
and 57), as with the G228S mutation, was also
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59. Attenuated viruses with a S227N mutation led to higher
hemagglutinin inhibition titers in ferrets (60). Thus,
enhanced binding to a2-3 ligands, especially to 6-sulfated
GalNAc, could lead to an increas ed uptake into an tigen-
presenting cells and subsequent antibody production.
60. E. Hoffmann, A. S. Lipatov, R. J. Webby, E. A. Govorkova,
R. G. Webster, Proc. Natl. Acad. Sci. U.S.A. 102, 12915
61. The 2003 isolates contain Ala
, Arg
, whereas Viet04 has Thr
(which introduces a
glycosylation site at Asn
), Lys
, Arg
, and Ser
62. Consor tium for Funct ional Glyco mics (www:// functio nalglycomi cs.
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66. W. L. Delano (2002); (
67. The work was supported in part by National Institute of
Allergy and Infectious Diseases grant AI058113 (I.A.W.,
T.T., J.K.T.); National Institute of General Medical Sciences
grants GM062116 (to J.C.P., I.A.W.) and GM060938 (to
J.C.P.); and partial support from NIH grants to I.A.W.
(CA55896 and AI42266). We thank P. Palese and L. Glaser
(Mount Sinai School of Medicine, New York) for providing
the full-length clone of A/Vietnam/1203/2004; the staff of
the Advanced Light Source Beamline 8.2.2 for the
beamline assistance; X. Dai, S. Ferguson, P. Carney, and
J. Vanhnasy (The Scripps Research Institute) for expert
technical assistance; and R. Stanfield and M. Elsliger (The
Scripps Research Institute) for helpful discussions. This is
publication 17916-MB from The Scripps Research Insti-
tute. Coordinates and structure factors have been
deposited in the Protein Data Bank (code 2FK0) and will
be released on publication.
Supporting Online Material
Materials and Methods
Figs. S1 to S6
Tables S1 to S4
3 January 2006; accepted 28 February 2006
Published online 16 March 2006;
Include this information when citing this paper.
Ultrafast Laser–Driven Microlens
to Focus and Energy-Select
Mega–Electron Volt Protons
Toma Toncian,
Marco Borghesi,
Julien Fuchs,
Emmanuel d’Humie
Patrizio Antici,
Patrick Audebert,
Erik Brambrink,
Carlo Alberto Cecchetti,
Ariane Pipahl,
Lorenzo Romagnani,
Oswald Willi
We present a technique for simultaneous focusing and energy selection of high-curre nt, mega–electron
volt proton beams with the use of radial, transient electric fields (10
to 10
volts per meter) triggered
on the inner walls of a hollow micr ocylinder by an inte nse subpicoseco nd laser pulse. Because of
the transient nature of the focusing fields, the proposed method allows selection of a desired range
out of the spectrum of the polyenergetic proton beam. This technique addresses curren t dra wbacks of
laser-accelerated proton beams, such as their broad spectrum and divergence at the source.
he recent development of ultra-intense
laser pulses (1) has opened up oppor-
tunities for applications in many areas,
including particle acceleration (2–5), inertial fu-
sion energy (6), generation of intense x-ray
pulses (7), laser-driven nuclear physics (8), and
laboratory astrophysics (9). In particular, the
acceleration of mega–electron volt ions from
the interaction of high-intensity laser-pulses
with thin solids has major applicative prospects
because of the high beam quality of these ion
bursts (10, 11). Such proton beams are already
applied to produce high-energy density matter
(12) or to radiograph transient processes (13),
and they offer promising prospects for tumor
therapy (14), isotope generation for positron
emission tomography (15), fast ignition of fu-
sion cores (16), and brightness increase of con-
ventional accelerators. However, because these
proton beams are polyenergetic and divergent
at the source, reduction and control of their di-
vergence and energy spread are essential re-
quirements for most of these applications.
Heinrich Heine Universita
sseldorf, D-40225 Du
German y .
School of Mathematics and Physics, The Queen’s
University of Belfast, Belfast BT7 1NN, Northern Ir eland, UK.
Laboratoire pour l’Utilisation des Lasers Intenses, UMR
7605 CNRS-CEA-Ecole Polytechnique-Universite´ Paris VI,
91128 Palaiseau, France.
Centre de Physique The´orique,
UMR 7644 CNRS-Ecole Polytechnique, 91128 Palaiseau,
*To whom correspondence should be addressed. E-mail:
on July 27, 2010 www.sciencemag.orgDownloaded from
... A review study by Miranda de Graaf and Ron A M Fouchier reported that although the sequence of the three listed factors in RBS is different between subtypes and hosts, they have similar structures [25]. Avian influenza viruses bind to sialic acid (SA), a-2, 3-Gal receptor, while human influenza viruses desire SA-a-2,6, Gal receptors [65,66]. Xu et al. ...
... Kord reported six glycosylation sites in the HA gene and one potential glycosylation site at position 165-167 (aa166) [14]. Yegani also identified seven glycosylation sites at positions 26,39,156,181,302,499 and 558 in the HA gene and four sites at positions 44,68,126 and 215 (excluding four sites due to deletion in the stem region in amino acid [49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65][66][67][68] in the NA gene [13]. goose/ Guangdong/1/1996 virus have seven glycosylation site at positions 50,58,63,68,88,146 and 235 in NA and seven glycosylation site at positions 27,39,181,209,302, 500, and 559 in HA gene. ...
Full-text available
The highly pathogenic avian influenza (HPAI) H5N1 virus has received considerable attention during the past 2 decades due to its zoonotic and mutative features. This Virus is of special importance due to to the possibility of causing infection in human populations. According to it’s geographical location, Iran hosts a large number of aquatic migratory birds every year, and since these birds can be considered as the host of the H5 HPAI, the country is significantly at risk of this virus. the In this study, the molecular characteristics of hemagglutinin (HA) and neuraminidase (NA) genes of the H5N8 strain were identified in Malard county of Tehran province and Meighan wetland of Arak city, Markazi province were investigated. Based on the analysis of the amino acid sequence of the HA genes, the cleavage site of the gene includes the PLREKRRKR/GLF polybasic amino acid motif, which is a characteristic of highly pathogenic influenza viruses. The HA gene of two viruses had T156A, S123P, S133A mutations associated with the increased mammalian sialic acid-binding, and the NA gene of two viruses had H253Y mutations associated with the resistance to antiviral drugs. Phylogenetic analysis of the HA genes indicated the classification of these viruses in the b subclade. Although the A/Goose/Iran/180/2016 virus was also an H5N8 b virus, its cluster was separated from the A/Chicken/Iran/162/2016 virus. This means that the entry of these viruses in to the country happened through more than one window. Furthermore, it seems that the introduction of these H5N8 HPAI strains in Iran probably occurred through the West Asia–East African flyway by wild migratory aquatic birds.
... Cet attachement est un premier facteur pour la spécificité d'hôte et le tropisme tissulaire, puisque la bonne reconnaissance conformationnelle des HA et acides sialiques dépend de la séquence en acides aminés de HA et de la forme de liaison de l'acide sialique avec la chaîne glucidique qui le supporte. Ainsi, les liaisons d'acides sialiques les plus communes sont en α-2,3 et α-2,6, et leur distribution tissulaire dépend de l'espèce hôte (Baigent and McCauley, 2003;Stevens et al., 2006;Swayne, 2008). Suite à l'attachement spécifique du virus à la membrane hôte, celui-ci est internalisé et les HA peuvent alors être clivées par des protéases généralistes de l'hôte, permettant la libération de l'ARN viral dans le cytoplasme. ...
... Les VIA adaptés aux oiseaux présentent généralement une meilleure affinité pour les acides sialiques en α-2,3, tandis que les souches adaptées à l'humain et autres mammifères sont plus affines pour les formes α-2,6 (Kuiken et al., 2006). Or il se trouve une prédominance d'acides en α-2,6 dans la trachée humaine, et en α-2,3 dans les voies respiratoires profondes (alvéoles pulmonaires), tandis que chez les oiseaux se trouve une coexpression de formes α-2,3 et α-2,6 dans les tissus respiratoires supérieurs et inférieurs, et majoritairement α-2,3 dans les tissus digestifs, endothéliaux, rénaux et nerveux (Baigent and McCauley, 2003;França et al., 2013;Kuiken et al., 2006;Stevens et al., 2006;Swayne, 2008). Les VIA adaptés à chaque hôte s'attacheront donc préférentiellement dans l'appareil respiratoire supérieur plus exposé, mais par exemple un virus aviaire pourra infecter l'appareil respiratoire profond humain s'il y parvient (Kuiken et al., 2006;Long et al., 2019). ...
Les épizooties d'influenza aviaire hautement pathogène survenues dans le Sud-Ouest de la France durant les hivers 2015-2016, 2016-2017 puis 2020-2021 ont eu un impact sanitaire et économique durable sur la filière avicole régionale. Les deux derniers épisodes ont aussi eu un impact à l'échelle européenne sur le compartiment sauvage et en particulier l'avifaune aquatique migratrice par laquelle les virus auraient été introduits et diffusés sur le continent. En revanche, l'avifaune terrestre commensale des élevages a semblé épargnée cliniquement et les quelques études ciblant ces populations pour comprendre leur rôle potentiel d'hôtes relais ou de maintenance n'ont pas permis de détecter de virus. Malgré cette absence de preuves, des mesures préventives de restriction des contacts avec les volailles ont été prescrites. Ces mesures sont souvent perçues par les éleveurs comme contraignantes et contraires aux pratiques agricoles durables d'élevage en plein air. Dans ce contexte, l'objectif de cette thèse a été de mieux comprendre les rôles épidémiologiques de l'avifaune commensale des élevages vis-à-vis d'agents infectieux viraux et bactériens, en s'intéressant plus particulièrement aux interactions et à la transmission de marqueurs infectieux entre compartiments sauvage et domestique. Pour ce faire, ce travail a utilisé comme modèle un élevage de canards en plein air caractéristique de ceux du Sud-Ouest de la France, afin d'étudier finement l'interface sauvage-domestique. Dans un premier temps, une étude longitudinale sur plusieurs années a permis de caractériser au fil des saisons biologiques la population d'avifaune visiteuse et commensale de l'élevage. Cette étude écologique a révélé une grande richesse en avifaune sur l'élevage, avec la présence essentiellement des passereaux dont plusieurs espèces en déclin dans les milieux ruraux, et de rares mais parfois grands groupes d'avifaune aquatique. Plus spécifiquement ensuite, l'abondance et les interactions de l'avifaune vis-à-vis des canards domestiques ont été analysées, mettant en évidence des espèces beaucoup plus intensément connectées à ceux-ci. De fréquents contacts directs et indirects avec les canards ont été objectivés pour quelques espèces, dominées par les moineaux (Passer domesticus, P. montanus) et bergeronnettes grises (Motacilla alba). Dans un second temps, afin d'évaluer l'efficacité de ces contacts pour les transmissions infectieuses, les prélèvements biologiques nombreux et variés collectés à intervalles répétés sur les différents compartiments de l'élevage (avifaune cible, canards, environnement) ont été analysés. Nous avons ciblé en priorité les virus influenza, ainsi que des virus et bactéries marqueurs des différentes voies de transmission. Leurs prévalences et identifications ont été comparées entre compartiments pour rechercher des similitudes suggérant des échanges infectieux. Les résultats ont montré que les abondances des espèces sauvages et leurs contacts avec l'élevage ne semblaient pas corrélés au profil infectieux, en regard des fortes charges virales ou bactériennes détectées chez les canards ou dans leur environnement partagé. Dans un dernier temps, afin de consolider les hypothèses suggérées par les résultats précédents, des travaux complémentaires ont été mis en œuvre, visant d'une part à détecter finement l'ADN d'hôtes aviaires dans l'environnement de l'élevage, et d'autre part à caractériser les déplacements inter-sites des bergeronnettes grises par suivi télémétrique. Ce travail montre que l'avifaune commensale semble jouer un rôle épidémiologique minime pour les agents étudiés, hors contexte épidémique, à l'interface avec les canards élevés en plein air. Certaines mesures de restriction des contacts pourraient alors être réévaluées afin d'en réduire les contraintes et l'impact écologique potentiel, tandis que la contamination environnementale par les élevages devrait être aussi considérée dans les transmissions infectieuses à l'interface.
... The flexible nature of HA makes rational medication creation extremely difficult. Both antigenic drift and shift may also help to increase the diversity of HA (Wilson et al. 1981;Stevens et al. 2006). The current study discussed how HA protein can be a potential target in the influenza virus by using various flavonoids. ...
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Influenza viruses cause acute respiratory illnesses in birds, humans, and other mammals, and are a major public health concern around the world. Pandemic flu could be caused by an unforeseen human adaptation of an influenza subtype or strain rather than currently circulating influenza viruses. The need for plant metabolites-based new anti-influenza drugs appears to be urgent. Blocking Haemeagglutinin (HA) protein is one of the most appealing drug targets to halt the growth of the virus. The influenza virus can acquire resistance to currently existing therapies, therefore necessitating the development of new medications. The plant's bioactive metabolites, flavanoids are having potential medicinal efficacy. The current study aimed to identify certain flavonoids (Diosmetin, Tangeritin, and Anthocyanidins) that might interact with the HA protein of the influenza virus and help in inhibiting its growth. We used PyRx v0.8 for virtual screening and docking studies. The highest binding affinity docked structures were analyzed using PyMOL and Discovery Studio Visualizer. The present study revealed that these naturally occurring compounds interacted with HA protein, resulting in the minimization of energy in the range.
... Гемагглютинин синтезируется в качестве одноцепочечного предшественника (HA0) в эндоплазматической сети, где он собирается в виде тримера, а затем экспортируется на поверхность клетки через аппарат Гольджи. На клеточной поверхности HA0 расщепляется специфическими протеазами-хозяевами на HA1 и HA2 [7][8][9][10][11]. ...
На данный момент значительную обеспокоенность вызывает распространение в мире эпизоотий вируса гриппа птиц. Вирус гриппа обладает наиболее высокой генетической вариабельностью и вероятностью появления новых штаммов, способных создавать большие эпидемии. Эволюция вируса гриппа протекает очень быстро, следовательно, первостепенной задачей исследователей является антигенное картирование подтипов гемагглютинина, а также выявления особенностей антигенной структуры. Рентгеновская кристаллография является часто используемым методом при определении трехмерной структуры белка. Целью данных исследований являлось получение векторных конструкций для экспрессии белка первой субъединицы вируса гриппа типа А для дальнейшей экспрессии в бактериальной системе. В результате проведенных исследований были синтезированы специфические праймеры для амплификации гена первой субъединицы гемагглютинина. При дизайне олигонуклеотидов для наибольшей специфичности учитывали все критерии, предъявляемые праймерам для ПЦР. Для праймеров были выбраны эндонуклеазы рестрикции NcoI и XhoI, сайты которых отсутствуют в последовательности самого гена и присутствуют в мультиклональном участке плазмиды. Также в ходе проведенных исследований была получена генетическая конструкция для экспрессии белка первой субъединицы гемагглютинина в клетках E.coli. Полученный рекомбинантный белок будет использован для дальнейших работ по кристаллографии и трехмерному моделированию белка.
... The virus enters susceptible cells through HA-mediated binding to host receptors following the cell membrane fusion. Since most of the neutralizing antibody targets are in the HA globular head domain, this region is an essential target for conventional influenza vaccines [21,22]. Despite a high incidence of annual seasonal influenza infections, vaccination is still considered an effective strategy to prevent influenza in humans. ...
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Influenza viruses are responsible for millions of cases globally and significantly threaten public health. Since pandemic and zoonotic influenza viruses have emerged in the last 20 years and some of the viruses have resulted in high mortality in humans, a universal influenza vaccine is needed to provide comprehensive protection against a wide range of influenza viruses. Current seasonal influenza vaccines provide strain-specific protection and are less effective against mismatched strains. The rapid antigenic drift and shift in influenza viruses resulted in time-consuming surveillance and uncertainty in the vaccine protection efficacy. Most recent universal influenza vaccine studies target the conserved antigen domains of the viral surface glycoproteins and internal proteins to provide broader protection. Following the development of advanced vaccine technologies, several innovative strategies and vaccine platforms are being explored to generate robust cross-protective immunity. This review provides the latest progress in the development of universal influenza vaccines.
H1N1 reassortants between the swine Eurasian avian-like (EA) and H1N1 2009 pandemic (H1N1 pdm/09) viruses have been circulating stably in pig populations for more than ten years, and they may have contributed to increased human infections. Whether these H1N1 viruses acquire adaptive mutations to increase their pathogenicity towards a new host is unknown. To address this problem, mouse-adapted (MA) variants of swine-origin EA H1N1 influenza virus isolated from dogs (A/canine/Guangxi/LZ57/2015[LZ57-MA]) were generated by serial lung-to-lung passages in BALB/c mice. These exhibited greater virulence and replication capability than the wild-type virus (LZ57-WT). Of the six adaptive mutations, two were mapped to the ribonucleoprotein (RNP) complex (PB2-E578D and PA-T97I), two to hemagglutinin (HA-N198D and HA-A227E) and two to the non-structural protein 1 (NS1) and nuclear export protein (NS1-A53D and NEP-R42K, respectively). Reverse genetic substitution of the viral genes and mutation experiments demonstrated that the mutations in PA-T97I could enhance the polymerase activity, but a significant downregulation of activity was seen with PB2-E578D, which was consistent with a decrease in virulence. However, HA and NS, which are genes that act synergistically, were found to be determinants of virulence in mice. The reassortant viruses bearing HA mutations (N198D and A227E) were acquired during adaptation enhanced early-stage viral replication in mammalian cells. The single-point mutations in the NS genes had limited effects on virulence. Furthermore, a combination of HA (N198D and A227E) with NS(A53D) in the rLZ57-WT backbone resulted in efficient replication and a significant increase in virulence. The results suggest that these substitutions could compensate for the polymerase function and contribute to enhanced virulence, which highlights a major role for mutations in the HA and NS genes.
Viral infections have been the causes of global pandemics, including the ongoing coronavirus disease 2019, which prompted the investigation into the infection mechanisms to find treatment and aid the vaccine design. Betacoronaviruses use spike glycoprotein on their surface to bind to host receptors, aiding their host attachment and cell fusion. Protein‐glycan interaction has been implicated in the viral entry mechanism of many viruses and has recently been shown in SARS‐CoV‐2. Here, we reviewed the current knowledge on protein‐glycan interactions that facilitate SARS‐CoV‐2 host entry, with special interest in sialoglycans present on both the virions and host cell surfaces. We also analyze how such information provides opportunities and challenges in glyco‐based inhibitors.
Through their specific interactions with proteins, cellular glycans play key roles in a wide range of physiological and pathological processes. One of the main goals of research in the areas of glycobiology and glycomedicine is to understand glycan-protein interactions at the molecular level. Over the past two decades, glycan microarrays have become powerful tools for the rapid evaluation of interactions between glycans and proteins. In this review, we briefly describe methods used for the preparation of glycan probes and the construction of glycan microarrays. Next, we highlight applications of glycan microarrays to rapid profiling of glycan-binding patterns of plant, animal and pathogenic lectins, as well as other proteins. Finally, we discuss other important uses of glycan microarrays, including the rapid analysis of substrate specificities of carbohydrate-active enzymes, the quantitative determination of glycan-protein interactions, discovering high-affinity or selective ligands for lectins, and identifying functional glycans within cells. We anticipate that this review will encourage researchers to employ glycan microarrays in diverse glycan-related studies.
H9N2 avian influenza viruses (AIVs) circulate globally in poultry and have become the dominant AIV subtype in China in recent years. Previously, we demonstrated that the H9N2 virus (A/chicken/Eastern China/SDKD1/2015) naturally harbors a mammalian-adaptive molecular factor (627K) in the PB2 protein and is weakly pathogenic in mice. Here, we focused on new markers for virulence in mammals. A mouse-adapted H9N2 virus was serially passaged in mice by infecting their lungs. As expected, infected mice showed clinical symptoms and died at passage six. A comparison between the wild-type and mouse-adapted virus sequences identified amino acid substitutions in the hemagglutinin (HA) protein. H9N2 viruses with the T187P + M227L double mutation exhibited an increased affinity to human-type (SAα2,6Gal) receptors and significantly enhanced viral attachment to mouse lung tissues, which contributed to enhancing viral replication and virulence in mice. Additionally, HA with the T187P + M227L mutation enabled H9N2 viral transmission in guinea pigs via direct contact. AIV pathogenicity in mice is a polygenic trait. Our results demonstrated that these HA mutations might be combined with PB2-627K to significantly increase H9N2 virulence in mice, and this enhanced virulence was achieved in other H9N2 AIVs by generating the same combination of mutations. In summary, our study identified novel key elements in the HA protein that are required for H9N2 pathogenicity in mice and provided valuable insights into pandemic preparedness against emerging H9N2 strains.
Recently, the H3N2 influenza outbreak has caused serious global public health concern for future control of the next influenza pandemic. Since using current anti-influenza drugs targeting neuraminidase (oseltamivir and zanamivir) and the proton M2 channel (amantadine and rimantadine) leads to drug resistance, it is essential to seek new anti-viral agents that act on additional viral targets. Hemagglutinin (HA), a glycoprotein embedded in the viral surface and playing a critical role in influenza the viral replication cycle has become an attractive target. This work investigates the molecular binding mechanism of HA H3N2 of influenza virus complexed with the fusion inhibitor, arbidol and its derivative (der-arbidol), by means of molecular dynamics simulation. The result showed that the arbidol derivative could form many and strong hydrogen bonds with the HA surrounding amino acids comprising GLU1032(1), LYS3071(1) and LYS3102(1) while arbidol makes this type of interaction with only LYS582(1). The introduction of hydroxyl group at the meta-position of the thiophenol ring was detected to replace the nearby water molecule, thus allowing the direct hydrogen bond formation between der-arbidol and GLU1032(1) of HA residue. Furthermore, the salt bridge networks established among residues GLU572(1)···ARG542(1)···GLU972(2) were considerably more stable in HA-Der-arbidol than that found in HA-Arbidol. The predicted protein-ligand binding free energies were in agreement with experimental data indicating that der-arbidol exhibits higher inhibitory potency against HA H3N2 of influenza virus. Detailed information could be useful for further designing and optimizing HA fusion inhibitors with improved efficiency.
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An outbreak of highly pathogenic avian influenza A (H5N1) has recently spread to poultry in 9 Asian countries. H5N1 infections have caused >52 human deaths in Vietnam, Thailand, and Cambodia from January 2004 to April 2005. Genomic analyses of H5N1 isolates from birds and humans showed 2 distinct clades with a nonoverlapping geographic distribution. All the viral genes were of avian influenza origin, which indicates absence of reassortment with human influenza viruses. All human H5N1 isolates tested belonged to a single clade and were resistant to the adamantane drugs but sensitive to neuraminidase inhibitors. Most H5N1 isolates from humans were antigenically homogeneous and distinct from avian viruses circulating before the end of 2003. Some 2005 isolates showed evidence of antigenic drift. An updated nonpathogenic H5N1 reference virus, lacking the polybasic cleavage site in the hemagglutinin gene, was produced by reverse genetics in anticipation of the possible need to vaccinate humans.
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Foot-and-mouth is one of the world's most economically important livestock diseases. We developed an individual farm–based stochastic model of the current UK epidemic. The fine grain of the epidemiological data reveals the infection dynamics at an unusually high spatiotemporal resolution. We show that the spatial distribution, size, and species composition of farms all influence the observed pattern and regional variability of outbreaks. The other key dynamical component is long-tailed stochastic dispersal of infection, combining frequent local movements with occasional long jumps. We assess the history and possible duration of the epidemic, the performance of control strategies, and general implications for disease dynamics in space and time.
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Highly pathogenic H5N1 influenza A viruses are now endemic in avian populations in Southeast Asia, and human cases continue to accumulate. Although currently incapable of sustained human-to-human transmission, H5N1 represents a serious pandemic threat owing to the risk of a mutation or reassortment generating a virus with increased transmissibility. Identifying public health interventions that might be able to halt a pandemic in its earliest stages is therefore a priority. Here we use a simulation model of influenza transmission in Southeast Asia to evaluate the potential effectiveness of targeted mass prophylactic use of antiviral drugs as a containment strategy. Other interventions aimed at reducing population contact rates are also examined as reinforcements to an antiviral-based containment policy. We show that elimination of a nascent pandemic may be feasible using a combination of geographically targeted prophylaxis and social distancing measures, if the basic reproduction number of the new virus is below 1.8. We predict that a stockpile of 3 million courses of antiviral drugs should be sufficient for elimination. Policy effectiveness depends critically on how quickly clinical cases are diagnosed and the speed with which antiviral drugs can be distributed.
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A novel trypsin-like protease associated with rat bronchiolar epithelial Clara cells, named Tryptase Clara, was purified to homogeneity from rat lung by a series of standard chromatographic procedures. The enzyme has apparent molecular masses of 180 +/- 16 kDa on gel filtration and 30 +/- 1.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Its isoelectric point is pH 4.75. Studies with model peptide substrates showed that the enzyme preferentially recognizes a single arginine cleavage site, cleaving Boc-Gln-Ala-Arg-4-methylcoumaryl-7-amide most efficiently and having a pH optimum of 7.5 with this substrate. The enzyme is strongly inhibited by aprotinin, diisopropylfluorophosphate, antipain, leupeptin, and Kunitz-type soybean trypsin inhibitor, but inhibited only slightly by Bowman-Birk soybean trypsin inhibitor, benzamidine, and alpha 1-antitrypsin. Immunohistochemical studies indicated that the enzyme is located exclusively in the bronchiolar epithelial Clara cells and colocalized with surfactant. An immunoreactive protein with a molecular mass of 28.5 kDa was also detected in airway secretions by Western blotting analyses, suggesting that the 30-kDa protease in Clara cells is processed before or after its secretion. Proteolytic cleavage of the hemagglutinin of influenza virus is a prerequisite for the virus to become infectious. Tryptase Clara was shown to cleave the hemagglutinin and activate infectivity of influenza A virus in a dose-dependent way. These results suggest that the enzyme is a possible activator of inactive viral fusion glycoprotein in the respiratory tract and thus responsible for pneumopathogenicity of the virus.
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The H5 hemagglutinin (HA) of a highly virulent avian influenza virus, A/Turkey Ontario/7732/66 (H5N9), was previously shown to have five neutralizing epitopes, and escape mutants within one epitope (group 1) were markedly attenuated (M. Philpott, B. C. Easterday, and V. S. Hinshaw, J. Virol. 63:3453-3458, 1989). To define the genetic changes related to these antigenic and biologic properties, the HA genes of mutants within each of the epitope groups were sequenced by using the polymerase chain reaction. The mutations in the attenuated group 1 mutants were located near the distal tip of the HA molecule in close proximity to the receptor-binding site, on the basis of alignment with the three-dimensional structure of the H3 HA. All group 1 mutations involved charged amino acids. The group 1 mutants, similar to the wild-type virus, spread systemically and were recovered from the spleens of infected chickens but, unlike the wild-type virus, failed to produce severe necrosis in the spleens. Viral replication in the spleens was investigated by in situ hybridization of spleen sections from chickens infected with the wild-type or attenuated mutants. Wild-type virus replication was demonstrated in large, mononuclear, macrophagelike cells; however, group 1 mutant virus was detected attached only to erythrocytes within the red pulp. These results suggest that the attenuated mutants differ in their cell tropism within the spleen.
Corrections to 1949 Proc. Phys. Soc. B 62 597
The haemagglutinin (HA) glycoproteins of influenza virus membranes are responsible for binding viruses to cells by interacting with membrane receptor molecules which contain sialic acid (for review see ref. 1). This interaction is known to vary in detailed specificity for different influenza viruses (see, for example, refs 2-4) and we have attempted to identify the sialic acid binding site of the haemagglutinin by comparing the amino acid sequences of haemagglutinins with different binding specificities. We present here evidence that haemagglutinins which differ in recognizing either NeuAc alpha 2 leads to 3Gal- or NeuAc alpha 2 leads to 6Gal- linkages in glycoproteins also differ at amino acid 226 of HA1. This residue is located in a pocket on the distal tip of the molecule, an area previously proposed from considerations of the three-dimensional structure of the haemagglutinin to be involved in receptor binding.
The RNA of the human influenza virus Singapore (H2N2) strain has been labeled in vivo by phosphorus-32 and separated by polyacrylamide gel electrophoresis into eight segments, which were correlated to the corresponding gene functions and/or proteins. The base sequence homology between the individual genes (segments) of the H2N2 virus and those of different influenza A strains has been determined by molecular hybridization. Segments 1, 5, 7, and 8 of the Singapore strain exhibit a base sequence homology of almost 100% as compared to the FM1 strain (HlNl), while the homology between the other segments is significantly lower (24–76%). For the Singapore and Hong Kong (H3N2) strains all segments except that coding for the hemagglutinin (HA, 24%) exhibit a homology close to 100%. The 32P-labeled segment 4 (HA-gene) of the avian influenza A strain duck Ukraine (Hav7Neg2) shows a homology of 92% to Hong Kong, while the homology of at least two other segments is significantly lower. These results are taken as an indication that the H2N2 subtype is derived from the HlN1 subtype by a recombination event retaining four H1N1 segments, while the other four segments are gained from another yet unknown strain. The H3N2 subtype is presumably derived from a H2N2 subtype, retaining seven segments of the H2N2 subtype, while the gene coding for the HA is obtained from the duck Ukraine or another highly related strain.
The interaction between influenza virus hemagglutinin and its cell-surface receptor, 5-N-acetylneuraminic acid (sialic acid), was probed by the synthesis of 12 sialic acid analogs, including derivatives at the 2-carboxylate, 5-acetamido, 4-, 7-, and 9-hydroxyl, and glycosidic positions. The equilibrium dissociation constants of these analogs were determined by nuclear magnetic resonance spectroscopy. Ligand modifications that reduced or abolished binding included the replacement of the 2-carboxylate with a carboxamide, the substitution of azido or N-benzyloxycarbonyl groups for the 5-acetamido group, and the replacement of the 9-hydroxyl with amino or O-acetyl moieties. Modifications having little effect on binding included the introduction of longer chains at the 4-hydroxyl position, the replacement of the acetamido methyl group with an ethyl group, and the removal of the 7-hydroxyl group. X-ray diffraction studies yielded 3 A resolution crystal structures of hemagglutinin in complex with four of the synthetic analogs [α-2-O-methyl-, 4-O-acetyl-α-2-O-methyl-, 9-amino-9-deoxy-α-2-O-methyl-, and α-2-O-(4′-benzylamidocarboxybutyl)-N-acetylneuraminic acid] and with the naturally occurring cell-surface saccharide (α2-3)sialyllactose. The X-ray studies unambiguously establish the position and orientation of bound sialic acid, indicate the position of the lactose group of (α2-3)sialyllactose, and suggest the location of an α-glycosidic chain (4′-benzylamidocarboxybutyl) that increases the binding affinity of sialic acid by a factor of about 3. Although the protein complexed with α-2-O-methylsialic acid contains the mutation Gly-135 → Arg near the ligand binding site, the mutation apparently does not affect the ligand's position. The X-ray studies allow us to interpret the binding affinities in terms of the crystallographic structure. The results suggest further experiments which could lead to the design of tight binding inhibitors of possible therapeutic value.
The evolution of the hemagglutinin (HA) of equine H3 influenza viruses was studied by analysis of nucleotide and deduced amino acid sequences, as well as reactivity of the HA molecule to ferret antisera and monoclonal antibodies. The evolutionary tree constructed from the nucleotide sequences clearly illustrated that the HA of equine H3 influenza viruses evolved in a single lineage, as has been shown for human influenza A viruses. Comparison of the nucleotide sequences of the HA genes of human, swine, avian, and equine viruses showed that the equine strains have evolved independently, without recent introduction of HA genes from other species. By contrast, the results of antigenic analysis indicated that equine H3 influenza viruses include multiple, cocirculating strains. This discrepancy suggests that antigenic analysis of viral proteins should be used with caution in studies of evolutionary pathways.