Structure of the Uncleaved Human H1 Hemagglutinin from the Extinct 1918 Influenza Virus

Abstract

The 1918 "Spanish" influenza pandemic represents the largest recorded outbreak of any infectious disease. The crystal structure of the uncleaved precursor of the major surface antigen of the extinct 1918 virus was determined at 3.0 angstrom resolution after reassembly of the hemagglutinin gene from viral RNA fragments preserved in 1918 formalin-fixed lung tissues. A narrow avian-like receptor-binding site, two previously unobserved histidine patches, and a less exposed surface loop at the cleavage site that activates viral membrane fusion reveal structural features primarily found in avian viruses, which may have contributed to the extraordinarily high infectivity and mortality rates observed during 1918.

Full text

DOI: 10.1126/science.1093373
, 1866 (2004); 303Science
et al.James Stevens,
VirusHemagglutinin from the Extinct 1918 Influenza
Structure of the Uncleaved Human H1
This copy is for your personal, non-commercial use only.
. clicking herecolleagues, clients, or customers by
, you can order high-quality copies for yourIf you wish to distribute this article to others
. herefollowing the guidelines
can be obtained byPermission to republish or repurpose articles or portions of articles
(this information is current as of June 21, 2010 ):
The following resources related to this article are available online at www.sciencemag.org
http://www.sciencemag.org/cgi/content/full/303/5665/1866
version of this article at:
including high-resolution figures, can be found in the onlineUpdated information and services,
http://www.sciencemag.org/cgi/content/full/1093373/DC1
can be found at: Supporting Online Material
found at:
can berelated to this articleA list of selected additional articles on the Science Web sites
http://www.sciencemag.org/cgi/content/full/303/5665/1866#related-content
http://www.sciencemag.org/cgi/content/full/303/5665/1866#otherarticles
, 14 of which can be accessed for free: cites 36 articlesThis article
115 article(s) on the ISI Web of Science. cited byThis article has been
http://www.sciencemag.org/cgi/content/full/303/5665/1866#otherarticles
33 articles hosted by HighWire Press; see: cited byThis article has been
http://www.sciencemag.org/cgi/collection/biochem
Biochemistry
: subject collectionsThis article appears in the following
registered trademark of AAAS.
is aScience2004 by the American Association for the Advancement of Science; all rights reserved. The title
CopyrightAmerican Association for the Advancement of Science, 1200 New York Avenue NW, Washington, DC 20005.
(print ISSN 0036-8075; online ISSN 1095-9203) is published weekly, except the last week in December, by theScience
on June 21, 2010 www.sciencemag.orgDownloaded from

6. L. D. Belmont, T. J. Mitchison, Cell 84, 623 (1996).
7. L. Cassimeris, Curr. Opin. Cell Biol. 14, 18 (2002).
8. G. Di Paolo, B. Antonsson, D. Kassel, B. M. Riederer, G.
Grenningloh, FEBS Lett. 416, 149 (1997).
9. P. Amayed, D. Pantaloni, M. F. Carlier, J. Biol. Chem.
277, 22718 (2002).
10. P. P. Budde, A. Kumagai, W. G. Dunphy, R. Heald,
J. Cell Biol. 153, 149 (2001).
11. H. Daub, K. Gevaert, J. Vandekerckhove, A. Sobel, A.
Hall, J. Biol. Chem. 276, 1677 (2001).
12. V. S. Kraynov et al., Science 290, 333 (2000).
13. M. A. Sells, A. Pfaff, J. Chernoff, J. Cell Biol. 151, 1449
(2000).
14. T. Wittmann, G. M. Bokoch, C. M. Waterman-Storer,
J. Cell Biol. 161, 845 (2003).
15. Materials and methods are available as supporting
material on Science Online.
16. O. Griesbeck, G. S. Baird, R. E. Campbell, D. A.
Zacharias, R. Y. Tsien, J. Biol. Chem. 276, 29188
(2001).
17. M. O. Steinmetz et al., EMBO J. 19, 572 (2000).
18. L. Beretta, T. Dobransky, A. Sobel, J. Biol. Chem. 268,
20076 (1993).
19. I. A. Leighton, P. Curmi, D. G. Campbell, P. Cohen, A.
Sobel, Mol. Cell. Biochem. 127-128, 151 (1993).
20. The fast diffusion of COPY (D
COPY
⬇ 13 to 18
␮m
2
/s as approximated by FRAP experiments) and
comparison of CFP-stathmin and YFP distribution
in live A6 cells do indicate a homogeneous distri-
bution of COPY. Supporting text is available on
Science Online.
21. H. Chneiweiss, J. Cordier, A. Sobel, J. Neurochem. 58,
282 (1992).
22. S. V. Drouva, B. Poulin, V. Manceau, A. Sobel, Endo-
crinology 139, 2235 (1998).
23. H. Imamura et al., Mol. Biol. Cell 9, 2561 (1998).
24. I. Okamoto et al., J. Biol. Chem. 274, 25525 (1999).
25. P. I. Bastiaens, I. V. Majoul, P. J. Verveer, H. D. Soling,
T. M. Jovin, EMBO J. 15, 4246 (1996).
26. U. Marklund, N. Larsson, H. M. Gradin, G. Brattsand,
M. Gullberg, EMBO J. 15, 5290 (1996).
27. T. Kuntziger, O. Gavet, A. Sobel, M. Bornens, J. Biol.
Chem. 276, 22979 (2001).
28. R. E. Carazo-Salas, E. Karsenti, Curr. Biol. 13, 1728
(2003).
29. P. T. Stukenberg, Curr. Biol. 13, R848 (2003).
30. C. M. Waterman-Storer, E. D. Salmon, J. Cell Biol.
139, 417 (1997).
31. T. Wittmann, G. M. Bokoch, C. M. Waterman-Storer,
J. Biol. Chem. 279, 6196 (2003); published electron-
ically 26 Nov 2003.
32. G. C. Brown, B. N. Kholodenko, FEBS Lett. 457, 452
(1999).
33. We thank R. Heald for the kind gift of the stathmin
mutants, T. Stauber for comments on the manuscript, T.
Zimmermann and J. Rietdorf for help with microscopy, and
V. Georget for helpful advice concerning cloning proce-
dures. We also thank I. Arnal for initial trials on the detec-
tion of stathmin-tubulin interactions.
Supporting Online Material
www.sciencemag.org/cgi/content/full/303/5665/1862/
DC1
Materials and Methods
SOM Text
Fig. S1
References
Movie S1
27 November 2003; accepted 4 February 2004
Structure of the Uncleaved
Human H1 Hemagglutinin from
the Extinct 1918 Influenza Virus
James Stevens,
1
Adam L. Corper,
1
Christopher F. Basler,
3
Jeffery K. Taubenberger,
4
Peter Palese,
3
Ian A. Wilson
1,2
*
The 1918 “Spanish” influenza pandemic represents the largest recorded outbreak
of any infectious disease. The crystal structure of the uncleaved precursor of the
major surface antigen of the extinct 1918 virus was determined at 3.0 angstrom
resolution after reassembly of the hemagglutinin gene from viral RNA fragments
preserved in 1918 formalin-fixed lung tissues. A narrow avian-like receptor-binding
site, two previously unobserved histidine patches, and a less exposed surface loop
at the cleavage site that activates viral membrane fusion reveal structural features
primarily found in avian viruses, which may have contributed to the extraordinarily
high infectivity and mortality rates observed during 1918.
Influenza is a viral infection of the respiratory
tract that affects millions of people annually.
Combined with subsequent infection from
bacterial pneumonia, influenza remains one
of the leading causes of death in the United
States, killing on average more than 50,000
people per year. However, the 1918 pandem-
ic killed over 500,000 people in the United
States and more than 20 million worldwide
(1), making it the largest and most destructive
outbreak of any infectious disease in recorded
history (2, 3). Why the 1918 virus was so
devastating is still a mystery. The pandemic
struck before viruses were known as the caus-
ative agent and, consequently, no intact virus
survived. Nevertheless, fragments of the viral
genome did survive in Alaskan victims
buried in the permafrost and in fixed and
archived autopsy material, which recently
enabled gene reassembly (4–7).
There are three types of influenza virus
(A, B, and C), and the 1918 virus is a
member of type A, which accounts for all
known major epidemics and pandemics.
Hemagglutinin (HA) is the surface glyco-
protein responsible for virus binding to the
host receptor, internalization of the virus,
and subsequent membrane-fusion events
within the endosomal pathway in the infect-
ed cell. HA is also the most abundant an-
tigen on the viral surface and harbors the
primary neutralizing epitopes for antibod-
ies. Fifteen avian and mammalian serotypes
of HA have been identified, but only three
have become adapted to humans in the last
century, resulting in the emergence of pan-
demic strains H1 in 1918, H2 in 1957, and
H3 in 1968 (see fig. S1 for sequences).
Recently, three small outbreaks arose from
avian subtypes (H5, H7, and H9) that man-
aged to make a direct leap to humans, but
their low transmissibility prevented major
new epidemics (8–10). However, the emer-
gence of future influenza virus pandemic
strains is likely (11), and their severity will
depend on the ability to contain and
combat infection by timely development of
an appropriate vaccine.
The mature HA forms homotrimers of
⬃220 kD, with multiple glycosylation
sites. Each monomer is synthesized as a
single polypeptide precursor (HA0) that is
subsequently cleaved into HA1 and HA2
subunits (12) by a candidate trypsin-type
endoprotease, “tryptase Clara,” that has
been isolated from rat bronchiolar epitheli-
al Clara cells (13). Structural information is
available only for influenza A HAs of the
human H3 (14), swine H9 (15), and avian
H5 subtypes (15), and for an influenza C
HA esterase fusion (HEF) protein (16).
Twenty-two years after the first structural
characterization of the HA from the 1968
H3 human pandemic (14), we now present
the HA crystal structure from a second
human subtype (H1) derived from reassem-
bly of the extinct 1918 influenza virus (4 ).
The ectodomain of the HA gene (fig.
S1) from the 1918 influenza virus A/South
Carolina/1/18 (18HA0) was cloned and ex-
pressed (fig. S2) in a baculovirus expres-
sion system (17, 18). 18HA0 crystallized at
pH 5.5 (table S1) (19), and its structure was
determined by molecular replacement
(MR) to 3.0 Å resolution (table S1) (20).
18HA0 is ⬃135 Å in length with two dis-
tinct domains (Fig. 1A). The cylindrical
trimer has a tightly intertwined “stem” do-
main at its membrane-proximal base, which
is composed of HA1 residues 11 to 51 and
276 to 329 and HA2 1 to 176 (Fig. 1A). The
dominant feature of this stalk region is the
three long parallel ␣ helices (⬃50 amino
acids in length), one from each monomer,
1
Department of Molecular Biology,
2
Skaggs Institute
for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, CA 92037,
USA.
3
Department of Microbiology, Mount Sinai
School of Medicine, One Gustave L. Levy Place, Box
1124, New York, NY 10029, USA.
4
Division of Molec-
ular Pathology, Department of Cellular Pathology and
Genetics, Armed Forces Institute of Pathology, Wash-
ington, DC 20306, USA.
*To whom correspondence should be addressed. E-
mail: wilson@scripps.edu
R EPORTS
19 MARCH 2004 VOL 303 SCIENCE www.sciencemag.org1866
on June 21, 2010 www.sciencemag.orgDownloaded from

that associate to form a triple-stranded
coiled coil. This region also contains the
cleavage site where host enzymes normally
cut HA0. The membrane-distal domain
consists of a globular “head,” which can be
further subdivided (Fig. 1B) into the R
region, containing the receptor-binding site
and major epitopes for neutralizing anti-
bodies, and the E region, with close struc-
tural homology to the esterase domain of
influenza C HEF (16).
The superimposition of other published
HAs onto the 18HA0 monomer (Fig. 1B) by
means of their HA2 domains [root mean
square deviations (RMSDs) in table S2], in-
dicates that 18HA0 is most closely related to
the avian H5 subtype (RMSD 2.3 Å), where-
as the human H3 subtype is the most diver-
gent (RMSD 4.1 Å). The HA1 receptor (R)
region of the human H3 subtype is displaced
most (RMSD ⬃7.4 Å) from its equivalent
18HA0 domain (Fig. 1B). This conformation-
al variability in the HA globular heads stems
primarily from a rigid-body rotation of the
HA1 receptor domains relative to the HA2
stem domains about the central threefold
axis, as described previously for H3, H5, and
H9 (21). 18HA0 is rotated to the same extent
(⬃17°) as avian H5 relative to H3, whereas
swine H9 is intermediate (⬃11°) (fig. S3, A
and B) (22). The individual HA1 subdomains
(R and E regions in Fig. 1B) superimpose
with low RMSDs (1.1 to 2.6 Å) (table S3).
The 18HA0 structure reveals a substan-
tially different conformation for the cleavage
site loop compared with the uncleaved H3
and cleaved H5 subtypes (Fig. 2, A to D). The
H3 cleavage loop projects out from the gly-
coprotein surface, exposing it to potential
proteases (Fig. 2A) (23), whereas the 18HA0
cleavage site abuts the HA surface (Fig. 2B)
(24). From Pro
A324
, the 18HA0 cleavage
loop extends toward the trimer interface so
that Arg
A329
now covers the electronegative
cavity that is normally occupied by the HA2
fusion peptide after cleavage activation (Fig.
2, E and F). The Arg
A329
side chain points
toward solvent as a result of repulsion from
Lys
F39
and Lys
F121
of the adjacent subunit.
Thus, Arg
A329
is substantially less exposed
than the equivalent Gln
329
(⬃15 Å farther
from the HA surface) that was mutated
from Arg to determine the crystal structure
of an uncleaved H3 subtype (Fig. 2, A and
D) (23). In cleaved HA structures at neutral
pH, the N-terminal HA2 fusion peptide in-
serts into a negatively charged cavity and
makes up to five hydrogen bonds from its
backbone amide groups to conserved HA2
ionizable residues (Asp
B109
and Asp
B112
).
The 18HA0 cleavage loop does not pene-
trate as far into this cavity (Fig. 2D, left)
and makes only one hydrogen bond,
Ser
A325
to Asp
B112
, between the loop and
the conserved acidic residues.
These different cleavage loop conforma-
tions in the H1 and H3 structures may be
influenced in part by nearby glycosylation
sites. In 18HA0, Asn
A20
and Asn
A34
are po-
sitioned above and to the side of the cleavage
loop (upper right, Fig. 2B), creating a cavity
in which a section of the HA0 loop (Ile
B10
to
Trp
B21
) can be accommodated. Equivalent
glycosylation sites (Asn
A22
and Asn
A38
)in
uncleaved H3 are farther from the cleavage
site loop (Fig. 2A and fig. S1) and, thus, may
exert less influence on its conformation. Our
attempts to cleave the 18HA0 trimer with
tryptase [molecular weight (MW) 135 kD]
from human lung failed, even after 20
hours of incubation at 28°C, yet cleavage
with trypsin (MW 43 kD) was complete
after 20 min [at neutral pH (25)]. Newly
synthesized viral proteins are exported to
the cell surface by way of the Golgi com-
plex, where the pH becomes more acidic
during progression through the secretory
pathway (26–28). During viral assembly,
the 18HA0 cleavage loop could adopt this
less exposed conformation to protect from
premature cleavage (and membrane-fusion
activation) by intracellular proteases.
From the cleavage site, the HA0 main
chain traverses the surface below the glyco-
sylation site at Asn
A20
, where it then forms
another previously unobserved miniloop
structure with Met
B17
at its tip. In H3 and H5,
the HA2 Trp
B21
indole points up toward the
distal end of the HA, allowing the main chain
to loop around toward the trimer interface.
However, in 18HA0, this miniloop alters the
Trp
B21
indole direction so that it now faces
the HA membrane proximal end (Fig. 2D,
right). Interestingly, the H5 cleaved structure
(21) is more similar to the cleaved H3 sub-
type (29) (RMSD 0.7 Å), even though its
nearby glycosylation sites map onto the H1
subtype reported here, whereas in the vicinity
of Trp
B21
(HA2), the 1918 H1 and avian H5
Fig. 1. Crystal structure of 1918 HA0 and comparison to other human, avian, and swine HAs. (A)
Overview of the 18HA0 trimer, represented as a ribbon diagram. For clarity, each monomer has
been colored differently [A (HA1), red; B (HA2), pink; C, dark gray; D, light gray; E, dark green; F, light
green]. Carbohydrates observed in the electron-density maps are colored orange and labeled with
the asparagine to which they are attached. E95 is not labeled because it is positioned immediately
behind C95. The locations of the three receptor-binding and the cleavage sites are indicated on only
one monomer. The basic patch is indicated in the light blue ellipse and consists of HA1 residues
His
C298
, His
C285
, His
C47
, Lys
C50
, and His
C275
(shown from left to right). This figure was generated
with Deepview (48) and rendered with Pov-Ray 3.5 (www.povray.org). (B) Structural comparison of
the 18HA0 monomer (red) with human H3 (green), avian H5 (orange), and swine H9 (blue) HAs.
Structures were first superimposed on the HA2 domain of 18HA0 through the following residues:
18HA0: A11 to A51, A276 to A324, and B1 to B160; H3 (PDB ID code: 2hmg): A11 to A51, A276
to A324, and B1 to B160; H5 (PDB ID code: 1jsm): A1 to A41, A276 to A324, and B1 to B160; and
H9 (PDB ID code: 1jsd): A1 to A41, A267 to A315, and B1 to B160. Figure B was generated with
VMD (47) and rendered with Tachyon (49).
R EPORTS
www.sciencemag.org SCIENCE VOL 303 19 MARCH 2004 1867
on June 21, 2010 www.sciencemag.orgDownloaded from

residues are virtually identical (Fig. 3, A and
B). Thus, local sequence differences do not
easily explain the different cleavage loop
conformations. However, a major difference
is the HA0 crystallization conditions (pH 5.5
for H1 and pH 7.5 for H5). At low pH, HA0
is reported to be stable (30), and the 18HA0
structure confirms this assertion. Once
cleaved, HA is metastable at low pH and
undergoes irreversible conformational chang-
es, a prerequisite for membrane fusion and
infection (31). What exactly triggers this
change is still not clear because only the pre-
and postacidification conformations have
been determined (14, 32).
However, the differences between H1 and
H5 structures around HA2 Trp
B21
and the
cleavage site may hint at a possible mecha-
nism for fusion that until now was not appar-
ent from other structures crystallized at pH
7.5. In H1 and H5 structures, HA2 Trp
B21
is
surrounded by three pH-sensitive histidines
(His
A18
, His
A38
, and His
B111
) that form a
largely uncharged pocket at neutral pH (Fig.
3A). These histidines are conserved in human
H1, H2, and H5 sequences (www.flu.lanl.
gov), but only at two positions in 1999 H9
sequences (His
A38
and His
B111
), and at one
position (His
A18
) in human H3. Below pH
6.0, this pocket becomes positively charged,
and may account for the conformation differ-
ences observed between 18HA0 and H5 (Fig.
3B). In 18HA0, His
A38
points toward the tip
of the novel loop (Ile
B10
to Trp
B21
) and the
His
A18
backbone hydrogen bonds to the main
chain at Trp
B21
. Such differences may re-
veal a mechanism (yet to be tested experi-
mentally) for destabilization or even expul-
sion of the cleaved fusion peptide from the
electronegative cavity in H1 HAs. In this
uncleaved structure, the glycoprotein may
not undergo its full rearrangement because
of the physical connection of the HA2 fu-
sion peptide to HA1.
The primary event in influenza infection
is the binding of the virus to the host receptor.
The HA receptor-binding site is situated in a
shallow pocket in the membrane-distal HA1
domain. The nature of the receptor sialic acid
linkage to the vicinal galactose is the primary
determinant in lung epithelial cells that dif-
ferentiates avian viruses from mammals (spe-
cies barrier). Avian viruses preferentially
bind to receptors with an ␣2,3 linkage,
whereas human-adapted viruses are specific
for the ␣2,6 linkage (33–35). In particular,
residues 226 and 228 have been linked to
receptor specificity (36, 37) and are Gln
226
and Gly
228
in avian viruses but Leu
226
and
Ser
228
in human-adapted H3 viruses (Fig. 4).
On the contrary, in 18HA and other human
H1 viruses, avian-type residues predominate.
Despite these binding-sequence correlations,
human H1s, such as A/PR/8/34 and A/FM/1/
47, can bind sialic acid receptors with both
␣2,3 and ␣2,6 linkages, albeit with reduced
affinity for the latter (38, 39). The only dif-
ference between swine- and swine-avian–
adapted H1 viruses is a Glu
190
3 Asp
190
mu-
tation (4), that, although subtle, leads to a
slight increase in the pocket size (upper left
Fig. 2. Structural comparison of the 18HA0 cleavage site with other HAs. HA2 domains for human H3
HA0 (PDB ID code: 1ha0) and cleaved avian H5 HA1/HA2 (PDB ID code: 1jsm) (50) were aligned with
18HA0. The cleavage sites are colored (A) green for human H3 HA0, (B) red for 18HA0 and (C) orange
for H5 HA1/HA2. R
A329
Q, Arg
A329
3 Gln
A329
.(D) Overlay of cleavage loops of H3 HA0, H1 HA0, and
avian H5 HA1/HA2. The two views differ by a rotation of 90° about the threefold vertical axis. (E)
Surface views showing the trimer interface and the position of the cleavage loop. (F) Removal of the
cleavage loop reveals the electronegative cavity that it masks. Arg
329
is colored blue and N-acetyl-
glucosamines, indicating the nearby glycosylation sites, are colored gold. (A) to (D) were generated as
in Fig. 1, and (E) and (F) were generated with MSMS (46) through the program VMD (47).
Fig. 3. Structural com-
parisons of the environ-
ment around HA2 Trp
21
in 18HA0 and H5 HA1/
HA2. The avian H5
structure (PDB ID code:
1jsm) was aligned with
the 18HA0 model for
comparison, as in Fig. 2.
In the avian structure
(A), His
A18
and His
A38
are ⬃3.7 Å apart,
whereas in 18HA0 (B),
the same residues are
⬃13.5 Å apart. The
Trp
B21
“flip” in 18HA0 is
stabilized by close proximity to the side chains of Trp
B14
and Ala
B36
. This figure was generated in the same
way as Fig. 1A.
R EPORTS
19 MARCH 2004 VOL 303 SCIENCE www.sciencemag.org1868
on June 21, 2010 www.sciencemag.orgDownloaded from

side in Fig. 4) that could perhaps increase
affinity for ␣2,6 linkages.
Comparison of 18HA0 structure with
other subtypes reveals that the receptor-
binding site is more akin to avian than to
human HAs (Fig. 4). The 18HA0 pocket is
narrower than human H3 and swine H9
HAs, consistent with previous reports that a
reduced width of the avian receptor-
binding site enhances interaction of Gln
226
with Ala
138
with ␣2,3-linked disaccharides
(15, 40). The question of how 18HA so
efficiently infected humans remains open
and will require crystal structures with
bound ligands. Other, as yet unidentified,
18HA properties may also facilitate infec-
tion. A noteworthy feature is a second
patch of exposed ionizable histidines on the
HA1 chain adjacent to the vestigial esterase
domain (E region in Fig. 1B). Four HA1
histidines (His
A47
, His
A275
, His
A285
, and
His
A298
) and a lysine (Lys
A50
) (Fig. 1A
and fig. S4) contribute to a very basic
patch, not observed in other HA structures
(fig. S5) (41, 42). For example, H3 sub-
types have a glycosylation site at position
285 that masks this region. Other viruses,
such as the vesicular stomatitis virus, are
reported to depend on histidine protonation
for membrane fusion (43). Thus, the pH-
sensitive electrostatic properties of this region
in 18HA may also assist in the membrane-
fusion event, giving the virus a selective advan-
tage during infection. Clearly, experimental
testing of such a proposal is required.
Four antigenic sites for H1 HAs, includ-
ing 18HA, have been identified (Ca, Cb,
Sa, and Sb) (4, 44). In 18HA0, with the
exception of Ca, all are exposed for anti-
body recognition (fig. S6). The Ca site is
proximal to the oligosaccharide at HA1
Asn
95
, which may interfere with antibody
recognition of both subregions, Ca1 and
Ca2. In other reported HA structures, only
H9 has a glycosylation site at this position
(21). Otherwise, the closest relative (94%
identity) to the 1918 HA is the 1930 swine
virus (A/swine/Iowa/15/30) that is believed
to have evolved from the 1918 virus (45).
Because the life-span of swine is short,
immunological drift is much slower, and as
a result, any differences between these
1918 and 1930 viruses are minimized. Un-
fortunately, because no virus samples exist
for comparison from before 1918, it is dif-
ficult to reconstruct the data necessary to
fully explain the pathogenicity of this virus.
However, statistics reveal that people over 65
years old in 1918 were no more at risk than
for a normal pandemic (2), which suggests
that people born before ⬃1855 may have
acquired some resistance to a related H1 virus
or other cross-reactive subtype (44).
The publication of the first sequences
(4) of the 1918 HA did not reveal any
characteristics that were obviously respon-
sible for the extreme pathogenicity of the
1918 pandemic, such as the polybasic res-
idues that make avian viruses so lethal.
Notwithstanding, recent data suggest that,
when expressed on a mouse-adapted WSN
viral backbone (A/WSN/33, H1N1 virus),
18HA is more virulent than a control H1
(A/New Caledonia/20/99) (17). The struc-
tural analysis here reveals a viral antigen
with a number of previously unobserved
features that may have contributed to al-
tered cleavage properties and/or fusion
propensity. Such characteristics may have
endowed the virus with unusual mecha-
nisms, which have not been seen in subse-
quent infections, that enhanced host-cell
infection, particularly in those individuals
with no previous exposure to an antigeni-
cally similar virus, which could have pro-
vided some antibody protection. Finally,
previously unobserved aspects of the ex-
pression system used here provide impor-
tant methodological advances for future
production of unprocessed HAs and other
homotrimeric viral coat proteins, such as
human immunodeficiency virus–1 gp41,
for which additional structural information
is urgently needed.
References and Notes
1. A. H. Reid, J. K. Taubenberger, T. G. Fanning, Microbes
Infect. 3, 81 (2001).
2. What distinguished this pandemic from all others was
the high proportion of deaths among young adults. For
a typical influenza epidemic, a plot of age versus death
rate is usually U-shaped, meaning that the very young
and old are in the high-risk groups. For the 1918 pan-
demic, the graph was “W”-shaped, with a sharp peak
corresponding surprisingly to a high death rate among
15 to 34 year olds (3). Mortality rates were severe, over
2.5%, compared with 0.1% for more modern epidemics.
Some isolated populations, such as communities of
Alaskan Eskimos, experienced mortality rates above
70%. Most of these deaths occurred in young adults,
between 15 and 34 years of age, around 20 times as
high as in previous years, with 99% of excess deaths
among people under 65 years of age.
3. A. H. Reid, J. K. Taubenberger, Lab. Invest. 79, 95 (1999).
4. A. H. Reid, T. G. Fanning, J. V. Hultin, J. K. Tauben-
berger, Proc. Natl. Acad. Sci. U.S.A. 96, 1651 (1999).
5. A. H. Reid, T. G. Fanning, T. A. Janczewski, J. K. Tauben-
berger, Proc. Natl. Acad. Sci. U.S.A. 97, 6785 (2000).
6. C. F. Basler et al., Proc. Natl. Acad. Sci. U.S.A. 98,
2746 (2001).
7. A. H. Reid, T. G. Fanning, T. A. Janczewski, S. McCall,
J. K. Taubenberger, J. Virol. 76, 10717 (2002).
8. K. Subbarao et al., Science 279, 393 (1998).
9. M. Peiris et al., Lancet 354, 916 (1999).
10. J. C. de Jong et al., Ned. Tijdschr. Geneeskd. 147,
1971 (2003).
11. R. J. Webby, R. Webster, Science 302, 1519 (2003).
12. D. C. Wiley, J. J. Skehel, Annu. Rev. Biochem. 56, 365
(1987).
13. H. Kido, K. Sakai, Y. Kishino, M. Tashiro, FEBS Lett.
322, 115 (1993).
14. I. A. Wilson, J. J. Skehel, D. C. Wiley, Nature 289, 366
(1981).
Fig. 4. Structural comparison of
HA receptor-binding sites. (left)
18HA0 receptor-binding site
showing key conserved HA1 res-
idues that determine receptor
specificity. The H3 and H5 sub-
types are shown for comparison.
(right) Corresponding solvent-
excluded surfaces [probe 1.4 Å,
calculated with the program
MSMS (46)] of the receptor-
binding sites showing the surface
cavity for binding the host re-
ceptor sialic acid (51). Clearly,
the narrower 1918 HA0 binding
site (top) resembles the avian
H5 structure (bottom), rather
than more open human H3
(middle) or swine H9 binding
site (25). The Glu
190
3 Asp
190
(E190D) mutation slightly in-
creases the width of the 1918
H1 binding site compared with
avian H5. Such a small change
may allow accommodations of
different conformations of ␣2,6-
versus ␣2,3-linked sugars. This fig-
ure was generated with VMD (47)
and rendered with Tachyon (49).
R EPORTS
www.sciencemag.org SCIENCE VOL 303 19 MARCH 2004 1869
on June 21, 2010 www.sciencemag.orgDownloaded from

15. Y. Ha, D. J. Stevens, J. J. Skehel, D. C. Wiley, Proc.
Natl. Acad. Sci. U.S.A. 98, 11181 (2001).
16. P. B. Rosenthal et al., Nature 396, 92 (1998).
17. T. M. Tumpey et al., Proc. Natl. Acad. Sci. U.S.A. 99,
13849 (2002).
18. Materials and methods are available as supporting
material on Science Online.
19. 18HA0 at a concentration of 10 to 15 mg/ml was
used to grow crystals in sitting drops with a precip-
itant solution of 1.68 M sodium dihydrogen phos-
phate, 0.32 M dipotassium hydrogen phosphate, and
0.1 M phosphate-citrate, with pH 5.5 (18).
20. Swine H9 HA [Protein Data Bank (PDB) identification
(ID) code: 1jsh] was used as the initial MR model. The
final R factor R
cryst
and free R factor R
free
values (table
S1 legend) are 27.0 and 29.6%, respectively, with only
three residues (0.7%) per monomer (Ile
B10
, Met
B17
, and
Asn
B60
) in the disallowed regions of the Ramachandran
plot. Only Met
B17
has good density and is positioned at
the tip of a loop, proximal to the membrane-fusion
loop. The crystal asymmetric unit contains one ho-
motrimer (502 HA0-encoded residues per monomer)
with an estimated solvent content of 66%, based on a
Matthews’ coefficient (V
m
), of 3.57 Å
3
/dalton. For com-
parison with previous structures, the HA0 sequence is
numbered in the same way as the H3 subtype (14) and
labeled HA1 11 to 329 and HA2 1 to 175, even though
the covalent bond still links HA1 to HA2. Thus, the H1
1918 HA0 structure begins at residue 11 because of an
insertion of 10 residues in H3. Insertions in H1 relative
to H3 are labeled by the preceding residue with a letter
(e.g., Asn
19A
). The three HA0 chains in the trimer are
labeled A, C, and E (HA1) and B, D, and F (HA2). The
electron density maps revealed only two disordered
regions around residues 78 to 81 of chains HA1 and 10
to 14 of HA2 that, although in disparate regions of the
monomer, are in close proximity to other symmetry-
related molecules in the crystal. Although B values were
high in this region, main-chain atoms could be inter-
preted from the electron density in both regions, except
at Gly
12
and Gly
13
in the HA2 chain. Occupancy and B
values were set to zero for side-chain atoms that were
uninterpretable from the electron density.
21. Y. Ha, D. J. Stevens, J. J. Skehel, D. C. Wiley, EMBO J.
21, 865 (2002).
22. Angles of rotation reported here for the H5 and H9
subtypes are less than those in (21). Our method of
analysis, as described in the legend for Fig. 1, was
different but reports a similar trend and reveals a
rotation for the 18HA0 as seen for the avian subtype.
23. J. Chen et al., Cell 95, 409 (1998).
24. Electron density of the main chains around Arg
329
was well defined and could be traced through to
residue 10 in HA2, at which point the electron den-
sity became disordered through to residue 14.
25. J. Stevens et al., data not shown.
26. The pH ranges from almost neutral on exiting the
endoplasmic reticulum to pH 5.9 within the trans-
Golgi network and as low as pH 5.4 in the secretory
vesicles (27, 28).
27. N. Demaurex, W. Furuya, S. D’Souza, J. S. Bonifacino,
S. Grinstein, J. Biol. Chem. 273, 2044 (1998).
28. M. Grabe, G. Oster, J. Gen. Physiol. 117, 329 (2001).
29. RMSD was calculated by overlapping C␣’s at A312 to
A324 and B1 to B22 of H3 (PDB ID code: 2hmg) with
residues A309 to A321 and B1 to B22 of H5 (PDB ID
code: 1jsm).
30. C. Bo¨ttcher, K. Ludwig, A. Herrmann, M. van Heel, H.
Stark, FEBS Lett. 463, 255 (1999).
31. J. J. Skehel et al., Proc. Natl. Acad. Sci. U.S.A. 79, 968
(1982).
32. P. A. Bullough, F. M. Hughson, J. J. Skehel, D. C. Wiley,
Nature 371, 37 (1994).
33. R. J. Connor, Y. Kawaoka, R. G. Webster, J. C. Paulson,
Virology 205, 17 (1994).
34. G. N. Rogers, B. L. D’Souza, Virology 173, 317 (1989).
35. J. J. Skehel, D. C. Wiley, Annu. Rev. Biochem. 69, 531
(2000).
36. G. N. Rogers et al., Nature 304, 76 (1983).
37. C. T. Hardy, S. A. Young, R. G. Webster, C. W. Naeve,
R. J. Owens, Virology 211, 302 (1995).
38. G. N. Rogers, J. C. Paulson, Virology 127, 361 (1983).
39. Y. Suzuki et al., Biochim. Biophys. Acta 903, 417
(1987).
40. The widths of the binding pockets were calculated by
measuring the distance from Gly
A134
C␣ to Gln
A222
C␣ for 18HA0 (13.9 Å), Gly
A134
C␣ to Gln
A226
C␣ for
human H3 (15.2 Å), Gly
A130
C␣ to Gln
A222
C␣ for
avian H5 (14.0 Å), and Gly
A128
C␣ to Gln
A216
C␣ for
swine H9 (15.2 Å).
41. Only one other sequence in the current influenza
database of human H1, H2, H3, H5, and H9 subtypes
possesses the same patch (H1; A/Alma-Ata/1417/84
virus) (42).
42. A. B. Beklemishev et al., Mol. Gen. Mikrobiol. Virusol.
1, 24 (1993).
43. F. A. Carneiro et al., J. Biol. Chem. 278, 13789 (2003).
44. G. G. Brownlee, E. Fodor, Philos. Trans. R. Soc. London
Ser. B 356, 1871 (2001).
45. R. E. Shope, J. Exp. Med. 63, 669 (1936).
46. M. F. Sanner, A. J. Olson, J. C. Spehner, Biopolymers
38, 305 (1996).
47. W. Humphrey, A. Dalke, K. Schulten, J. Mol. Graph.
14, 33 (1996).
48. N. Guex, M. C. Peitsch, Electrophoresis 18, 2714
(1997).
49. J. E. Stone, thesis, University of Missouri (1998).
50. Residues 311 to 324 and 350 to 357 from the human
H3 subtype (PDB ID code: 1ha0) and A308 to A321
and B21 to B28 from the avian H5 subtype (PDB ID
number: 1jsm) were aligned with residues A311 to
A324 and B21 to B28 of 18HA0.
51. Residues A95 to A99, A128 to A161, A179 to A200,
and A220 to A230 from 18HA0; A95 to A99, A128 to
A161, A179 to A200, and A220 to A230 from the
human H3 subtype (PDB ID code: 2hmg); A87 to A92,
A124 to A156, A175 to A196, and A216 to A226
from the avian H5 subtype (PDB ID code: 1jsm); A87
to A92, A122 to A150, A169 to A190, and A210 to
A220 from the swine H9 subtype (PDB ID code: 1jsd)
were aligned and surfaces were generated with
MSMS (46) through the program VMD (47).
52. I.A.W. is supported by NIH grants CA55896 and
AI42266 and National Institute of General Medical
Sciences (NIGMS) grant P50-GM 62411. P.P. and
C.F.B. are both supported by NIH grants. C.F.B. is an
Ellison Medical Foundation New Scholar in Global
Infectious Diseases. P.P. is an Ellison Medical Founda-
tion Senior Scholar. J.K.T. is supported by NIH grant
AI50619 and by the intramural funds of the Armed
Forces Institute of Pathology. This work was carried
out in the framework of a multidisciplinary influenza
consortium with a pending NIH grant AI058113-01.
We thank the staff of the Stanford Synchrotron
Radiation Laboratory (SSRL) Beamline 9-2 for the
beamline assistance; X. Dai and T. Horton ( The
Scripps Research Institute) for expert technical assist-
ance; and R. Stanfield, M. Elsliger, and D. Zajonc ( The
Scripps Research Institute) for helpful discussions.
This is publication 16185-MB from The Scripps Re-
search Institute. Coordinates and structure factors
have been deposited in the PDB (ID code 1RD8).
Supporting Online Material
www.sciencemag.org/cgi/content/full/1093373/DC1
Materials and Methods
Figs. S1 to S6
Tables S1 to S3
References and Notes
6 November 2003; accepted 7 January 2004
Published online 5 February 2004;
10.1126/science.1093373
Include this information when citing this paper.
Conserved Genetic Basis of a
Quantitative Plumage Trait
Involved in Mate Choice
Nicholas I. Mundy,
1
* Nichola S. Badcock,
2
Tom Hart,
2
Kim Scribner,
3
Kirstin Janssen,
4
Nicola J. Nadeau
1
A key question in evolutionary genetics is whether shared genetic mechanisms
underlie the independent evolution of similar phenotypes across phylogeneti-
cally divergent lineages. Here we show that in two classic examples of melanic
plumage polymorphisms in birds, lesser snow geese (Anser c. caerulescens) and
arctic skuas (Stercorarius parasiticus), melanism is perfectly associated with
variation in the melanocortin-1 receptor (MC1R) gene. In both species, the
degree of melanism correlates with the number of copies of variant MC1R
alleles. Phylogenetic reconstructions of variant MC1R alleles in geese and skuas
show that melanism is a derived trait that evolved in the Pleistocene.
The genetic basis of independent origins of
the same phenotype is important to models of
phenotypic evolution. There are few data,
especially for vertebrates, because the loci
underlying phenotypic evolution in natural
populations are rarely known. The lesser
snow goose (Anser c. caerulescens) and arc-
tic skua (or parasitic jaeger, Stercorarius
parasiticus) have prominent melanic plum-
age polymorphisms (Fig. 1) showing clinal
variation in the frequency of melanic morph
phenotypes across their arctic breeding rang-
es (1, 2). In both species, there is quantitative
variation in the degree of melanism among
adult individuals with the melanic phenotype
(“blue” snow geese and “intermediate” and
“dark” skuas) and discrete separation be-
tween these and the nonmelanic phenotypes
(“white” geese and “pale” skuas).
These polymorphisms influence mate
choice. In snow geese, mate color preference
follows parental color, leading to assortative
1
Department of Zoology, University of Cambridge,
Cambridge CB2 3EJ, UK.
2
Department of Biological
Anthropology, University of Oxford, Oxford OX2
6QS, UK.
3
Department of Fisheries and Wildlife and
Department of Zoology, Michigan State University,
East Lansing, MI 48824, USA.
4
Department of Molec-
ular Biotechnology, University of Tromsø, N-9037
Tromsø, Norway.
*To whom correspondence should be addressed. E-
mail: nim21@cam.ac.uk
R EPORTS
19 MARCH 2004 VOL 303 SCIENCE www.sciencemag.org1870
on June 21, 2010 www.sciencemag.orgDownloaded from