Professional Documents
Culture Documents
1985
0 1985 by The American Society of Biological Chemists, Inc. Printed in U.S.A.
Human and animal influenza A isolates of the H3 origin observed for virus isolates bearing the H3 hemagglu-
serotype preferentiallybind SAa2,6Gal or SAa2,3Gal tinin (4). Thehuman isolates of this serotype exhibit strong
linkages(where SA representssialicacid), respec- preferential binding to oligosaccharides terminating in
tively, on cell-surface sialyloligosaccharides. Previ- SAa2,6Ga11 sequences, while avian and equine H3 isolates
ously, we have demonstrated selection of SAa2,3Gal- preferentially bind SAa2,3Gal sequences. Furthermore, bind-
specific receptor variants of several human viruses
which differed from the parent viruses by a single ing of cell-surface receptors by the human H3 isolates is very
amino acid at residue 226 of the hemagglutinin which sensitive to inhibition by a glycoprotein presentin horse
is located in the receptor binding pocket (Rogers, G. serum, a*-macroglobulin, whereas avian and equine isolates
N., Paulson, J. C., Daniels, R. S., Skehel, J. J., Wilson, are not inhibited (7,8). Thus,by growing human H3 isolates
I. A., and Wiley, D. C. (1983) Nature 304, 76-78). in the presence of horse serum, it has been possible to select
In this report, the selection in the reverse direction variants with.receptor properties that closely resemble those
was accomplished starting with a SAa2,3Gal-specific of avian and equine H3 isolates, i.e. that recognize oligosac-
avian virus, A/duck/Ukraine/l/63 (H3N7), yielding charides terminated by the SAa2,3Gal linkage and are insen-
SAa2,bGal-specific variants that exhibit the receptor sitive to inhibition by equine a*-macroglobulin. Sequence
binding properties characteristic of the human isolates. analysis of the genes for the hemagglutinins of these viruses
Selection was again mediated at residue 226 of the revealed that they differed by a single nucleotide resulting in
hemagglutinin, in this case changing from Gln in the an amino acid change at residue 226 from leucine in the
parent virusto Leu in the variants. parental (SAa2,bGal-specific)phenotype to glutamine in the
Although the SAa2,BGal-specific avian virus var-
iants were stableto passage in MDCK cells, they ex- variant with the avian type (SAa2,3Gal-specific) receptor
hibited dramatic reversion to the SAa2,3Gal-specific specificity (7). In the three-dimensional structure of the H3
phenotype of the parent virus during a single passage hemagglutinin reported by Wilson et al. (13), it was deter-
in chicken embryos. Thiswasincontrasttothe mined that amino acid 226 is located in the receptor binding
SAa2,6Gal-specific human virus isolates which were pocket in the distal globular region of the molecule. How the
stable to passage in both hosts. The reversion of the difference of a single amino acid a t this site can mediate
avian virus variants ineggs provides compelling evi- dramatic changes in receptor binding specificity is still not
dence for host-mediated selection of influenza virus clear.
receptor variants. This report describes a novel approach to the selection of
receptor variants from an avian influenza virus, A/duck/
Ukraine/l/63(H3N7), distinguished from the parental,
SAa2,3Gal-specific, virus by their ability to bind SAa2,6Gal
Influenza viruses exhibit considerable diversity in their sequences of cell-surface oligosaccharides. As reported for
ability to recognize specific sialyloligosaccharidestructures as receptor variants of the human H3 isolates (7), thehemagglu-
cell-surface receptor determinants (1-4), and it has become tinins of these avian variants differ from the parent by a
increasingly evident from laboratory models (5-8) that such single amino acid, at residue 226, located in the receptor
specificity can provide the basis for changes in receptor bind-
binding pocket. Unlike the human H3 isolates, however, the
ing that have been reported to occur during host adaptation
SAa2,6Gal-specificvariants of A/duck/Ukraine/l/63 rapidly
(9-12). Indirect evidence that receptor binding properties may
be important in the ecology of influenza viruses comes from revert to the parental, SAa2,3Gal-specific, phenotype when
the correlation between receptor specificity and species of grown in the egg allantois but notwhen grown in mammalian
cell culture. While the mechanism for this receptor shift
* This work was supported by United States Public Health Service remains unclear, these results appear to present a very clear
Research Grant AI-16165 to J. C. P., National Institutes of Health
Grant AI-13654 to D. C. W., and National Science Foundation Grant The abbreviations SAa2,BGal and SAa2,3Gal refer to the termi-
PC-771398 to D. C. W. (computing hardware). The costs of publica- nal sialic acid (SA) linkage commonly found on glycoprotein oligo-
tion of this article were defrayed in part by the payment of page saccharides linked to asparagine and to threonine or serine, respec-
charges. This article must therefore be hereby marked “advertise- tively (25, 26). Unless otherwise specified, SA refers to N-acetylneu-
ment” in accordance with 18 U.S.C. Section 1734 solely to indicate raminic acid (NeuAc). Other abbreviations used include: NeuGc, N-
this fact. glycolylneuraminic acid; Gal, galactose; GalNAc, N-acetylgalactosa-
11 Recipient of an American Cancer Society Faculty Research mine; GlcNAc, N-acetylglucosamine. pfu, plaque-forming units; HA,
Award. hemagglutination; HAI, hemagglutination inhibition assays.
7362
Host-mediated Selection of Influenza
Virus Receptor Variants 7363
example of host-mediated selection based on receptor speci- has proven to be a selective inhibitor of hemagglutination of
ficity. viruses specific for SAa2,3Gal, including human glycophorin
which is presumably the predominant receptor on erythro-
MATERIALS ANDMETHODS cytes. Consequently, selection of SAa2,6Gal variants from
Cells-Madin-Darby canine kidney (MDCK) cells (ATCC CCL34) SAaZ,SGal-specific avian isolates required a different ap-
were passaged and maintained in Eagle's minimum essential medium proach. The procedure used, depicted in Fig. 1,involves selec-
supplemented with 5% fetal calf serum and 100 units/ml of penicillin tive adsorption andelution of the SAa2,3Gal-specificA/duck/
G, 100 pg/ml streptomycin sulfate, and 0.25 pg/ml amphotericin B. Ukraine11163 with erythrocytes enzymatically modified to
Virus Growth-A/duck/Ukraine/1/63 (H3N7) was generously pro-
vided by Dr. Robert G. Webster, St. Jude Children's Research Hos- contain SAa2,6Gal sequences. First, Virus was adsorbed to
pital, Memphis, TN. Cloned variants of A/Memphis/102/72 (H3N2) cells under conditions of large viral excess as previously
were produced from a seed stock, also obtained from Dr. Webster, by described (7). The bound virus was eluted from cells by
passage in the absence (M1/5) or presence (Ml/HS8) of horse serum addition of C. perfringens sialidase (250 milliunits/ml) and
as previously described (8). incubation at 37 "C for 3 h. The virus released from erythro-
As standard procedure for growth in MDCK cell culture, monolay-
ers were inoculated with virus, multiplicity of infection around 0.001,
cytes was then grown in MDCK cell culture to amplify var-
and incubated at 34 "C for 36-48 h in Eagle's minimum essential iants. After four such cycles, the eluted virus was plaqued on
medium containing 2.5% bovine serum albumin and 20 pg/ml trypsin. MDCK cells without intermediate amplification. Primary
At harvest, the medium was removed and centrifuged a t 750 X g for clones were initially screened for sensitivity to inhibition of
10 min to remove cell debris. Virus preparations were stored a t -70 "C growth in MDCK cells in the presence of horse serum and,
and assayed in parallel for hemagglutination activity and infectivity finally, for receptor specificity by adsorption to derivatized
on MDCK cell monolayers in the presence of 20 pg/ml trypsin (14).
For growth of virus in ouo,0.1 ml (lo' pfu/ml) of MDCK cell erythrocytes. Of 64 primary clones, 5 were found to be specific
lysates, diluted in phosphate-buffered saline containing antibiotics for SAaZ,6Gal linkages.
(15), was injected into theallantoic cavity of 10- to 11-day-oldchicken The receptor binding characteristics of five wild type clones
embryos and incubated a t 37 "C for 48 h. At harvest, eggs were cooled and five cloned variants from A/duck/Ukraine/l/63 were
for 1 h a t -20 "C; the allantoic fluid was collected and clarified at compared to the parent virus, and the results are shown in
7500 X g for 20 min. Virus was collected by centrifugation (5000 X g
for 18 h), resuspended in phosphate-buffered saline, and stored a t Table I. Each of the virus preparations was examined for its
-25 "C. ability to agglutinate native, sialidase-treated (asialo) and
Preparation of Deriuatized Erythrocytes-The procedures for the enzymatically resialylated erythrocytes containing SAa2,3Gal
enzymatic modification of human erythrocyte oligosaccharides have or SAa2,6Gal sequences and for sensitivity to equine a*-
been previously described (4, 16). Briefly, native human erythrocytes
were treated with Vibrio cholera sialidase (GIBCO) to remove sialic
macroglobulin, the major glycoprotein inhibitor of horse
acid and abolish viral adsorption and hemagglutination. The sialic serum. The parent strain of A/duck/Ukraine/l/63-agglutin-
acid determinants were restored in a single, defined sequence by
treatment of sialidase-treated (asialo) erythrocytes with CMP-["C]
salic acid (New England Nuclear) and one of two highly purified
mammalian sialyltransferases. The sialyltransferases employed in
this study include the Galp1,GalNAc a2,3-sialyltransferase,l puri-
fied from porcine submaxillary glands (17), and the GalB1,4GlcNAc
cu2,6-sialyltransferase,purified from rat liver (18). The terminal S
sialyloligosaccharide sequence elaborated and the amount of sialic
KEY :
E
acid incorporated for each of these enzymes are, respectively, L
SAa2,3Galp1,3GalNAc,100-110 nmol/ml of packed erythrocytes, and
SAa2,6Galpl,4GlcNAc, 35-40 nmol/ml of packed erythrocytes.
Hemagglutination (HA) and Hemagglutination Inhibition (HAI)
Assays-HA and HA1 titers were determined in a Cooke microtiter
.(%. SAa2,6Gal RBC
RESULTS
MDCKCELLS
Selection of Receptor Variants of A/Duck/Ukraine/l/63-
FIG. 1. Selection of receptor variants by adsorption to de-
While horse serum glycoprotein inhibitors have proven useful rivatized erythrocytes. The schematic depicts a simplified eryth-
in the isolation of SAa2,3Gal-specific variants from human rocyte (RBC) enzymatically modified to contain sialic acid (SA) in
(SAa2,6Gal-specific) viruses, no glycoprotein yet examined the SAa2,6Gal linkage. Receptor variants which bind the SAa2,gGal
linkage adsorb to the erythrocytes, allowing separation from the
Sialyltransferases are distinguished by prefixes indicating their predominant parent virus with the SAa2,3Gal-specific receptor phe-
preferred acceptor sequence and the isomeric linkage formed in the notype. Subsequent elution of bound variants with bacterial sialidase
product. Thus, Galp1,BGalNAc a2,3-sialsltransferase catalvzes the followed by growth in MDCK cell culture yields a virus population
general reaction CMP-SA + Galj31,3GalNAc+SAa2,3Ga@l,3Gal- enriched in the SAa2,6Gal-specific hemagglutinin. Details of the
NAc +CMP. procedure are given in the text.
7364 Host-mediated Selection
Influenza
ofVirus Receptor Variants
TABLEI
Characterization of receptor variants derived from A/duck/Ukraine/l/63 (H3N7)
Agglutination of erythrocytes* Hemagglutination
ViNS" Amino acid at
inhibition'
Native Asialo SAa2,3Gal SAa2,BGal (eauine a,") 226dresidue
HA titer HA1 titer
A/duck/Ukraine/l/63 64 0 64 0 160 Gln
(parent)
Receptor variants
UK6 32 0 64 0 80
UK19 64 0 128 0 80 Gln
UK25 64 0 128 0 80 Gln
UK48 64 0 128 0 80
UK62 64 0 128 0 80
UK3 128 0 0 128 5120
UK16 64 0 0 64 2560
UK43 32 0 0 32 5120 Leu
UK49 64 0 0 128 2560 Leu
UK50 32 0 0 32 5120
a Receptor variants of A/duck/Ukraine/l/63 were obtained as described inthe text, plaque-purified threetimes,
and grown inMDCKcell culture. Hemagglutination titrations wereperformedwithMDCKcelllysates after
removal of cell debris by low speed centrifugation (700 X g for 10 min).
* Human erythrocytes were either unmodified (Native),treated with V. cholera sialidase (asialo),or sialidase-
treated cells resialylated with CMP-sialic acid and purified sialyltransferases as described under "Materials and
Methods." The sialyloligosaccharide structures examined are SAa2,3Galpl,3GalNAc (SAcr2,3Gal)and
SAa2,6Galp1,4GlcNAc (SAa2,gGal)commonly found as terminal sequenceson 0- and N-linked oligosaccharides,
respectively. HA titers were determined as described and are expressed asthe reciprocal of the greatest dilutionof
virus that produced agglutination(0 = <2).
Hemagglutination inhibition by purified, heat-inactivated (30 min at 56 "C) equine crz-macroglobulin (nz-M)
was performed as described. The initial concentration of inhibitor (3 mg/ml) was roughly the same concentration
found in unfractionated serum (22). Results are expressed as the reciprocal of the highestdilution of az-
macroglobulin causing inhibitionof native erythrocyte agglutination by 4 hemagglutinating units of virus.
Deduced from the nucleotide sequences of the HAI region of the hemagglutinin genes. Nucleotide sequences
were determined usingthe dideoxynucleotide chain termination method with a primer extension system containing
virus RNA, reverse transcriptase, and 5'-32P-labeledsynthetic oligonucleotide primers (7). The only amino acid
sequence changes detected were at residue 226 as a result of changes in the triplet nucleotides 754-756 from CAG
(ghtamine, G k ) to CTG (leucine,Leu).
ated erythrocytes derivatizedto contain theSAaB,3Gal link- bindingproperties,revertingtothe wild type SAa2,3Gal-
age did not agglutinate SAa2,6Gal-derivatized cells and was specific, inhibitor-insensitive phenotype.T o further examine
not sensitive to hemagglutination inhibition by equine ap- this phenomenon, variants which had been plaque-purified
macroglobulin. The five wild type clones exhibited receptor (four successive plaque to plaque passes) and passaged once
properties identical to those of the parent strain. In contrast, in MDCK cultures were seeded at various concentrations,
five variants agglutinatedcells modified to containSAa2,GGal ranging from lo7 to 10 plaque forming units, in embryonated
linkages did not agglutinate SAa2,3Gal-derivatized cells and eggs and MDCK monolayers and grown for various lengths
were sensitive to hemagglutination inhibition by equine az- of time. Table I1 shows hemagglutination titrations, using
macroglobulin. The receptor binding propertiesof these var- derivatized red blood cells, of representative SAa2,3Gal-spe-
iants of A/duck/Ukraine/l/63, therefore, are very similar t o cific and SAa2,6Gal-specific cloned viruses after growth in
those previously associated with the corresponding receptor the egg allantois (20 h a t 37 "C) or MDCK cell culture (har-
type isolated for the human H3 isolates (4, 7,8). vested at peak yield). The virus preparations were concen-
The complete amino acid sequences of the HAI region of trated approximately lOO-fold, by differential centrifugation,
the hemagglutinins expressed by the parent strain and rep- prior t o assay to eliminate potential inhibitorsof agglutina-
resentative SAa2,3Gal- and SAa2,6Gal-specific clones were tion thatmay be present in allantoic fluid. Progeny from both
deduced from thenucleotide sequences of their RNAgenes as of the SAa2,3Gal-specific isolates (UK19 and UK25) prefer-
previouslydescribed (7). The only aminoacid sequence entially agglutinated
erythrocytes modified tocontain
changes detectedwere at residue 226. As summarized in Table SAa2,3Gal determinants whether grown in MDCK cells or
I, amino acid 226 was glutamine in the parent strain and in the embryonated egg. The weak agglutination of SAa2,6Gal-
the cloned wild type viruses whilethe cloned variants specific containingerythrocytes observed in both MDCK and egg
for SAa2,6Gal determinants hadleucine at position 226. This grown virus is typical of concentrated preparations of A/
residue is located in the distal portionof the molecule in the duck/Ukraine/l/63(4).The SAa2,6Gal-specific variants
receptor pocket (13), and the presence of leucine or glutamine (UK43 and UK49), on the other hand, retained specific- their
at amino acid 226 was previously observed to account for the ity for SAa2,BGal sequences when grown in MDCK cells, but
different binding propertiesof the SAa2,BGal-specific human exhibited an apparently dose-dependent gradient of reversion
H 3 isolates and their SAa2,3Gal-specific receptor variants, from the original SAa2,6Gal-specificphenotype t o a wild type
respectively (7). SAa2,3Gal-specific virus when grown in eggs. This receptor
Changes in Receptor Specificity Associated with Growth in shift isespecially evident at the higher infectious doses where
Eggs-During growth of the avian receptor variants in em- one of the variants, UK43, agglutinatedonly SAa2,3Gal-
bryonated eggs for the purpose of sequence determination, derivatized cells and showed no agglutination of erythrocytes
the SAa2,6Gal-specific variants underwent a shift inreceptor containing SAa2,6Galsequences.
Host-mediated Selection of Influenza Virus Receptor Variants 7365
TABLEI1 the SAa2,3Gal-binding phenotype grew more rapidly in ouo
Receptor specificities of Ukraine variants afterpassage in the egg than the SAa2,gGal variants (Tables I1 and 111) and that
crllantois and MDCK cell culture selection could occur simply by the wild type virus outgrowing
Infec- UK25 I
I
UK19 II UK49 I
I
UK43 and variants.
Host tious Absence of Receptor Shift when Other Receptor Variants of
dose” a2,3 a2,6 012.3
a2,3 a2.6 a2,6 a2,6 a2,3
the H3 Serotype Are Grown in Ovo-A change in receptor
HA titerb
PfU specificity upon transfer from MDCK cell culture to growth
MDCK’ 107 4,096 128 4,096 128 o 4,096 a 4,096 in ouo is not a phenomenon observed with all SAa2,6Gal-
lo6 8,192 128 0 4,096
specific viruses of theH3 serotype. Indeed, numerous
lo6 8,192 128 0 8,192
104 8,192 128 0 16,384 SAa2,6Gal-specific human isolates have been routinely pas-
103 8,192 256 16 7,182 saged in ovo with retention of receptor specificity (4, 7). To
lo2 8,192 128 0 16,384 illustrate this, TableIV compares the receptor specificities of
10 8,192 256 0 16,384 the SAa2,6Gal- and SAa2,3Gal-specific A/duck/Ukraine/l/
EGG^ 107
63 viruses with the corresponding receptor specific viruses
lo6 derived from the human virus, A/Memphis/102/72 (€9,after
105 growth in MDCK cells or eggs. The SAa2,3Gal-specific var-
10‘ iants of A/duck/Ukraine/l/63 and A/Memphis/102/72 both
lo3 retain the specificity of MDCK grownparents asindicated by
lo2 16 agglutination of erythrocytes bearing SAa2,3Gal sequences.
10 1 As reported above for the UK43 and UK49 clones (Tables I1
“Virus seed stocks were prepared by growth of plaque-purified and 111), three other SAa2,6Gal-specific variants of Alduckl
virus (4 successive plaque to plaque passes) in MDCK cell cultures.
Infectious titers (plaque-forming units/ml) of the cell lysates were
Ukraine11163 grew in MDCK cultures, with retention of
determined as described under “Material and Methods.” These prep- specificity, but revert to the SAa2,SGal-specific wild type
arations hadlog pfu/HA ratios of 6.3 (UK19), 6.2 (UK25),6.0 (UK43), when grown in theallantoic cavity of fertile eggs. In contrast,
and 5.7 (UK49). Receptor specificity was confirmed by hemaggluti- SA2,GGal-specific clones of the human virus A/Memphis/
nation titrations with derivatized erythrocytes, and results are shown 102/72 retain theirspecificity for the SAa2,6Gal linkage when
in Table I. grown in either MDCK cell culture or the embryonated egg.
Hemagglutination titers toward resialylated SAa2,3Gal (a2,3)
and SAa2,6Gal (a2,6) erythrocytes were assessed for each virus Differences in Receptor Binding between Human andAvian
preparation as described in Table I. All preparations were concen- SAa2,6Gal-specific Variants-Since human isolates of the H3
trated 100-fold prior to assay. subtype specific for SAa2,BGal determinants readily replicate
e Seed stocks were diluted as shown and grown on MDCK mono- in eggs and the A/duck/Ukraine/l/63 variants specific for
layers in the presence of trypsin (20 pg/ml) a t 34 “C. Virus was SAa2,6Gal sequences do not, attempts were made to identify
harvested a t optimum times for each infectious dose, as determined possible differences in receptor binding between them. Asialo
by visual assessment of the cytopathic effect.
Appropriate dilutions of virus seed stocks were injected (0.2 ml) human erythrocytes weremodified to containterminal
into theallantoic cavity of 10-day-oldchicken embryos and incubated SAa2,6Gal linkages of two naturally occurring sialic acids N-
20 h at 37 “C. acetylneuraminic acid (NeuAc) and N-glycolylneuraminic
14. Tobita, K., Sugiura, A., Enomoto, C., and Furoyama, M.(1975) 26. Yoshima, H., Furthmayr, H., and Kobata, A. (1980)J. Bwl. Chem.
Immunology 162,9-14 255.9713-9718
15. Hinshaw, V. S., Webster, R. G., and Turner, B. (1978)J. Gen. 27. NaeveIC. W., Hinshaw, V. S., and Webster, R. G . (1984)J. Virol.
Virol. 41. 115-127 5 1,567-569