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Journal of Virology, August 1999, p. 6743-6751, Vol. 73, No. 8
Department of Virology and Molecular Biology,
St. Jude Children's Research Hospital, Memphis, Tennessee
381011; Center for Macromolecular
Crystallography, Department of Microbiology, University of Alabama at
Birmingham, Birmingham, Alabama 352942;
Department of Virology, Istituto Superiore di Sanita, Rome,
Italy3; Department of Biochemistry,
School of Pharmaceutical Science, University of Shizuoka, Shizuoka
422, Japan4; and Department of
Pathobiological Sciences, School of Veterinary Medicine, University
of Wisconsin, Madison, Wisconsin 537065
Received 29 January 1999/Accepted 4 May 1999
Influenza A viruses possess two glycoprotein spikes on the virion
surface: hemagglutinin (HA), which binds to oligosaccharides containing
terminal sialic acid, and neuraminidase (NA), which removes terminal
sialic acid from oligosaccharides. Hence, the interplay between these
receptor-binding and receptor-destroying functions assumes major
importance in viral replication. In contrast to the well-characterized
role of HA in host range restriction of influenza viruses, there is
only limited information on the role of NA substrate specificity in
viral replication among different animal species. We therefore
investigated the substrate specificities of NA for linkages between
N-acetyl sialic acid and galactose (NeuAc Influenza virions contain two
glycoproteins on their surface, hemagglutinin (HA) and neuraminidase
(NA), which recognize sialic acid on host cell glycoconjugates. HA
binds to cell surface sialyloligosaccharides and then mediates the
entry of the virus into the cell (23), while NA prevents the
aggregation of progeny virions by removing sialic acid on the
oligosaccharides of newly synthesized HA and NA polypeptides
(14). It also facilitates the elution of progeny virions
from infected cells by removing sialic acid from host cell glycoconjugates.
The HA receptor specificity of influenza viruses differs according to
the host species of origin (3, 15). That is, HA preferentially recognizes the sialic acid-galactose linkages expressed on cells of the host from which the virus was isolated. For example, duck intestinal epithelial cells express primarily
N-acetylneuraminic acid (NeuAc) bound to galactose through
an Influenza viruses also infect animals (e.g., pigs) that express high
levels of N-glycolylneuraminic acid (NeuGc) bound to galactose in cellular glycoconjugates. NeuGc has not been detected on
human tracheal epithelial cells but is expressed at high levels on both
swine and equine tracheal epithelial cells (19, 19a), suggesting that it may play a role in influenza virus infection of
these hosts. This notion is supported by the observation that swine
influenza viruses efficiently bind glycoconjugates that contain NeuGc,
while human viruses do not (19).
In comparison with the HA receptor specificity, information on the NA
substrate specificity of influenza A viruses is limited in terms of
correlations between the host animals from which the viruses were
isolated and the structural basis for substrate recognition. To address
these issues, we first compared the NA specificities for linkages
between sialic acid and galactose (NeuAc Viruses and cells.
Influenza A viruses used in this study
were obtained from the repository at St. Jude Children's Research
Hospital and the Istituto Superiore di Sanita. Abbreviations (used on
the figures) and subtypes of the viruses used are enclosed in
parentheses: A/duck/Hong Kong/7/75 (dk/HK/7/75) (H3N2), A/mallard/New
York/6750/78 (mal/NY/6750/78) (H2N2), A/gull/Delaware/2838/87
(gull/Del/2838/87) (H3N2), A/swine/Italy/1850/77 (sw/It/1850/77)
(H3N2), A/swine/Belgium/1/79 (sw/Bel/1/79) (H3N2),
A/swine/Finestere/55/80 (sw/Fin/55/80) (H3N2), A/swine/Netherlands/3/80
(sw/Ned/3/80) (H3N2), A/swine/Netherlands/12/85 (sw/Ned/12/85) (H3N2),
A/swine/Belgium/1/83 (sw/Bel/1/83) (H3N2), A/swine/Ukkel/1/84
(sw/Uk/1/84) (H3N2), A/swine/France/5027/87 (sw/Fr/5027/87) (H3N2),
A/swine/Italy/635/87 (sw/It/635/87) (H3N2), A/Singapore/1/57
(Sing/1/57) (H2N2), A/Ann Arbor/6/60 (AA/6/60) (H2N2), A/England/12/62
(Eng/12/62) (H2N2), A/Tokyo/3/67 (Tokyo/3/67) (H2N2), A/Berkeley/1/68
(Berk/1/68) (H3N2), A/Aichi/2/68 (Aichi/2/68) (H3N2), A/Hong Kong/1/68
(HK/1/68) (H3N2), A/Hong Kong/8/68 (HK/8/68) (H3N2), A/Korea/426/68
(Korea/426/68) (H2N2), A/Memphis/1/68 (Mem/1/68) (H3N2),
A/Netherlands/20/69 (Ned/20/69) (H3N2), A/Netherlands/84/68 (Ned/84/68)
(H3N2), A/Udorn/307/72 (Udorn/307/72) (H3N2), A/Bangkok/1/79 (Bang/1/79) (H3N2), and A/Los Angeles/2/87 (LA/2/87) (H3N2). 293T cells
were cultured in Dulbecco's modified Eagle medium supplemented with
10% fetal calf serum.
NA substrate specificity assays.
For the NA substrate
specificity assays, a concentrated stock of each virus was prepared.
Each virus was grown in 60 11-day-old embryonated chicken eggs. The
allantoic fluid was clarified by centrifugation at 5,000 × g for 15 min at 4°C. Each virus was pelleted by centrifugation
at 65,000 × g for 1 h at 4°C and resuspended in
1 ml of phosphate-buffered saline. Portions of the solution were stored
as aliquots at
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Amino Acid Residues Contributing to the Substrate
Specificity of the Influenza A Virus Neuraminidase
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
2-3Gal and
NeuAc2-6Gal) and for different molecular species of sialic acids
(N-acetyl and N-glycolyl sialic acids) in
influenza A viruses isolated from human, avian, and pig hosts.
Substrate specificity assays showed that all viruses had similar
specificities for NeuAc2-3Gal, while the activities for
NeuAc2-6Gal ranged from marginal, as represented by avian and early
N2 human viruses, to high (although only one-third the activity for
NeuAc2-3Gal), as represented by swine and more recent N2 human
viruses. Using site-specific mutagenesis, we identified in the earliest
human virus with a detectable increase in NeuAc2-6Gal specificity a change at position 275 (from isoleucine to valine) that enhanced the
specificity for this substrate. Valine at position 275 was maintained
in all later human viruses as well as swine viruses. A similar
examination of N-glycolylneuraminic acid (NeuGc)
specificity showed that avian viruses and most human viruses had low to
moderate activity for this substrate, with the exception of most human viruses isolated between 1967 and 1969, whose NeuGc specificity was as
high as that of swine viruses. The amino acid at position 431 was found
to determine the level of NeuGc specificity of NA: lysine conferred
high NeuGc specificity, while proline, glutamine, and glutamic acid
were associated with lower NeuGc specificity. Both residues 275 and 431 lie close to the enzymatic active site but are not directly involved in
the reaction mechanism. This finding suggests that the adaptation of NA
to different substrates occurs by a mechanism of amino acid
substitutions that subtly alter the conformation of NA in and around
the active site to facilitate the binding of different species of
sialic acid.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
2,3 linkage (NeuAc2-3Gal) (7), which is
preferentially recognized by duck virus HA (15). Likewise,
human virus HA preferentially binds NeuAc2-6Gal (3, 5, 15,
16), the primary linkage of sialic acid expressed on human
tracheal epithelial cells (4). Similarly, the NA specificity for NeuAc2-3Gal and NeuAc2-6Gal depends on the viral isolate examined (1, 24). The specificity of human virus N2 NA has changed over the years since its introduction from an avian virus (1). The earliest human N2 viruses, isolated in 1957, showed enzymatic activity against only NeuAc2-3Gal, while N2 viruses isolated in 1967 and 1968 showed limited activity against
NeuAc2-6Gal and still retained primary activity against
NeuAc2-3Gal. By 1972, human virus N2 NA had developed similar
specificities for both NeuAc2-6Gal and NeuAc2-3Gal. From this
finding, it was concluded that the N2 NA of human viruses had acquired
the ability to recognize the same linkage of sialic acid that is
preferentially recognized by the HA of the viruses, probably to
facilitate the efficient release of progeny virus from cells and to
prevent self-aggregation of virions.
2-3Gal versus NeuAc2-6Gal) and for different molecular species of sialic acids (NeuAc versus NeuGc) among avian, swine, and human viruses. We then
determined the amino acid residues contributing to these specificities
by using chimeric NAs and site-specific mutagenesis.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
20°C.
-D-N-acetylneuraminic acid (Sigma) in 0.2 mM potassium phosphate (pH 5.9) for 30 min at
37°C. The reaction was stopped by adding 200 µl of 0.1 M
glycine-25% ethanol (pH 10.7) per well. The fluorescence of released
4-methylumbelliferone was determined by use of a fluorometer
(Labsystems Fluoroskan II) with excitation at 355 nm and emission at
460 nm. One unit of NA enzymatic activity was defined as 1 µmol of
released NeuAc, equivalent to the amount of released
4-methylumbelliferone per min at 37°C.
2-3 (NeuAc2-3Gal) or an 2-6
(NeuAc2-6Gal) ketosidic linkage (Sigma) and (ii) GM3 gangliosides
with either the NeuAc or the NeuGc species of sialic acid. NeuAc-GM3
was purified from human liver (18), and NeuGc-GM3 was
purified from equine erythrocytes (6). Reactions were
performed by use of 50 µl of CaS buffer with 0.1 mM substrate and 10 mU of viral NA per ml. The NeuAc2-6Gal specificities of several human viruses were also determined with 100 mU of NA per ml. Reactions with GM3 gangliosides were performed with 0.1% sodium deoxycholate (SDC), required to solubilize the gangliosides. All reactions were
performed in duplicate at 37°C and various time points, typically between 15 and 60 min, and were stopped by heating at 100°C for 15 min. The amount of liberated sialic acid was determined by the
periodate-thiobarbituric acid assay (22). The colored
chromophore was extracted into 750 µl of n-butanol-5%
HCl (vol/vol), and the absorbance was measured at 549 nm. Although
these assays and subsequent assays described below were done at
multiple times, we typically selected results obtained at either 15 or
30 min, depending on the interval that fell within the linear response
range of the sialidase activity-versus-time plot. The data reported are
representative of two or three separate assays.
Cloning of NA genes. Full-length cDNAs of the NA genes from A/Singapore/1/57 (H2N2), A/England/12/62 (H2N2), and A/Tokyo/3/67 (H2N2) were amplified by PCR with oligonucleotides 5'-TCTCTTCGAGCAAAAGCAGGAGTGAAAATG-3' and 5'-ATTAACCCTCACTAAAAGTAGAAACAAGGAGTTTTTTTC-3' and Pfu DNA polymerase (Stratagene). The full-length PCR products were cloned into the pCRII vector of the TA cloning kit (Invitrogen) according to the provided instructions. The sequence of each gene was determined with N2-specific primers (sequences available upon request) and an automated sequencer (Applied Biosystems Inc., Foster City, Calif.). The NA genes of A/Singapore/1/57, A/England/12/62, and A/Tokyo/3/67 were then subcloned into the EcoRI restriction enzyme site of the plasmid expression vector pCAGGS/MCS (8, 13) to generate pCAT3DKSING57NASAP, pCAT3DKENG62NASAP, and pCAT3TOKYO67NASAP, respectively. Each gene was similarly subcloned into the EcoRI site of pUC19 to generate pUCT3DKSING57NASAP, pUCT3DKENG62NASAP, and pUCT3TOKYO67NASAP, respectively. The sequence of each cloned NA gene was confirmed to be identical to that of the wild-type gene by sequence analysis and thus did not contain errors introduced by PCR.
Generation of chimeric constructs. Using shared unique restriction enzyme sites in pUCT3DKSING57NASAP and pUCT3DKENG62NASAP, we generated five chimeric constructs in which portions of the A/England/12/62 NA gene were replaced with the corresponding region of the A/Singapore/1/57 NA gene. Sites for MamI, NheI, EcoRV, and HindII at N2 gene nucleotide positions 98, 626, 869, and 1213, respectively, and Asp718 in the pUC19 multiple cloning site (MCS) were used to generate the chimeras (see Fig. 2A). Two additional chimeras were generated to investigate individual amino acid differences between the NAs of A/Singapore/1/57 and A/England/12/62. To replace Lys258 of A/Singapore/1/57 with Glu258 of A/England/12/62, we substituted the pUTCDKENG62NASAP sequence for the NheI-BlpI fragment of pUCT3DKSING57NASAP, resulting in the pUCT3DKSING57-258E construct. Similarly, Ile275 of A/Singapore/1/57 was replaced with Val275 of A/England/12/62 by replacing the BlpI-EcoRV fragment of pUCT3DKSING57NASAP with pUTCDKENG62NASAP to generate pUCT3DKSING57-275V. Each chimeric NA was then subcloned into the EcoRI site of pCAGGS/MCS to generate pCAT3DKSING57-258E and pCAT3DKSING57-275V, respectively.
Additional chimeric constructs (designated 6 to 10) in which portions of the A/England/12/62 N2 NA gene were replaced with the corresponding region of the A/Tokyo/3/67 N2 NA gene exactly as described above were generated (see Fig. 4A).Site-directed mutagenesis of specific NA residues.
To
introduce three specific point mutations, representing nucleotides in
A/Tokyo/3/67, into A/England/12/62, we synthesized two mutagenic
primers for PCR amplifications. The first mutagenic oligonucleotide,
5'-GTCATAGTTGACAGCGATAATCGGTCAGG-3', contains the
HindIII site of the NA gene at position 1213. The
reverse primer, 5'-ATGACCATGATTACGCCAAGCTTGC-3', binds
nucleotides 445 to 469 of the pUC19 MCS. PCR was done with
Pfu DNA polymerase for 30 cycles at an annealing temperature
of 60°C and with pUCT3DKENG62NASAP as a template. A PCR fragment
containing nucleotide mutations G1221
A and T1227C was generated,
resulting in amino acid mutations Asn401Asp and Trp403Arg,
respectively. This fragment was subcloned into the
HindII site (nucleotide position 1213 of the NA gene) and SalI (MCS) site of pUCT3DKENGNASAP to generate
pUCT3DKENG-401D,403R. Using the second mutagenic primer,
5'-GTCATAGTTGACAGTAATAATTGGTCAGG-3', and pUCT3TOKYO67NASAP
as a template, we generated a PCR fragment containing nucleotide
mutations G1221A and C1227T, resulting in amino acid mutations
Asp401Asn and Arg403Trp, respectively. This fragment was
subcloned into pUCT3DKENG62NASAP and had the net effect of introducing
Gln431Lys into the A/England/12/62 NA protein, generating the mutant
NA construct pUCT3DKENG-431K. pUCT3DKENG-401D,403R and
pUCT3DKENG-431K were each subcloned into the EcoRI
site of pCAGGS/MCS to generate pCAT3DKENG-401D,403R and
pCAT3DKENG-431K, respectively. Introduction of only the desired mutation in each final construct was confirmed by sequence analysis of
the entire region generated by PCR.
Substrate specificity of cell-expressed NA.
The pCAGGS/MCS
expression plasmid construct for each NA gene or chimeric construct was
expressed in 293T cells. Cells at 70 to 80% confluency were
transfected with 2 µg of each plasmid DNA per well of a six-well
tissue culture plate by use of Lipofectamine (Life Technologies Inc.).
After incubation for 40 h at 37°C, the cells were washed from
the plate, washed once with phosphate-buffered saline, and then
resuspended in CaS buffer. Dilutions of the cell suspensions were
prepared in CaS buffer and assayed for NA enzymatic activity with the
substrate
2'-(4-methylumbelliferyl)--D-N-acetylneuraminic acid as described above. On the basis of the calculated activity of
each expressed NA, 0.5 mU of NA activity per reaction was used to
determine the substrate specificity of each expressed NA. The required
volume of cell suspension was divided into aliquots, which were placed
in 1.5-ml tubes. The cells were then pelleted for 30 s at
16,000 × g. After the supernatant fluids were
aspirated, the cells were resuspended at 4°C in 50 µl of CaS buffer
containing either 0.1 mM sialyllactose or GM3 ganglioside and 0.1%
SDC. The remainder of the assay was performed as described for the
concentrated viruses. The reported data represent duplicate assays and
duplicate reactions within each assay.
NA amino acid sequence analysis. The amino acid sequences of avian, swine, and human N2 NAs were compared at positions 275 and 431. For sequences available from GenBank, the accession numbers are reported. Additional human sequences were obtained from a published sequence analysis (11). Sequences not available from GenBank or literature sources were determined by automated sequencing. The identity of amino acid 275, inferred from the nucleotide sequence, was determined with oligonucleotide 5'-GTAATGACTGATGGAAGTGC-3', which binds nucleotides 737 to 756 (human virus N2 NA sequence numbering). Similarly, the identity of amino acid 431 was determined with oligonucleotide 5'-GTAATGACTGATGGAAGTGC-3', which binds nucleotides 1148 to 1164.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Specificities of avian, human, and swine virus N2 NAs for
NeuAc2-3Gal and NeuAc2-6Gal.
A drift in the substrate
specificity of human virus N2 NAs, due to continuous replication of N2
viruses in humans since the introduction of the N2 NA from an avian
virus in 1957, has led to a higher specificity for NeuAc2-6Gal
(1). To understand the molecular basis of this change, we
examined the substrate specificities of a panel of human N2 viruses
isolated between 1957 and 1987 to identify the point at which NA first
showed a detectable increase in specificity for NeuAc2-6Gal. In
these studies, we compared avian virus N2 NAs for their ability to
recognize the NeuAc2-3Gal and NeuAc2-6Gal substrates. Swine virus
N2 NAs were also of interest because they were introduced from humans and because pig trachea contains both NeuAc2-3Gal and NeuAc2-6Gal (7). Using equal amounts of viral NAs, as determined with
the substrate
2'-(4-methylumbelliferyl)--D-N-acetylneuraminic
acid, we found that all of the viral NAs had high activity for
NeuAc2-3Gal. Within the linear response range of the NA activity
assay with NeuAc2-3Gal, the levels of sialic acid released,
expressed as the optical density at 549 nm of the sialic acid
derivative generated by the Warren assay (22), were in the
range of 0.130 to 0.255, with a random distribution of the viruses from
all three hosts within this range.
2-6Gal linkage was dependent on the
host species and, for the human viruses, on the year of isolation (Fig.
1A). Avian viruses showed very low
activity for the NeuAc2-6Gal linkage, while the two swine viruses
showed high activity for that substrate. Human viruses isolated in 1957 and 1960, soon after the first appearance of the N2 NA in human viruses, showed low activity for NeuAc2-6Gal, similar to that of the
avian viruses. To verify and extend these results, we performed additional assays with 100 mU of NA per ml to improve quantitation of
the released sialic acid (Fig. 1B) and added A/England/12/62 to the
analysis to provide an intermediate isolate. The first detectable
increase in specificity for NeuAc2-6Gal among human virus N2 NAs was
observed with A/England/12/62. There was a gradual increase in
specificity for NeuAc2-6Gal that continued until a maximal level of
activity was observed by 1972 (Fig. 1). There is some isolate-specific
variation in specificity in later isolates, as in the lower specificity
of A/Bangkok/1/79 than of A/Udorn/307/72 and A/Los Angeles/2/87, but an
overall trend toward higher activity for NeuAc2-6Gal, compared to
that of the 1957 and 1960 isolates, is maintained. These results are
similar to those of Baum and Paulson (1), who showed that
the human virus N2 NA acquired specificity for NeuAc2-6Gal, with
activities for NeuAc2-6Gal and NeuAc2-3Gal becoming approximately
equal, by 1972. In contrast to this latter finding, the maximal
activity of the N2 NA for NeuAc2-6Gal in our study was only about
30% that for NeuAc2-3Gal. In addition, our findings also indicate
that NeuAc2-6Gal specificity was preserved in pigs after the
introduction of the N2 NA from humans.
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Region of NA important for NeuAc2-6Gal substrate
specificity.
To identify the region of the human virus N2 NA
protein that confers increased specificity for NeuAc2-6Gal, we
studied chimeric constructs representing A/England/12/62, the virus
showing the first incremental increase in specificity, and
A/Singapore/1/57, one of the first N2 NA-containing human viruses. In
the five constructs, sequential portions of A/England/12/62 N2 NA were
replaced by equivalent regions from A/Singapore/1/57 N2 NA (Fig.
2A). Each chimera NA and the parental
virus NA were individually expressed in 293T cells, and the substrate
specificity of the expressed protein was determined. The parental virus
and chimeric NAs all exhibited similar specificities for NeuAc2-3Gal
(data not shown). As was observed with the NA from concentrated virus,
the N2 NA of A/Singapore/1/57 showed very low specificity for
NeuAc2-6Gal, in contrast to N2 NA of A/England/12/62, whose
specificity for this linkage was approximately 2.5- to 3.0-fold higher
(Fig. 1B and 3B). Chimeras 1, 3, 4, and 5 had NeuAc2-6Gal specificities similar to that of parental virus
A/England/12/62, although chimera 2 had a lower specificity for
NeuAc2-6Gal, similar to that of parental virus A/Singapore/1/57.
This result suggested that the region of A/England/12/62 NA responsible
for the higher NeuAc2-6Gal specificity has been replaced by the
corresponding region of A/Singapore/1/57 NA in chimera 2. Conceivably,
the lower NeuAc2-6Gal specificity of chimera 2 could have been
caused by a global perturbation in the NA structure due to transfer of
an A/Singapore/1/57 NA sequence into A/England/12/62 NA. This
possibility is unlikely, as the NA activity for NeuAc2-3Gal remained
similar to that of the parental viruses and the other chimeras.
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; pCAT3DKENG62NASAP) were replaced with the
sequences of A/Singapore/1/57 N2 NA (
; pCAT3DKSING57NASAP) by use of
shared restriction sites to generate chimeras 1 to 5. Amino acid
mutations K258
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Amino acid residues affecting NeuAc2-6Gal specificity.
The
region exchanged in chimera 2 (amino acid residues 204 to 283)
contained two amino acid differences between the two parental virus NAs
(Table 1). In A/England/12/62 NA, a
glutamate had replaced lysine at position 258, and a valine had
replaced isoleucine at position 275. To determine if one or both of the
two residues are responsible for the higher NeuAc2-6Gal specificity
of A/England/12/62 NA, we generated two site-specific A/Singapore/1/57
NA mutants. Residues 258 and 275 of A/Singapore/1/57 NA were separately
exchanged with those of A/England/12/62 NA, introducing valine at
position 275 and lysine at position 258 to yield constructs
pCAT3DKSING57-275V and pCAT3DKSING57-258E (pCAGGS/MCS
expression vector constructs), respectively. In 293T cells, the
expressed proteins had high levels of sialidase activity for
NeuAc2-3Gal, as did the parental virus NAs (data not shown).
Compared with the situation for A/Singapore/1/57 NA, the amino acid
substitution at position 258 did not affect NA activity for
NeuAc2-6Gal (Fig. 2C). On the other hand, the substitution of valine
for isoleucine at position 275 increased NA activity for NeuAc2-6Gal
to about 65% that of A/England/12/62 NA, indicating that the residue
at this position plays a significant role in determining the
specificity of the N2 NA for NeuAc2-6Gal.
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Substrate specificities of avian, swine, and human virus NAs for
NeuGc.
Another species of sialic acid that is expressed at high
levels on the cells of some host species infected by influenza A viruses is NeuGc attached to galactose by an 2-3 linkage
(NeuGc2-3Gal). This type of sialic acid is found on swine cells but
not on human cells at the sites of virus infection (19). To
examine the role played by NeuGc2-3Gal in influenza virus infection,
we compared the specificities of avian, swine, and human virus NAs for
NeuAc-GM3 and NeuGc-GM3, which contain NeuAc2-3Gal and
NeuGc2-3Gal, respectively. The N2 NAs of the avian, swine, and human
viruses all had similarly high specificities for NeuAc-GM3 (data not
shown). This result was expected and mirrors the high specificities
shown by the NAs for the NeuAc2-3Gal-containing sialyllactose
substrate (both substrates contain the same species and linkage of
sialic acid). In contrast, there were species-dependent differences in
the recognition of NeuGc-GM3 by the NAs (Fig. 3A). Avian viruses had
low to moderate specificity for NeuGc, whereas swine viruses had high
specificity, as previously reported (19). Human virus N2 NAs
showed an unexpected trend in specificity. Early human N2 viruses,
isolated between 1957 and 1962, exhibited low to moderate specificity
for NeuGc, as did avian viruses. However, A/Aichi/2/68, A/Hong
Kong/1/68, and A/Memphis/8/68 showed high specificity for NeuGc, much
like the results for swine viruses. By 1972, the NeuGc specificity of
the human viruses had returned to low to moderate levels like those
observed before 1968.
Amino acids affecting NeuGc substrate specificity. A/Tokyo/3/67 had an approximately 2.6-fold higher specificity for NeuGc than did A/England/12/62 (Fig. 3B). This difference was used to identify amino acid residues in NA responsible for the higher NeuGc specificity of A/Tokyo/3/67. Taking advantage of the high homology between the NA genes of A/England/12/62 and A/Tokyo/3/67, we generated five chimeric constructs (chimeras 6 to 10) in which portions of the A/England/12/67 NA gene were replaced with the corresponding regions of the A/Tokyo/3/67 NA gene (Fig. 4A). Each chimeric and parental NA was then expressed in 293T cells, and the substrate specificities of the cell-expressed NAs were determined with NeuAc-GM3 and NeuGc-GM3, using equal amounts of NA activity.
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Asp and Trp403Arg
did not appreciably affect NeuGc specificity.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We have identified two amino acid residues, at positions 275 and
431, near the enzymatic active center of N2 NA, that are involved in
the ability of NA to recognize NeuAc2-6Gal and NeuGc, respectively.
Residue 275 is an isoleucine in avian virus NAs as well as NAs of early
human N2 viruses isolated in 1957 (Table 2). Isoleucine at this position is
associated with high activity for NeuAc2-3Gal and very low activity
for NeuAc2-6Gal. A change to valine results in higher NeuAc2-6Gal
activity without affecting NeuAc2-3Gal activity. In A/England/12/62,
valine at position 275 is associated with the first increment in the
gradual increase in NeuAc2-6Gal specificity that characterizes human
N2 viruses. This single amino acid change is maintained in the
sequences of all human H2N2 and H3N2 viruses isolated after 1962 (Table
2). Hence, the change in the identity of residue 275 may be the first important step in the shift of N2 NA toward increased specificity for
NeuAc2-6Gal. Valine at position 275 is also found in the N2
sequences of swine viruses, which have higher NeuAc2-6Gal specificity than do avian viruses (Fig. 1A) (24).
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The increase in NeuAc2-6Gal specificity in human virus N2 NAs, due
to the change at residue 275, was followed by further increases in
specificity for that substrate (Fig. 1) (1). The lack of
additional mutations at this position suggests that the gradual shift
to higher NeuAc2-6Gal specificity seen in human virus N2 NAs
resulted from successive amino acid changes in other key residues that
each subtly altered the conformation of the active site to better
accommodate this linkage of sialic acid. However, there appears to be a
limit to this process of adaptation, as maximal specificity for
NeuAc2-6Gal was apparently attained by 1972 (Fig. 1A)
(1). Whether further adaptation is not possible because of
an adverse effect on enzymatic activity or whether there is simply
insufficient selective pressure from the environment to select variants
with still higher levels of NeuAc2-6Gal specificity is uncertain.
Couceiro et al. (4) have suggested that NeuAc2-6Gal on
human epithelial tracheal cells, together with NeuAc2-3Gal in
bronchial mucus secretions, may exert selective pressure to determine
human virus HA specificity (4). Similarly, we propose that
in addition to acquiring NeuAc2-6Gal specificity, it would be
advantageous for human virus NA to maintain appreciable NeuAc2-3Gal specificity to support movement of the virus in bronchial mucus.
The mutation at residue 275 may not be the only one required to promote
an increase in NeuAc2-6Gal specificity in early human N2 viruses.
The mutation of isoleucine at residue 275 in A/Singapore/1/57 NA to
valine resulted in about 65% of the NeuAc2-6Gal specificity shown
by A/England/12/62 NA. This result suggests that an additional mutation(s) may be necessary to maximize binding to the NeuAc2-6Gal substrate or to compensate for conformational changes induced in the
rest of the enzyme as a consequence of the change at residue 275. Analysis of the N2 NA sequences of other viruses showed that A/Ann
Arbor/6/60 NA already had valine at position 275, although it showed
the same low NeuAc2-6Gal specificity as A/Singapore/1/57 NA (Fig. 1B
and Table 2). Since A/Ann Arbor/6/60 NA has several amino acid
differences that are not shared by either A/Singapore/1/57 NA or
A/England/12/62 NA, one or more of these differences may counter the
effect of valine at position 275 on the activity of the enzyme for
NeuAc2-6Gal.
Site-specific mutagenesis demonstrated that a lysine at position 431 determines the highest NeuGc specificity, although A/swine/Italy/635/87 NA, which contains a glycine at this position, also has high NeuGc specificity (Table 2). That swine viruses have high NeuGc specificity is not unexpected, because swine tracheal epithelial cells express high levels of NeuGc (19). Examination of other swine N2 virus sequences indicated that lysine and glycine are not the only residues that can occupy position 431. Arginine is present at this position in A/swine/Kanagawa/2/78 (12) but represents only a conservative change from lysine, and the substrate specificity of the isolate was not examined. Proline was observed at position 431 in avian and human viruses; it was found in all avian N2 virus sequences examined as well as those of human N2 viruses isolated from 1957 to 1960. Other human viruses contain glutamine or glutamate which, like proline, is associated with low to moderate NeuGc specificity. A close relationship between lysine at position 431 and high NeuGc specificity is further supported by the fact that A/Korea/426/68 has glutamine at this position and is the only human virus isolated from 1967 to 1969 with low activity for this sialic acid.
The mechanism by which amino acid changes affect NA substrate
specificity is expected to involve very small shifts in NA structure that subtly affect the enzymatic active site. The high degree of
conservation among active-site residues (2, 20, 21) suggests
that amino acid substitutions within the active site which could permit
the binding of alternative linkages or species of sialic acid are not
likely to occur, a prediction substantiated by site-specific
mutagenesis (9). Indeed, both of the amino acid
substitutions found to affect the specificity of NA for NeuAc2-6Gal and NeuGc occur at positions close to the enzymatic active site and not
at those believed to be involved in the reaction mechanism. Residue 275 is directly adjacent to glutamate 276 and glutamate 277, both of which
are highly conserved and directly involved in the binding of sialic
acid to the enzymatic active site (21).
The mechanism of NA specificity for the 2-3 or 2-6 glycosidic
linkage appears to entail restrictions on accessibility to the active
site. In the crystal structure of an intramolecular trans-sialidase which has strict specificity for
NeuAc2-3Gal (10), the side chain of a tryptophan lies
above the C-2 position, excluding binding to a substrate with a
NeuAc2-6Gal linkage. Hence, it is important to consider possible
interactions with the substrate of side chains in the upper portion of
the active site. When there is a valine at position 275 below the
active site, as in the structure shown in Fig. 5, the side chain of
glutamate 276 in the next position becomes involved in good contacts
with the glycerol group in NeuAc. The pyranose ring of NeuAc is
horizontal in the active site, allowing the remaining moiety of the
2-3Gal or 2-6Gal oligosaccharide to extend freely to the open
region of that site. If valine 275 is replaced with a bulkier residue, such as isoleucine, glutamate 276 will have to be lifted a little to
accommodate the substitution. Such a conformational change may cause
the pyranose ring of NeuAc to tilt toward the right side of the active
site. The side chain of aspartic acid 151 is near the glycosidic
oxygen, right above the pyranose ring of NeuAc. Such geometry may allow
free access to a substrate with a NeuAc2-3Gal linkage but may impede
access to a substrate with a NeuAc2-6Gal linkage owing to steric
hindrance of the remaining moiety of the 2-6Gal oligosaccharide by
the side chain of aspartic acid 151.
The side chain of lysine 431, which confers NeuGc specificity in N2 NA, is far above the active site (Fig. 5). There do not seem to be any direct contacts of this residue with the substrate sialic acid. The only possible interactions would be with the second or third sugar moiety of NeuAc in an oligosaccharide substrate; such interactions would be unlikely to impose any restrictions on NeuGc substrates. A substitution at this position with glycine or perhaps arginine does not seem to change enzyme specificity for NeuGc, compared to that of NAs with lysine at position 431. Glutamate or glutamine can decrease NeuGc specificity, an effect that may result from long-range charge-charge interactions between the negatively charged glutamate (or the partially negatively charged glutamine) and the positively charged arginines in the active site. Substitutions with glutamate or glutamine could also alter the active-site conformation through long-range effects. Proline 431, as found in avian and early (1957 to 1960) human viruses, could restrict the conformational flexibility required for NeuGc binding.
Human tracheal epithelial cells do not possess detectable levels of NeuGc (19). Hence, one would not anticipate any pressure during viral replication in humans for the selection of N2 NA variants with higher NeuGc specificity. An unexpected observation was that most of the H2N2 and H3N2 human viruses isolated between 1967 and 1969 had high NeuGc specificity and possessed lysine at position 431, like swine viruses. The only exception was A/Korea/426/68 (Fig. 3A). One explanation for this finding is that some event occurring between 1967 and 1969 favored the emergence of human viruses which had high NeuGc specificity and which rapidly replaced viruses with glutamine at position 431. A possible scenario (although highly speculative) to explain these observations is the transmission of H2N2 viruses from humans to pigs or some other animal species with high NeuGc content on its cells prior to 1967 such that NA acquired increased specificity for NeuGc in the new host. These viruses were then transmitted back to humans. During this period, the same N2 NA virus was reassorted with an avian H3 virus to generate the lineage of human H3N2 viruses (17) with high NeuGc specificity. Although this scenario would explain our observations, evidence to confirm this scenario (i.e., the presence of N2 viruses in pigs prior to 1967) does not exist due to a lack of surveillance of influenza virus infections in swine at that time. After 1969, high NeuGc specificity was not maintained in human viruses, probably due to a lack of NeuGc on the human cells that are infected by influenza viruses. Alternatively, the appearance of viruses with high NeuGc specificity in humans in 1967 to 1969 might not have had any biologic significance and might have been the result of point mutations selected by immunologic pressure.
Cross-species transfer of influenza viruses will continue to be a source of potentially serious disease in animals, including humans. The active site of the NA molecule is highly conserved among the nine NA subtypes; however, during the 20th century, only two subtypes, N1 and N2, have been identified among human viruses, suggesting that specific requirements must be met before a new subtype can emerge and support influenza virus growth in humans. Understanding the role that NA plays in cross-species transfer of influenza viruses and the adaptive mechanism that it uses to efficiently support virus growth in new hosts will greatly aid in understanding how subtypes of viruses become established and are maintained. Such knowledge will also enhance surveillance efforts by helping to identify avian virus subtypes that potentially pose the most serious threat to humans and domestic animals, such as pigs.
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ACKNOWLEDGMENTS |
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We thank Krisna Wells and Scott Krauss for excellent technical assistance, Clayton Naeve and the St. Jude Children's Research Hospital for preparing oligonucleotides and for computer support, and John Gilbert for editing the manuscript.
Support for this work was provided by National Institute of Allergy and Infectious Diseases Public Health Service Research grants, a Cancer Center Support (CORE) grant from the National Cancer Institute, and the American Lebanese Syrian Associated Charities (ALSAC).
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, 2015 Linden Drive West, Madison, WI 53706. Phone: (608) 265-4925. Fax: (608) 265-5622. E-mail: kawaokay{at}svm.vetmed.wisc.edu.
Present address: DVP/OVRR/CBER/FDA, 29 Lincoln Drive, Bethesda, MD 20892.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Virology, August 1999, p. 6743-6751, Vol. 73, No. 8
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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