From the Laboratory of Molecular Biophysics,
Department of Biochemistry, University of Oxford, South Parks Road,
Oxford OX1 3QU, United Kingdom, the ¶ Faculty of Biomedical and
Life Sciences, Division of Virology, University of Glasgow, Church
Street, Glasgow G11 5JR, United Kingdom, and the
Complement
Biology Group, Department of Medical Biochemistry, University of Wales
College of Medicine, Heath Park,
Cardiff CF4 4XX, United Kingdom
Received for publication, December 10, 2002
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ABSTRACT |
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Decay-accelerating factor (CD55), a
regulator of the alternative and classical pathways of complement
activation, is expressed on all serum-exposed cells. It is used by
pathogens, including many enteroviruses and uropathogenic
Escherichia coli, as a receptor prior to infection. We
describe the x-ray structure of a pathogen-binding fragment of human
CD55 at 1.7 Å resolution containing two of the three domains required
for regulation of human complement. We have used mutagenesis to map
biological functions onto the molecule; decay-accelerating activity
maps to a single face of the molecule, whereas bacterial and viral
pathogens recognize a variety of different sites on CD55.
The innate immune system provides the first line of defense
against invading pathogens. A key aspect of this defense is the complement system; this consists of classical, alternative, and lectin
pathways that converge, via a well characterized cascade of protein
intermediates, to target cells for destruction. Regulators of
complement activation (RCA1
proteins), acting at key points in the cascade, have evolved to prevent
the pathological consequences of inappropriate activation.
Decay-accelerating factor (CD55) is a
glycophosphatidylinositol-anchored member of the RCA protein family
that protects autologous cells from complement damage (1-3). CD55
regulates all three pathways by binding and consequently accelerating
the decay of the C3 convertases (2), the enzymes that link the three
activation pathways and the downstream effectors. This central role in
regulating complement has led to the development of transgenic animals
that express human CD55 as a potential source of organs for
xeno-transplantation.
A number of additional activities have been associated with CD55, which
is the location of the Cra and Tca blood group
antigens (4). Recent studies have shown that it is involved in
coordinating the innate immune response to bacterial pathogens by
enhancing natural killer cell activity (5). Additionally, CD55 binds
CD97, a member of the epidermal growth factor (EGF)-TM7 protein family,
the expression of which is rapidly up-regulated upon activation of T
and B cells (6).
Perhaps as a consequence of its ubiquitous expression, CD55 has been
exploited as an attachment molecule by bacterial and viral pathogens
(7). The fimbriae of uropathogenic strains of E. coli bind
CD55, as do many echoviruses (EV) and other members of the enterovirus
genus of the Picornaviridae, a family of small, non-enveloped positive-strand RNA viruses associated with a range of
human diseases including meningitis, pleurodynia, exanthema, respiratory disease, and acute hemorrhagic conjunctivitis.
CD55, like the related complement control proteins CD46 and CR1, is
composed of a series of short consensus repeat (SCR) domains, each of
~60 residues folded into a characteristic Deletion of individual SCRs and exchange with homologous domains from
related proteins has broadly defined the regions of CD55 necessary for
the various biological functions. Complement control requires SCR2-4
(10), whereas CD97 binding only involves SCR1 (6). Among the
CD55-binding enteroviruses, there are clear differences in the SCR
domains bound (a few exclusively need SCR1 (11, 12)), whereas all the
echoviruses require at least paired SCR domains, consisting of SCR3
linked to either SCR2 and/or SCR4 (13).
Understanding the functional biology of CD55 requires a more detailed
knowledge of the three-dimensional structure. Since the majority of
CD55 interactions involve multiple domains, the relative spatial
orientation of each SCR must be critical. This cannot be accurately
modeled from the existing structural data base. For this reason, we
have determined the structure of the two membrane-proximal SCR domains
of CD55 (SCRs 3 and 4; designated CD5534) in a range of
different crystal environments to a maximal resolution of 1.7 Å. A
panel of CD55 mutants were produced based upon this structure, and the
ability of the variant proteins to function in complement assays and
pathogen interactions was determined. This structure and the functional
analysis of variant CD55 proteins provide detailed insights at high
resolution into the domain architecture of CD55, the natural
interactions of the protein in the complement system, and the range of
pathogen interactions.
Protein Production and Crystallization--
C-terminally
histidine-tagged CD5534 was expressed in yeast, and
crystals were grown from sitting drops equilibrated against a mother
liquor containing polyethylene glycol 4000 and NaCl at low pH as
described previously (13, 14). Mutants were expressed in Chinese
hamster ovary cells as soluble, dimeric, glycosylated, Fc-linked
proteins containing CD551234 and purified to high
homogeneity using affinity chromatography (>95%). All proteins
displayed a wild-type phenotype in at least one biological assay
(convertase decay, virus binding (see "Results"), or interaction
with domain-specific monoclonal antibodies (data not shown)),
demonstrating that the mutations were localized in influence and did
not grossly disturb SCR folding.
Crystallographic Analysis--
Multiwavelength anomalous
dispersion x-ray diffraction data to 2.0 Å were collected on European
Synchrotron Radiation Facility, Grenoble, France, BM14 from a two-site
platinum (K2PtCl4) derivatized P1 crystal
frozen at 100 K. 360 degrees of data were collected at three
wavelengths chosen with respect to the platinum edge. A
three-site gold derivative data set (NaAuCl4) was collected at Synchrotron Radiation Source, Daresbury, UK, beamline 9.6. A highly
redundant 1.7-Å native data set was collected from a single crystal at
100 K at SRS beamline 14.1. Data were processed using the CCP4 suite of
programs (15). Phases were calculated using SHARP (16). Multiple cycles
of rebuilding in Xfit (17) and refinement in CNS (18) led to the model
(consisting of two copies of CD5534: P and Q and 177 water
molecules placed in 2 Immunological Assays (Complement, HA, and Antibody
Binding)--
Classical and alternative pathway complement assays were
conducted essentially according to Harris et al. (20).
Classical pathway activity is given as the mean percentage of wild-type (WT) activity ± S.D. if it differs from WT by at least
three standard deviations, otherwise shown as WT. Activities were
determined from a minimum of three experiments. Alternative pathway
activity was determined in duplicate and is shown as WT (activity did
not differ from WT by more than a factor of 2) or factor difference from WT activity. Echovirus binding was quantified by a
hemagglutination inhibition (HAI) assay as described previously (13).
Input virus was normalized to 4 HA units (where 1 HA unit is the amount
of virus required to hemagglutinate 50 µl of 0.5% human
erythrocytes) and incubated with serial dilutions of purified dimeric
Fc-linked CD55. HA endpoints were scored visually and were repeated at
least three times and are presented in the same manner as the
alternative pathway data.
CD5534 was crystallized in a number of different
spaces (14) from identical crystallization conditions. The overall
structure of CD5534 (Fig.
1A) contains two copies of the
SCR fold observed in other RCA proteins (8), each consisting of five
antiparallel
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-strand structure held
together by two disulfide bridges (8). CD55 has four SCR domains,
attached to the cell membrane by a glycophosphatidylinositol anchor and
separated from it by a heavily O-glycosylated
serine/threonine-rich "stalk" (3, 9).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
difference density peaks) described below. The
two SCR domains from molecule P were used independently for sequential
molecular replacement into the other crystal forms using program EPMR
(19). For the two crystal forms at lower resolution (C2b and
P212121), SCR3 was rotated and
translated into position followed by a search for SCR4. In both cases,
the top solutions were seen to position the two SCRs so that the
C terminus of SCR3 and N terminus of SCR4 could be trivially joined.
Rigid body refinement where the two SCRs were allowed to move
independently was followed by least squares minimization and either
grouped B-factor refinement (two Bs per residue for crystal form C2b)
or setting of all atom Bs to the Wilson B value (for crystal form
P212121). Limited rebuilding in
Xfit and further refinement led to the models described below. For
crystal form C2a, the higher resolution of the data (2.0 Å) allowed a full rebuild and refinement using Xfit and CNS. The final
model statistics for the different crystal forms are as shown in Table
I. Contact distances were calculated
using the CCP4 program suite.
Structure solution
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strands with strands 1 and 4b and strands 3 and 5b
being "pulled together" by a conserved disulfide at opposing ends
of the domains. Strand 1 does not meet the strict criteria for
definition as a
-strand in these structures.
View larger version (43K):
[in a new window]
Fig. 1.
Fold and arrangement of SCR domains in
CD5534. This figure was drawn using AESOP
(M. E. M. Noble, unpublished program). A,
secondary structure and location of disulfide bonds in
CD5534. Strands are labeled according to the
convention defined by Norman et al. (35). Note that there is
no strand 1 as the hydrogen-bonding pattern of these residues does not
meet the strict criteria for definition of a -strand. B,
variation in orientation between SCR domains 3 and 4 in the five
independent copies of CD5534 found in the different crystal
forms (Table I). C, model for topology of CD55 in the
membrane based on our structure of CD5534.
The crystals yielded five independent, differently packed copies of
CD5534, with only one crystal contact being conserved between two of the crystal forms (see the supplemental data
available online). Little structural variation is seen at the
interface between SCR3 and -4, implying that this junction is
relatively rigid. This conclusion is supported by numerical calculation
of tilt and twist angles, which define the orientation of SCR3 with respect to SCR4 (21, 34). The tilt for all molecules is 8 ± 8o (Table I). The packing of the SCR3 strand 3-4 loop
against the SCR3-SCR4 linker appears fundamental to maintaining the
relative orientation of the SCR domains. Additional stability is
probably provided by the packing of the SCR4 strand 4-5 loop (Fig.
2), although precise definition of the
determinants of the interdomain rigidity is not trivial. Although
hydrogen bonds are important for internal stabilization of the loops
(Fig. 2C), only the SCR3 strand 3-4 loop is tied to the
linker via a hydrogen-bonding network since the linker forms a
continuation of strand 5b.
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A panel of SCR3 and -4 mutations was generated to functionally probe the various faces of the molecule revealed by the structure. These mutants were tested in complement control and virus binding assays and provide further evidence supporting the importance of the observed SCR3-4 organization to the function of CD55. Prior to analysis, all mutants were screened with domain-specific monoclonal antibodies to confirm that there was no gross disruption of structure.
The mutant proteins were found to have a range of abilities to regulate the alternative and classical pathways of complement (Table II and Fig. 3). Our data confirm the role for residues 125KKK127 and 147LF148 reported earlier (22). In light of our structure (see "Discussion"), we interpret these mutations as acting by altering the packing of domains 2 and 3 rather than directly mapping the site of convertase interaction. Our data also demonstrate that changes to several other residues on the "back" face of CD55 (as defined in Fig. 3) are able to alter the ability of the molecule to regulate both pathways of complement activation.
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Echoviruses agglutinate erythrocytes by binding CD55 (13, 23), and
hemagglutination inhibition (HAI) assays provide a means to readily
quantify the interaction of virus and receptor. Earlier work (13) has
grouped CD55-binding echoviruses into three broad categories based on
specific SCRs required for virus binding. A virus from each of these
groups was assayed for its ability to bind to the panel of CD55 mutant
proteins by HAI. Substitutions at the SCR2-3 interface
(125KKK127 or 147LF148)
had little or no influence on virus binding (with the exception of
EV11, which was somewhat sensitive to the
125KKK127 change), implying that either the
SCR2-3 interdomain angle or SCR2 makes only minor contributions to
virus interactions. SPR analysis of CD55 binding by EV11 also supports
the latter conclusion (24). However, despite the close genetic
relationship between decay-accelerating factor-binding enteroviruses
(13), EV12 and EV7 were independently influenced by substitutions on
opposite faces of CD55 (Fig. 3C and Table II). Further
differences were apparent in the interaction of EV with African green
monkey (AGM) CD55, which differs by 42 residues within SCR1-4. EV7
binds to human and AGM CD55 indistinguishably, whereas EV11 cannot bind AGM CD55, and EV12 binding to AGM CD55 is significantly reduced (Table
II). These results are in agreement with our earlier SPR and HAI
analysis using SCR-deleted forms of CD55 to broadly map the domains of
CD55 that interact with these viruses (13, 23, 24). They also
demonstrate that viruses that share the same SCR specificity for
binding may actually interact with distinctly different faces of the molecule.
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DISCUSSION |
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Molecular Architecture of CD55-- Using the extended linear structure of CD5534 to predict the topology of the four SCR domains in the full molecule produces a model that projects ~300 Å above the cell membrane (including an estimated ~100 Å for the serine/threonine-rich stalk). This is significantly extended, by almost 50%, when compared with previous models (22, 25). Alternative topologies are possible if the observed linearity of the SCR34 interface is not repeated at the junctions of the other SCR domains. A recent low resolution structure of CD55 complexed with EV7 (26) is essentially linear along the SCR2-3 junction but exhibits a distinct angle at the interface between SCR1 and -2. Until a high resolution structure of a four SCR domain CD55 molecule is determined, it is important that the functional data concerning SCRs 1 and 2 are interpreted in the light of a variety of architectural models, of which our structure-based model and earlier homology models of Kuttner-Kondo et al. (25) represent the extremes available.
Mapping Complement Regulation-- Alignment of mammalian CD55 sequences has identified two highly conserved regions in SCR3, a positively charged block (consisting of 125KKK127) and a hydrophobic patch (147LF148). Previous studies have shown that modification of these residues grossly perturbs complement control, and based on structural modeling, may form the primary sites of convertase interaction (22). Our data confirm the importance of these residues in influencing both classical and alternative pathway decay acceleration (Table II) and show that these regions are juxtaposed at the membrane-distal end of SCR3. Based on the observed linearity of the SCR3-4 interface (Fig. 1), the structure of the SCR3 interstrand loops located in this region, and the apparent linearity of the SCR2-3 junction (see above and Ref. 26), we predict that these mutations are buried at the SCR2-3 interface and exert their effect by altering the relative positions of the two domains (Fig. 3B). Based on the functional analysis of our mutants, we propose that the back face of CD5534 (as defined in Fig. 3) is the most likely site of direct contact with the convertases. Our mutational data suggest that the convertase interactions of both the classical and alternative pathway are broadly similar with the exception of E134A and F169A, which respectively solely affect the classical or alternative pathways. The E134A mutation does not significantly disrupt CD55 structure as the protein has native activity in alternative complement pathway assays, in virus binding (see below), and in binding domain-specific antibodies (data not shown). Our mapping of Glu134 to the "front" of the molecule suggests that CD55 may interact via an extended interface with the classical pathway convertase (which at 230 kD is much larger than CD55), the convertase wrapping around CD55 to contact both faces of the regulator.
CD55-Pathogen Interactions-- Many echoviruses use CD55 as a receptor, and earlier work (13) has grouped these viruses into families on the basis of which the CD55 domain(s) is critical for binding. Our results demonstrate that specific echovirus types bind to different sites on CD55 (even when the viruses are dependent on the same SCR for recognition); it is therefore likely that the binding determinants on the virus capsid will also differ (either in sequence or location). The cryo-electron microscopy-determined structure of EV7 complexed with CD55 shows the receptor binding across the 2-fold axes of symmetry (26), whereas previous mapping of EV11 mutations associated with the loss of CD55 binding has implicated a platform surrounding the 5-fold axes (27). Taken together, these observations further support the hypothesis that CD55 receptor usage may have evolved independently in these enteroviruses rather than being an ancestral trait (13).
The use of CD55 as a receptor has been coupled with symptomatic
infections by a variety of uropathogenic E. coli (28, 29) with recent evidence showing a link between the use of CD55 and stimulation of the host immune response via induction of MICA expression (5). The influence on E. coli binding of the two known single amino acid polymorphisms within the SCR domains has been
examined previously (28, 30) and suggests that those bacteria sensitive
to a change in SCR3 are insensitive to changes in SCR4 (and vice
versa). This strongly implies multiple, independent binding sites
on CD55 for different bacterial strains. Our structure shows that these
two polymorphisms lie on opposite faces of the molecule (Fig.
3D). Our structure-based mapping of CD55 biology provides
strong support for the hypothesis that convergent evolution is
responsible for the range of pathogens that bind CD55, implying a
distinct selective advantage for those that bind this molecule. The
precise nature of the evolutionary pressure for both viral and
bacterial pathogens to bind CD55 remains to be determined. The high
level expression of CD55 on serum-exposed cells means it could provide
an important initial attachment for the pathogen before interaction
with additional cell entry determinants (12, 31, 33). However, there
are many other widely expressed cell surface proteins, and an
attractive alternative hypothesis is that pathogens have evolved to
exploit the cellular role(s) of the molecule, perhaps to gain
immunological advantage. Our structure provides the basis for a better
understanding of the functional domains of CD55 and the interactions
with complement control proteins, bacterial fimbriae, and
enteroviruses. We are, however, aware that hypotheses proposed on the
basis of mapping mutational data will require validation by
determination of the structures of complexes of CD55 with its many ligands.
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ACKNOWLEDGEMENTS |
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We thank Bob Sim for discussion of complement assay data, Martin Noble for assistance with figure preparation, David Stuart for thoughtful reading of an earlier version of this manuscript and the staff of the SRS, Daresbury, UK and ESRF, Grenoble, France for assistance with data collection.
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FOOTNOTES |
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* This work was funded by Wellcome Trust Grant 059011 (to S. M. L.) and Grants 059011/A/99/Z and Medical Research Council Program Grant G9901250 (to D. J. E.) and by Arthritis Research Campaign Grant L0534 (to S. M. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains supplementary data concerning close
crystallographic and non-crystallographic contacts within the four
CD5534 forms.
The atomic coordinates and the structure factors (code 1H03, 1H03SF, 1H2Q, 1H2P, and 1H04) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY178110
§ Present address: ASTEX-Technology, Cambridge CB4 0WE, UK.
** To whom correspondence should be addressed. Tel.: 44-1865-275181; Fax: 44-1865-275182; E-mail: susan@biop.ox.ac.uk.
Present address: University of Southampton School of Medicine,
Biomedical Sciences Bldg., Southampton SO16 7PX, UK.
Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.M212561200
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ABBREVIATIONS |
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The abbreviations used are: RCA, regulators of complement activation; HA, hemagglutinin; HAI, hemagglutination inhibition; SCR, short consensus repeat; AGM, African green monkey; EV, echoviruses.
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