comprehensive mapping of poxvirus vcci chemokine binding

Chemokine Binding Profile of Poxvirus vCCI

1

Comprehensive Mapping of Poxvirus vCCI Chemokine Binding

Protein: Expanded Range of Ligand Interactions and Unusual

Dissociation Kinetics

Jennifer M. Burns, Daniel J. Dairaghi, and Thomas J. Schall*

From the Divisions of Discovery Biology and Molecular Pharmacology,

ChemoCentryx, San Carlos, California 94070

Running Title - Chemokine Binding Profile of Poxvirus vCCI

For Submission as a Brief Communication

*To whom correspondence should be addressed:

ChemoCentryx

1539 Industrial Road

San Carlos, CA 94070

Tel: 650-632-2900

Fax: 650-632-2910

Email: [email protected]

The abbreviations used are: vCCI, viral CC-chemokine inhibitor; HHV8, human

herpesvirus 8; CMV, cytomegalovirus; HHV6, human herpesvirus 6; MCV, poxvirus

Molluscum contagiosum; for chemokine abbreviations see reference (1)

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on November 5, 2001 as Manuscript M109884200 by guest on February 11, 2018

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Summary

In compiling a comprehensive map of the ligand binding capacity of elements

within the chemokine system, we have determined the spectrum of chemokines capable

of interacting with the poxvirus-encoded viral CC-chemokine inhibitor, vCCI. Greater

than 80 chemokines were tested in parallel for their ability to displace radiolabeled

signature chemokines from vCCI. Of these chemokines, 26 showed potential high

affinity interactions. These interactions revealed an expanded spectrum of binding

capacity for vCCI to now include molecules such as h MPIF-1 as ligands. In addition,

high affinity viral protein-protein interactions were revealed. For example, binding

between poxvirus vCCI and the herpesvirus vMIP-II from HHV8 occurs with IC50 ~10-50

nM. Unusual dissociation kinetics were observed between certain chemokines and vCCI.

Notably, many ligands displayed a precipitous displacement profile, suggesting marked

positive cooperativity of binding. Finally, heterologous competition provided evidence

for overlapping but distinct binding sites for the many chemokines that bind to vCCI.

The determination of the binding fingerprint and unusual binding interactions of vCCI

with a large number of chemokines suggests a finely honed evolutionary strategy of

chemokine sequestration during viral infection.

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Introduction

The ability of viruses to evade destruction by the host is achieved in part by the

molecular neutralization of immune defenses by virus-encoded components. Poxviruses

encode a secreted chemokine binding protein, viral CC-chemokine inhibitor (vCCI),

which likely represents one such mechanism, allowing the virus to achieve a molecular

pre-emption of the primary immune response. Also known as “T1” (in leporipoxviruses)

or “35kDa” (in orthopoxviruses), vCCI has been shown to bind to several human

chemokines, but it possesses no close molecular similarity to any endogenous proteins

(2). Since chemokines (for chemoattractant cytokines) are secreted proteins that serve to

direct the trafficking of leukocytes during the body’s response to infection and

inflammation, the sequestration of chemokines by poxviruses may serve an anti-

inflammatory function that is advantageous to the virus.

Chemokines comprise a seemingly vast array of structurally and functionally

related proteins: currently the number approaches ~ 50 distinct gene products identified

to date in humans (reviewed in 1). These are divided into four classes: CC, CXC, C, and

CX3C, based on the number and spacing of the N terminal cysteine residues in a

conserved structural motif. In addition, several viruses are known to make their own

versions of chemokines. These include vMIP-I, II, and III (3-7) made by HHV8, the

proteins vCXC-1 and 2 (8) produced by human CMV, and m131/139 (9,10) made by

murine CMV, the HHV6 viral chemokine U83 (11), and vMCC-1(6,12-14) from MCV.

Further adding to complexity in the chemokine system is the fact that postranslational

modifications of chemokines (e.g. by enzymatic modifications via CD26 (reviewed in 15)


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and other proteolytic processing steps, as well as alternative splicing events in some cases

(16-19)) create an even more diverse spectrum of chemokine variants, some of which

have different activities than the parental chemokine.

vCCI has been reported to bind preferentially to certain human CC chemokines

including such molecules as h RANTES(20,21), h MCP-1(20-23), h MIP-1α (20-22) and

h MIP-1β (20). However, there are discrepancies in the literature both in terms of

specificity and affinity of binding. For example, initially vCCI was thought to interact

broadly with CC and CXC chemokines(20); however, others(22,23) have suggested a

pattern of selectivity for CC-chemokines. Additionally, vastly disparate binding affinities

have been reported for the same chemokines ranging from low micromolar to low

nanomolar values (20,22,23). This may be due in part to the diverse methods of binding

analyses employed. To date there have been no studies attempting a comprehensive,

direct, and real-time comparison of the binding of many chemokines to vCCI under

similar analytical conditions.

In order to gain a thorough understanding of the biology of vCCI and its role in

viral pathogenesis, we undertook to map comprehensively its binding capacity, and to

investigate in detail the nature of its multipotential binding activity. Toward this end, we

employed the parallel interrogation of > 80 chemokines and chemokine variants from

human and viral sequences as well as those from other species, and assessed their ability

to bind to vCCI. We found that vCCI is capable of binding with high affinity (Kd ~5-

55nM) to a spectrum of ligands more wide ranging than previously reported. These

include chemokines encoded by other types of viruses as well. The ligand binding profile

determined here also reveals that vCCI does not bind to all CC-chemokines, nor does it


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interact with members of the CXC, C, or CX3C groups. Lastly we noted examples of

unusual binding interactions between vCCI and selected chemokines, suggesting that

vCCI interacts with different chemokines through similar yet distinct binding sites. Such

findings illuminate not only the biochemical characteristics of this unusual virally

encoded binding protein, but also may provide key insights into the virus’s ability to

evade the host immune response after infection.

Experimental Procedures

Binding Assays- We employed a variation of the recently developed DisplaceMax™

technique (6,24) to interrogate an array of chemokine interactions with vCCI and to

examine specific binding characteristics. These assays exploit the observation that vCCI

binds to the filter in radioligand filter binding assays. By including a radiolabeled

chemokine that interacts with vCCI it is then possible to detect vCCI binding to the filter.

Chemokines that compete for this binding then prevent vCCI-125I chemokine complexes

from forming and thus the decreasing cpm on the filter indicates competitor chemokine

bound to vCCI displacing the 125I chemokine. Eighty-three distinct chemokine elements

were incubated with 25 nM vCCI (R&D Systems) followed by the addition of

radiolabeled chemokine (125I h eotaxin, 125I h MIP-1α and 125I h MIP-1β, Amersham

Pharmacia Biotech) for 3hr at 4°C in the following binding medium (25 mM HEPES, 140

mM NaCl, 1 mM CaCl2, 5 mM MgCl2 and 0.2% bovine serum albumin, adjusted to pH

7.1). Where indicated, the detergents Tween-20 (Mallinckrodt Baker, Inc.) and Triton X-


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100 (Bio Rad) were included in the reaction at the indicated final concentrations. (Due to

the addition of high levels of detergent in the binding medium the ability to detect input

cpm and total cpm bound differs for this adaptation of the binding assay slightly as

follows: Tween-20 1%: ~49,000 cpm input, ~14,000 cpm total bound, ~775 cpm

nonspecific bound; Tween-20 0.1%: ~63,000 cpm input, ~14,000 cpm total bound, ~550

cpm nonspecific bound; Tween-20 0.01%: ~60,000 cpm input, ~16,000 cpm total bound,

~765 cpm nonspecific bound; Triton X-100 1%: ~54,000 cpm input, ~19,000 cpm total

bound, ~650 cpm nonspecific bound; Triton X-100 0.1%: ~62,000 cpm input, ~11,000

cpm total bound, ~600 cpm nonspecific bound; Triton X-100 0.01%: ~59,000 cpm input,

~9,600 cpm total bound, ~650 cpm nonspecific bound; binding medium only: ~51,000

cpm input, ~18,000 cpm total bound, ~1000 cpm nonspecific bound.) Following

incubation in all binding assays reactions were aspirated onto PEI-treated GF/B glass

filters (Packard) using a cell harvester (Packard) and washed twice (25 mM HEPES, 500

mM NaCl, 1 mM CaCl2, 5 mM MgCl2, adjusted to pH 7.1). Scintillant (MicroScint 10,

Packard) was added to the wells, and the filters were counted in a Packard Topcount

scintillation counter. Data were analyzed and plotted using Prism (GraphPad Prism

version 3.0a for Macintosh, GraphPad Software, www.graphpad.com).

Results and Discussion

Comprehensive Binding ‘Fingerprint’ of vCCI . A more thorough understanding of the

chemokine binding profile of vCCI could ultimately provide insight to the biologic

function of this protein in the in vivo setting. As a first approximation, we examined the


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chemokine binding profile of the poxvirus-encoded vCCI in an assumption blind manner.

We tested a starting panel of >80 purified chemokine elements and compared their ability

to displace selected ‘signature’ chemokines, such as 125I h MIP-1β, from vCCI in a

filtration based assay. As many as 26 chemokines, when used as cold competitors (at 200

nM final concentration), were found to inhibit > 60% of the specific binding of the

radiolabled signature chemokine to vCCI (Figure 1). The spectrum of ligands that bound

to vCCI included many interactions not previously reported. For example, multiple forms

of the CC chemokine h MPIF-1(25,26), including splice variants that possess an

additional coded exon (17), bound to vCCI. Also, two herpes virus encoded chemokines,

HHV8 vMIP-I and HHV8 vMIP-II, were very effective at displacing the signature

chemokine from vCCI. While most CC chemokines exhibited binding to vCCI, a few CC

chemokines, such as h MDC, h TARC and h TECK, did not. Qualitatively similar results

were seen with another signature chemokine, 125I h MIP-1α. In no case have we detected

evidence of vCCI binding to CXC, C, or CX3C chemokines.

Homologous Competition with Different Chemokines Suggests Distinct Binding

Interactions. We wished to investigate in greater detail the nature of the binding

interactions of vCCI with the potential high affinity ligands suggested from the

displacement binding fingerprint. Initial investigations employed homologous

competition binding experiments with selected radiolabeled chemokines. Representative

experiments tested whether radiolabeled h eotaxin or radiolabled h MIP-1α would be

displaced from vCCI over a concentration range of cold homologous competitor in order

to assess: (i) the relative binding affinities (as approximated by IC50 values) and (ii) the


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slope (Hill Coefficient) of the binding displacement curves. It has been established that

the Hill coefficients reveal something of the biophysical nature of receptor-ligand

interactions (24), where a Hill number of ~1 implies a single binding site with a simple

mass action displacement; Hill values >1 are indicative of cooperative binding, and Hill

values <1 suggest negative cooperativity. Homologous displacement with the

chemokines tested revealed a range of distinct interactions. For example, homologous

displacement of 125I h eotaxin from vCCI with cold h eotaxin (Figure 2A) revealed an IC50

of ~32nM and a Hill Coefficient of 1.2 (see Table 1). This was in marked contrast to the

interactions of 125I h MIP-1α: whereas competition for vCCI binding with cold h MIP-

1α showed a nearly identical IC50 (~32nM), the binding displacement curve had a

precipitous slope, with a very large Hill Coefficient (Figure 2B and Table 1). Thus, while

these two chemokines seem to bind to vCCI with similar affinities, eotaxin seems to

associate with simple one site kinetics while h MIP-1α displayed potentially marked

positive cooperativity.

Heterologous Competition. In addition to examining homologous binding competition,

heterologous binding interactions were examined. For example, we assessed the

susceptibility of 125I h eotaxin to be displaced by several other chemokines that appeared

to be interacting with vCCI at high affinity, (as suggested from the displacement

fingerprint (Figure 1)). Cold heterologous competitors tested included HHV8 vMIP-II

(Figure 3A), h SLC (Figure 3B) and h MCP-1 (Figure 3C). Strikingly, despite the fact

that in each case 125I h eotaxin is displaced by the cold competitors, the slope of the

dissociation curve is markedly different depending upon the competitor used. Indeed


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some ligands demonstrate a ‘catastrophic’ dissociation profile; for example, the

displacement of 125I h eotaxin by HHV8 vMIP-II (Figure 3A) reveals a very sharp

dissociation profile, with all of the h eotaxin displaced over a very small concentration

range of HHV8 vMIP-II. In contrast, the competition of 125I h eotaxin by h MCP-1 or h

SLC reveals a substantially different slope of the resultant binding curves. Table 1

summarizes both IC50 values and approximate Hill Coefficients for both homologous and

heterologous competition experiments. In particular the wide range of Hill Coefficients

over the course of the various experiments suggest that vCCI bound to its broad spectrum

of chemokines with overlapping but distinct characteristics.

Effects of Detergents on Binding Interactions. To test whether the high Hill Coefficients

seen in some of the displacement experiments reflect the formation of vCCI aggregates or

micelle-like structures, different detergent conditions were assessed in the binding

reaction. The addition of non-ionic detergents, such as Tween-20 (1% - 0.01%), had no

effect on the slope of the binding curve (Figure 4A and 4C). Triton X-100 did have some

effect in altering the slope of the curve when added to the binding buffer at

concentrations of 1% and 0.1%, however the slopes remain steep with Hill Coefficients >

1.5 (Figure 4B and 4C). These data suggest that vCCI exhibits very strong cooperative

binding with a select group of chemokines and this cooperative binding is not disrupted

by the addition of detergents.

In conclusion, we have developed a broad and comprehensive vCCI binding

profile allowing us to characterize vCCI-chemokine interactions. Unlike other binding

analyses, we endeavored to use a more native form of vCCI (e.g. not tethered through an


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epitopic tag) and we employed a filtration assay that allowed the proteins to interact

without interference by other cellular factors. The binding ‘fingerprint’ developed from

our studies suggests that vCCI is promiscuous, but not ubiquitous, in its binding of

chemokines. Twenty-six of the > 80 chemokine elements tested were identified as

potential high affinity vCCI ligands. Further investigation of these binding interactions

suggests that vCCI binds chemokines with overlapping but distinct sites. The finding that

several but not all chemokines tested had unusually steep binding slopes supports this

concept. The combination of such unusual binding and displacement characteristics

suggests that the virus has developed an adaptation to capture and sequester many

chemokines in the frame of limited protein topography. The selectivity for certain CC-

chemokines by vCCI and the distinct binding interactions with a subset of these proteins

indicates that the virus has adapted a function to alter its environment to the specific

needs of the virus. Collectively, these data suggest that vCCI may serve not so much as a

sink for certain chemokines in the local environment, but as a dynamic and selective

molecular switch that has evolved different strategies to regulate the local concentration

of a diverse spectrum of chemokines.


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FIG. 1. Ligand Binding Fingerprint of vCCI. Competitive binding profile of vCCI

binding to 125I h MIP-1β against a comprehensive array of purified viral, human and

murine chemokines. Chemokines are grouped into classes as follows: “potential high

affinity” (red bars) demonstrating greater than 75% inhibition, “potential moderate to

low affinity” (blue bars) showing between 75-60% inhibition, “little or no affinity”

(white bars) possessing less than 60% inhibition. Percent inhibition of binding is shown

and represents the mean of three determinations (~29,000 cpm input; ~8000 cpm total

bound; ~1000 cpm nonspecific bound). The standard deviation values for potential high

affinity ligand interactions are < 10% in all cases, however with the little to no affinity

interactions the standard deviations are ~40%; error bars are omitted for clarity. Intra-

assay variability was ~40% for lower affinity interactions, strongly suggesting that

binding values to the left of the zero meridian are not likely to be significant.

FIG. 2. Homologous Binding Competition. Profile of vCCI binding to 125I h eotaxin

(~32,000 cpm input; ~3000 cpm total bound; ~200 cpm nonspecific bound) (A) and 125I h

MIP-1α (~29,000 cpm input; ~9500 cpm total bound; ~250 cpm nonspecific bound) (B)

in competition with the identical unlabeled ligands. Data points represent the mean of

quadruplicate wells. Calculated IC50 and Hill Coefficients are shown in Table 1.

FIG. 3. Heterologous Binding Competition. Selected chemokine competitions with

vCCI and 125I h eotaxin demonstrate potential high, medium and low affinity interactions.

HHV8 vMIP-II represents a potential high affinity ligand for vCCI (~32,000 cpm input;

~1500 cpm total bound; ~150 cpm nonspecific bound) (A). By contrast, h SLC (B) and h


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MCP-1 (C) represent potential medium and low affinity ligands, respectively (~26,000

cpm input; ~750 cpm total bound; ~100 cpm nonspecific bound). Data points represent

the mean of quadruplicate wells. Calculated IC50 and Hill Coefficients are shown in Table

1.

FIG. 4. Addition of Detergents to Binding Assay. Addition of the non-ionic detergents

Tween-20 (A) and Triton X-100 (B) to binding medium at indicated concentrations to

attempt to disrupt the extreme cooperative binding indicated by the Hill coefficient. 125I h

MIP-1β competition with vMIP-II for binding to vCCI is shown here. The condition of

buffer alone is included in both panels for clarity. Data points represent the mean of

quadruplicate wells. The calculated IC50 and Hill Coefficients are indicated in the

accompanying table (C).


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-20% 0% 20% 60% 100%

h MPIF-1

h MPIF-46

h MIP-1α

m MIP-1γ

HHV8 vMIP-II

m MIP3β

HHV-8 vMIP-I

h MPIF-22

h HCC4

m MDC

h HCC-1

m MIP-1β

m MIP-1α

m MCP-1

m SLC

h MCP-3

h ELC

m C10

h MCP-1

h RANTES

h MCP-4

h SLC

h MIP-1β

h Eotaxin

h MIP-3α

m Eotaxin

h Finetaxin

h Met RANTES

m TECK

h TARC

r MIP3α

m Lymphotactin

h Leukotactin

h IP-10

h Fractalkine FL

h TECK

h MIP1δ

HHV-8 vMIP-III

h I-309

m GCP-2

m BLC

m Fractalkine CK

CMV UL146 T23

r CINC1

h Eotaxin 2

h ENA-70

h IL-8/EC

h MIG

h SDF-1β

h Lymphotactin

m RANTES

h ENA-74

h NAP2

h PF4

m MARC

m Fractalkine FL

h MDC

h IL-8/MO

vMCC-1

m TARC

h BLC-1

h SDF-1α

m CRG-2

r CINC-2β

m KC

h TECK

m MIG

h ENA-78

h GROγ

h GROβ

h PARC

h MCP-2

m CTACK

r CINC-2α

m SDF-1

h ITAC

h GCP-2

m MCP-5

h Fractalkine CK

m MIP-2

h GROα

h MIP-1α(70 aa)

CMV UL146 Y18

potentialhigh affinity

potentialmoderate tolow affinity

little orno affinity

Percent Inhibition of 125I MIP-1β BindingFigure 1


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-12 -11 -10 -9 -8 -7 -6 -50

500

1000

1500

2000

h eotaxin (log M)

-12 -11 -10 -9 -8 -7 -6 -50

5000

10000

15000

h MIP-1α (log M)

A

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-12 -11 -10 -9 -8 -7 -6 -50

500

1000

1500

2000

2500

HHV8 vMIP-II (log M)

-12 -11 -10 -9 -8 -7 -6 -50

250

500

750

1000

h SLC (log M)

-12 -11 -10 -9 -8 -7 -6 -50

250

500

750

1000

h MCP-1 (log M)

A

B

C


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-10 -9 -8 -7 -6 -50

50

1001% Detergent

0.1% Detergent

0.01% Detergent

Binding Medium Only


A

-10 -9 -8 -7 -6 -50

50

100


B

C Detergent Treatment IC50(nM) Hill Coefficient

Tween-20 1% 35 >4.0Tween-20 0.1% 18 >4.0Tween-20 0.01% 26 >4.0

Triton X-100 1% 16 2.8Triton X-100 0.1% 12 1.6Triton X-100 0.01% 17 >4.0

Binding Medium Only 21 >4.0


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Tab

le 1. Sum

mary of vC

CI-C

hemokine B

inding InteractionsIC50 (n

M)

Hill C

oefficien

t

Ho

mo

log

ou

s Co

mp

etition

125I h MIP

-1α/h M

IP-1α

32>

4.0125I h eotaxin/h eotaxin

321.2

Hetero

log

ou

s Co

mp

etition

:H

igh

Affin

ity 125I h M

IP-1α

/vMIP

-II26

>4.0

125I h MIP

-1β/h MIP

-1α23

>4.0

125I h MIP

-1β/h MP

IF40

>4.0

125I h MIP

-1β/m M

CP

-1/JE32

>4.0

125I h eotaxin/h MIP

-1α8

>4.0

125I h eotaxin/vMIP

-II15

2.8125I h eotaxin/r M

IP-3α

461.7

125I h eotaxin/h SLC

171.4

Hetero

log

ou

s Co

mp

etition

:L

ow

er Affin

ity 125I h M

IP-1α

/h MP

IF75

3.3125I h M

IP-1β/vM

IP-II

502.8

125I h MIP

-1β/r MIP

-3α130

1.5125I h eotaxin/h M

CP

-1147

0.4


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Chemokine Percent Inhibitionm MIP-1γ 100%

HHV-8 vMIP-II 100%h MIP-1α 98%

h MPIF-46 95%m MIP-1β 89%h MIP-1α (70 aa) 88%

rh MPIF-1 85%

rh MPIF-22 76%m MIP-1α 74%

h HCC4 64%m MIP3β 63%

h SLC 62%

m SLC 61%

h HCC-1 60%

h MCP-4 59%

h MCP-1 56%

h MCP-3 54%

HHV-8 vMIP-1 52%

m MIP-2 52%

m MDC 48%r CINC-2 α 45%

m MCP-1/JE 45%� h TECK 44%

CMV UL146 T23 43%

r CINC-1 42%

h Fractalkine FL 37%� h TARC 37%

h I-309 35%

h MCP-2 35%h MIP-3β 33%

h NAP2 33%

h RANTES 32%

h PARC 31%

h BLC-1 31%

m GCP-2 30%

h PF4 30%h SDF-1α 28%h GROβ 28%h MIP-1β 27%

h Finetaxin 26%

h IL-8 (EC) 26%

m KC 25%

HHV-8 vMIP-III 25%h SDF-1β 24%r MIP3α 24%

h Eotaxin 24%h GROα 24%

m Eotaxin 24%

h IP-10 22%

h ENA-78 21%

h Lymphotactin 21%� h MDC 19%

m MARC (MCP-3) 19%h GROγ 18%h MIP-3α 18%

m C10 18%

h MPIF-46 18%

h IL-8 17%h MIP-1δ 17%

h Leukotactin 15%

h ENA-74 15%

h Finetaxin 14%

h Fractalkine-CK 13%

m TECK 13%

CMV UL146 Y18 12%r CINC-2β 11%

h Met RANTES 11%

h GCP-2 11%

vMCV TypeII 9%

h ITAC 9%

m SDF-1 8%

CMV UL147 peak 1 8%

m BLC 7%

m MCP-5 6%

m CRG-2 6%

m Fractalkine-CK 6%

h Eotaxin 2 5%

m Fractalkine FL 5%

h ENA-70 3%

m ESkine 3%

CMV UL147 peak 2 1%

m RANTES -2%

m Lymphotactin -2%

m MIG -2%

m TARC -11%

Table of Percent Inhibition of 125I MIP-1α Binding with vCCI for Reviewers

SUPPLEMENTARY DATA

bold text = chemokines that compete

with both 125I MIP-1α and 125I MIP-1β for binding to vCCI

� = chemokines that do not compete with

either 125I MIP-1α and 125I MIP-1β for binding to vCCI


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Tab

le 1. Su

mm

ary of vC

CI-C

hem

okin

e Bin

din

g In

teraction

s for R

eviewers

IC50 (n

M)

95% C

on

fiden

ce In

tervals (nM

) H

ill Co

efficient

Ho

mo

log

ou

s Co

mp

etition

125I h MIP

-1α/h M

IP-1α

3225 to 41

>4.0

125I h eotaxin/h eotaxin32

17 to 621.2

Hetero

log

ou

s Co

mp

etition

:H

igh

Affin

ity 125I h M

IP-1α

/vMIP

-II26

21 to 33>

4.0125I h M

IP-1β/h M

IP-1α

234.3400e-32 to 1.2060e+

16>

4.0125I h M

IP-1β/h M

PIF

4016 to 109

>4.0

125I h MIP

-1β/m M

CP

-1/JE32

26 to 40>

4.0125I h eotaxin/h M

IP-1α

80.02 to 3435

>4.0

125I h eotaxin/vMIP

-II15

11 to 202.8

125I h eotaxin/r MIP

-3α46

27 to 771.7

125I h eotaxin/h SLC

179 to 31

1.4

Hetero

log

ou

s Co

mp

etition

:L

ow

er Affin

ity 125I h M

IP-1α

/h MP

IF75

57 to 973.3

125I h MIP

-1β/vMIP

-II50

38 to 632.8

125I h MIP

-1β/r MIP

-3α130

58 to 2801.5

125I h eotaxin/h MC

P-1

1470.3 to 5424

0.4

SU

PP

LEM

EN

TAR

Y D

ATA


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h E

o/vM

IP-II

-11

-10

-9-8

-7-6

0

1000

2000

3000

h vM

IP-II (lo

g M

)

h E

o/h

Eo

-10

-9-8

-7-6

0

1000

2000

3000

h eo

taxin (lo

gM

)

h E

o/h

MIP

-1α

-10

-9-8

-7-6

0

500

1000

1500

h M

IP-1α

(log

M)

h E

o/r M

IP-3α

-12.5

-10.0

-7.5

-5.0

0

500

1000

1500

r MIP

-3α

(log

M)

h E

o/h

SL

C

-12.5

-10.0

-7.5

-5.0

0

500

1000

1500

h S

LC

(log

M)

h E

o/h

MC

P-1

-12.5

-10.0

-7.5

-5.0

0

500

1000

1500

h M

CP

-1 (log

M)

h M

IP-1β/r M

IP-3α

-12.5

-10.0

-7.5

-5.0

0

5000

10000

15000

r MIP

-3α

(log

M)

h M

IP-1β/m

MC

P-1

-12.5

-10.0

-7.5

-5.0

0

5000

10000

15000

m M

CP

-1 (log

M)

h M

IP-1α

/vM

IP-II

-12.5

-10.0

-7.5

-5.0

0

5000

10000

15000

vMIP

-II (log

M)

h M

IP-1α

/h M

IP-1α

-12.5

-10.0

-7.5

-5.0

0

10000

20000

h M

IP-1α

(log

M)

h M

IP-1β/h

MP

IF

-12.5

-10.0

-7.5

-5.0

0

2500

5000

7500

10000

h M

PIF

(log

M)

h M

IP-1α

/h M

PIF

-12.5

-10.0

-7.5

-5.0

0

10000

20000

h M

PIF

(log

M)

h M

IP-1β/h

MIP

-1α

-12.5

-10.0

-7.5

-5.0

0

2500

5000

7500

10000

h M

IP-1α

(log

M)

h M

IP-1β/v

MIP

-II

-12.5

-10.0

-7.5

-5.0

0

1000

2000

3000

4000

vMIP

-II (log

M)

SU

PP

LE

ME

NT

AR

Y D

AT

A

Co

rrespo

nd

ing

Grap

hs R

eferred to

in T

able 1 fo

r Review

ers


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Jennifer M. Burns, Daniel J. Dairaghi and Thomas J. Schallrange of ligand interactions and unusual dissociation kinetics

Comprehensive mapping of poxvirus vCCI chemokine binding protein: expanded

published online November 5, 2001J. Biol. Chem.

10.1074/jbc.M109884200Access the most updated version of this article at doi:

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comprehensive mapping of poxvirus vcci chemokine binding

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