Poxvirus Secreted Chemokine-binding Proteins

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Poxvirus Secreted Chemokine-binding Proteins Grant McFadden1,* and Richard Moyer 2 1

The John P. Robarts Research Institute and Department of Microbiology and Immunology, The University of Western Ontario, 1400 Western Road, London, Ontario, N6G 2V4, Canada 2 Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, PO Box 100266, Gainesville, FL 32610-0266, USA * corresponding author tel: (519) 663-3184, fax: (519) 663-3847, e-mail: [email protected] DOI: 10.1006/rwcy.2000.14016.

SUMMARY

BACKGROUND

Unlike the classic viroceptors (virus-encoded receptor homologs) that bind and inhibit cytokines such as TNF, interferons, and IL-1 , the viral proteins that bind and modulate chemokines were discovered by screening analysis for novel cytokine inhibitors expressed by poxviruses. Currently, two classes of chemokine-binding proteins have been described which interact with chemokines in different fashion. The first class, exemplified by the M-T1 protein of myxoma virus and the secreted 35K proteins from a variety of orthopoxviruses, are 35±45 kDa secreted glycoproteins that bind and inhibit the ability of a broad spectrum of CC chemokines to engage their cognate receptors. Binding occurs in a relatively species-independent fashion. The second class is typified by the M-T7 protein of myxoma virus, which was first described as an inhibitor of IFN but has a second property of binding to a wide spectrum of chemokines via the conserved glycosaminoglycanbinding domains of these ligands. Both classes of proteins affect the pathogenesis of virus infections but are believed to interfere with chemokine biological functions by different mechanisms.

Discovery In 1997, the discovery of secreted chemokine-binding proteins suggested a novel virus strategy for modulating chemokine functions during virus infection (Graham et al., 1997). Subsequent studies revealed that these poxvirus-encoded chemokine-binding proteins (CBPs) were specific inhibitors of the CC family of chemokines (Smith et al., 1997; AlcamõÂ et al., 1998; Lalani et al., 1998). Members of this growing family of poxvirus proteins, designated the T1/35 kDa family of secreted viroceptors, have been uncovered in myxoma virus, Shope fibroma virus, cowpox virus, rabbitpox virus, raccoonpox virus, variola virus, and certain strains of vaccinia virus (see Figure 1). The discovery of the poxvirus chemokine-binding proteins has been reviewed in detail by Lalani and McFadden (1997).

Alternative names Chemokine-binding proteins (CBPs), viral chemokine-binding proteins (vCKBPs), viral chemokine inhibitors (vCCIs), and T1/35 kDa family.

2144 Grant McFadden and Richard Moyer

Structure The poxvirus CBPs constitute a family of secreted glycoproteins that exhibit considerable sequence similarity to each other. All the CBPs of this class share conserved sets of sequence motifs, particularly a characteristic cysteine-spacing arrangement (See Figure 1). However, to date no close cellular homologs have been described for this family. The crystal structure for one member of this family, namely the secreted 35 kDa protein from cowpox (vCCI), has recently been determined and shown to exhibit a novel sandwich fold that reveals a potentially new strategy to bind and inhibit CC chemokines (Carfi et al., 1999).

Main activities and pathophysiological roles The members of the T1/35 kDa CBP family inhibit a broad spectrum of CC chemokines, as measured by receptor-binding analysis, calcium flux measurements, and chemotaxis assays (Smith et al., 1997; AlcamõÂ et al., 1998; Lalani et al., 1998). Expression of these viral proteins modulates the influx and activation of monocytes/macrophages and other classes of reactive leukocytes into sites of virus replication in infected animals (Martinez-Pomares et al., 1995; Graham et al., 1997).

GENE

Accession numbers Myxoma virus (M-T1): U62677 Shope fibroma virus (S-T1): M17433 Rabbitpox virus (35K): U64724 Vaccinia virus Lister (C23L): D00612 Vaccinia virus Copenhagen (C23L/B29R): M35027 Variola virus Somalia 1977 (G3R): U18341 Cowpox virus (ORF B): L08906 Cowpox virus (D1L): Y11842

PROTEIN

Accession numbers Myxoma virus: 2076755 Shope fibroma virus: 139626 Rabbitpox virus: 2076757

Vaccinia virus Lister: 137559 Vaccinia virus Copenhagen: 335577 Variola virus Somalia 1977: 885856 Cowpox virus (orf B): 333518 Cowpox virus (D1L): 3096963

Sequence See Figure 1.

Description of protein The first member of this family (designated 35 K) was identified as the major secreted glycoprotein from cells infected with rabbitpox virus (Martinez-Pomares et al., 1995) and certain vaccinia virus strains (e.g. Evans, Lister) but not others (e.g. Western Reserve, WR) (Patel et al., 1990). Only later did functional studies reveal that members of this family could bind and inhibit a broad spectrum of CC chemokines in vitro (Graham et al., 1997; Smith et al., 1997; AlcamõÂ et al., 1998; Lalani et al., 1998). In addition to the expression and secretion of CBPs from poxvirusinfected cells, members of the T1/35 kDa family have also been overexpressed in active form using baculovirus vectors and as Fc fusion constructs (Smith et al., 1997; AlcamõÂ et al., 1998).

Relevant homologies and species differences No cellular homologs of the T1/35 kDa family have been reported, and a comparison of the chemokineinhibitory properties of members from vaccinia, rabbitpox, cowpox, and myxoma has revealed no obvious species-specificity against representative CC chemokines (Smith et al., 1997; Lalani et al., 1998).

Affinity for ligand(s) The affinities for T1/35 kDa family members for a variety of human and murine chemokines have been measured, and Kd values generally range in the picomolar to low nanomolar range for many of the representative CC chemokines tested (Smith et al., 1997; AlcamõÂ et al., 1998). Thus, the viral proteins have considerably greater affinities for CC chemokines than these ligands have for their own cellular receptors.

Poxvirus Secreted Chemokine-binding Proteins 2145

Figure 1 Alignments of the poxvirus T1/35 kDa family of chemokine-binding proteins. Amino acids are boxed if they are conserved in both myxoma virus and Shope fibroma virus, and at least four of the orthopoxvirus members. Myx, myxoma virus; SFV, Shope fibroma virus; RPV, rabbitpox virus; VVlis, vaccinia virus (Lister); VVcop, vaccinia virus (Copenhagen); VAR, variola virus; CPV, cowpox virus. Conserved cysteines are marked with an asterix. Adapted from Graham et al. (1997).

Myx MT1 SFV ST1 RPV 35kDa VVlis35kDa VVcop35kDa VAR 35kDa CPV 35kDa

1 1 1 1 1 1 1

Myx MT1 SFV ST1 RPV 35kDa VVlis35kDa VVcop35kDa VAR 35kDa CPV 35kDa

M M M K Q Y I M K Q Y I

K R V V

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L Y A A M M K Q Y I V L A C M C L A A A M K Q I V L A C I C L A A V

A V A A H A A

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P P P P P

A A A A T

S S S S S

L L L L L

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L F S S S S F

A A S S S S

T T S S S S

K K S S S S

G G S S S S S

I I S S S L S

C C C C C C C

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Q Q E E E E E

G D E E E E E

E E E E E E E

D D N N N N N

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R R H H H H H

Y Y H H H Y H

M M M M M M M

31 32 41 41 27 38 40

G G G G G G G

I I I I I I I

D D D D D D D

A V V V V V V

V V I I I I I

A V I I I I I

K K K K K K K

I V V V V V V

T T T T T T T

K K K K K K K

R K Q Q Q Q Q

D D D D D

Q Q Q Q Q

T T T T T T T

T S P P P P P

G G T T T T T

S S N N N N N

D D D D D D D

T T K K K K K

P V I I I I I

C C C C C C C

Q Q Q Q Q Q Q

G A S S S S S

L L V V V V V

R R T T T T T

T T E E E E E

T T I I I I V

I F T T T T T

E E E E E E E

S A S S S S S

A A E E E E E

Y H S S S S D

T K D D D D E

E G P P P P S

D D D D D E

E G P P P -

N A -

E N -

D D -

D S -

Myx MT1 SFV ST1 RPV 35kDa VVlis35kDa VVcop35kDa VAR 35kDa CPV 35kDa

69 70 77 77 63 72 75

G -

A -

T L -

G S E E E E E

T T V V V V V

E E E E E E V

Q Y S S S S K

P V E E E E G

D D D D D D D

D D D D D D -

L Y S S S S -

S S T T T T -

E E S S S S -

E E V V V V -

Y E E E E E -

E E D D D D -

Y Y V V V V -

D -

E E -

N Y -

D D -

E E -

S S -

F F -

L L -

T E -

G G -

F F -

V V D D D D -

I I P P P P -

G G P P P P P

S S T T T T T

T T T T T T T

Y Y Y Y Y Y Y

H Y Y Y Y Y Y

T T S S S S T

I I I I I I V

V V I I I I V

G G G G G G G

G G G G G G G

Myx MT1 SFV ST1 RPV 35kDa VVlis35kDa VVcop35kDa VAR 35kDa CPV 35kDa

109 107 103 103 89 98 91

G G G G G G G

L L L L L L L

S S R R R R T

V V M M M M M

T T N N N N D

F F F F F F F

G G G G G G G

F F F F F F F

T T T T T T T

G G K K K K K

C C C C C C C

P P P P P P P

T T Q Q Q Q K

V V I I I I I

K K K K K K S

A S S S S S S

I V I I I I I

S S S S S S S

E E E E E E E

H Y S S S S Y

V A A A A A S

K K D D D N D

G G G G G G G

R R N N N N N

H I T T T A T

V V V V V V V

Y F N N N N N

V I A A A A A

R R R R R R R

L L L L L L L

S S S S S S S

S S S S S S S

D D V V V V V

A A S S S P S

P P P P P P P

W W G G G G G

R R Q Q Q Q Q

D D G G G G G

T T K K K K K

N N D D D D D

Myx MT1 SFV ST1 RPV 35kDa VVlis35kDa VVcop35kDa VAR 35kDa CPV 35kDa

149 147 143 143 129 138 131

P P S S S S S

V M P P P P P

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M I I I I I I

N N T T T T T

R R H R R R R

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D E K K K K K

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S S S S S S S

V I I I I I I

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I I I I I I I

K K R R R R K

C C C C C C C

S S S S S S S

R N E E E E E

V E E E E E E

N T E E E E E

V V K K K K K

T S D D D D D

E E S S S S S

T T D D D D N

T T I I I I I

Y Y K K K Q K

G G T T T T T

T L H H H H H

A A P P P P P

A S V V V V V

L L L L L L L

V A G G G E G

Myx MT1 SFV ST1 RPV 35kDa VVlis35kDa VVcop35kDa VAR 35kDa CPV 35kDa

189 187 183 183 169 178 171

P P S S S S S

R H N N N N N

I I I I I I I

T T S S S S S

Q Q H H H H H

A A K K K K K

T T K K K K K

R E V V V V V

S S S S S

R R Y Y Y Y Y

S G E E E E E

H N D D D D D

I I I I I I I

I I I I I I I

G G G G G G G

S S S S S S S

T T T T T T T

L L I I I I I

V V V V V V V

D D D D D D D

T T T T T T T

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C C C C C C C

V V V V V V V

K E K K K K K

S N N N N N N

L L L L L L L

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M M M M M M M

C C C C C C C

K R K K K K K

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T T S S S S S

Myx MT1 SFV ST1 RPV 35kDa VVlis35kDa VVcop35kDa VAR 35kDa CPV 35kDa

228 226 223 223 209 218 211

S S S S S S S

D D E E E D E

L L L L L L L

S S E E E E E

A K V V V V V

R R K K K K K

D D D D D D D

S S G G G G G

L L F F F F F

K K K K K K K

V V Y Y Y Y Y

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N N D D D D D

G G G G G G G

S S S S S

A A A V A

S S S S S

K E K E E E E

L L G G G G D

L L A A A V A

E D T T T T A

D D D D D D D

D D D D D D D

I T T T T T T

L F S S S S S

V S L L L L L

L I I I I I I

R H D D D D N

T T S S S S S

P P T T T T A

T K K K K K K

L L L L L L L

K K K K K K I

A A A A A S A

C C C C C C C

N N V V V V V

260 258 258 258 244 253 246

2146 Grant McFadden and Richard Moyer

Regulation of receptor expression The viral proteins are expressed as standard early poxvirus genes (Macaulay and McFadden, 1989), and the processed proteins are efficiently secreted as 35±40 kDa glycoproteins (Patel et al., 1990).

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY The deletion of the 35 kDa gene from rabbitpox causes only minor effects on the overall lethality of this virus in infected rabbits but histological analysis reveals an increase in inflammatory cell influx in the first few days of infection (Martinez-Pomares et al., 1995; Graham et al., 1997). It is thought that the increased numbers of inflammatory cells remain poorly activated due to the combined activities of other anti-immune proteins expressed and secreted by the virus. When purified and tested alone, the vaccinia 35 K protein of blocked eotaxin-induced eosinophil infiltrates in a guinea pig skin model of inflammation (AlcamõÂ et al., 1998).

References AlcamõÂ , A., Symons, J. A., Collins, P. D., Williams, T. J., and Smith, G. L. (1998). Blockade of chemokine activity by a soluble chemokine binding protein from vaccinia virus. J. Immunol. 160, 624±633.

Carfi, A., Smith, C. A., Smolak, P. J., McGrew, J., and Wiley, D. C. (1999). Structure of a soluble secreted chemokine inhibitor vCCI (p35) from cowpox virus. Proc. Natl Acad. Sci. USA 96, 12379±12383. Graham, K. A., Lalani, A. S., Macen, J. L., Ness, T. L., Barry, M., Liu, L.-Y., Lucas, A., Clark-Lewis, I., Moyer, R. W., and McFadden, G. (1997). The T1/35 kDa family of poxvirus secreted proteins bind chemokines and modulate leukocyte influx into virus infected tissues. Virology 229, 12±24. Lalani, A. S., and McFadden, G. (1997). Secreted poxvirus chemokine binding proteins. J. Leukoc. Biol. 62, 570±576. Lalani, A. S., Ness, T. L., Singh, R., Harrison, J. K., Seet, B. T., Kelvin, D. J., McFadden, G., and Moyer, R. W. (1998). Functional comparisons among members of the poxvirus T1/ 35 kDa family of soluble CC-chemokine inhibitor glycoproteins. Virology 250, 173±184. Macaulay, C., and McFadden, G. (1989). Tumorigenic poxviruses: Characterization of an early promoter from Shope fibroma virus. Virology 172, 237±246. Martinez-Pomares, L., Thompson, J. P., and Moyer, R. W. (1995). Mapping and investigation of the role in pathogenesis of the major unique secreted 35-kDa protein of rabbitpox Virus. Virology 206, 591±600. Patel, A. H., Gaffney, D. F., Subak-Sharpe, J. H., and Stow, N. D. (1990). DNA sequence of the gene encoding a major secreted protein of vaccinia virus, strain Lister. J. Gen. Virol. 71, 2013± 2021. Smith, C. A., Smith, T. D., Smolak, P. J., Friend, D., Hagen, H., Gerhart, M., Park, L., Pickup, D. J., Torrance, D., Mohler, K., Schooley, K., and Goodwin, R. G. (1997). Poxvirus genomes encode a secreted soluble protein that preferentially inhibits chemokine activity yet lacks sequence homology to known chemokine receptors. Virology 236, 316±327.