SERP-1, a Secreted

Poxviral Serpin
Grant McFadden1,* and Richard Moyer2
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: mcfadden@rri.on.ca
DOI: 10.1006/rwcy.2000.03017.

SUMMARY While SERP-1 is secreted, the orthopoxvirus serpin

More than half a dozen poxvirus-encoded members
of the serpin superfamily of serine proteinase inhib-
itors have been discovered, but only one of these,
SERP-1 of myxoma virus, functions as an extracell-
ular immunomodulator. SERP-1 is expressed as a
secreted glycoprotein that protects myxoma-infected
cells from clearance by macrophages and other
inflammatory cells. Biochemically, SERP-1 protein
binds and inhibits a spectrum of serine proteinases,
such as plasmin, urokinase and tissue plasminogen
activator, but its precise mode of action in virus-
infected lesions remains to be elucidated. The purified
SERP-1 protein alone functions as a potent antiin-
flammatory reagent in various animal models of
inflammation.
SPI-3 has a similar, if not identical, spectrum of inhib-
ited proteinases (R. Moyer and P. Turner, unpub-
lished data). SERP-1 was first discovered in 1990, as a
single copy gene in a related poxvirus designated
malignant rabbit fibroma virus, and as a repeated
copy gene in the terminal inverted repeat sequences
of myxoma virus (Upton et al., 1990). Expression
studies indicated that the processed SERP-1 protein
is secreted as a 55±60 kDa glycoprotein that forms
inhibitory complexes with a variety of cellular serine
proteinases (Lomas et al., 1993; Nash et al., 1998).
Alternative names
SPI-4 (proposed by Turner et al., 1995).

BACKGROUND
Discovery
At least eight poxvirus genes have been described that
are members of the serpin superfamily of serine pro-
teinase inhibitors (Turner et al., 1995; Messud-Petit
et al., 1998). The only one of these that expresses a
protein secreted from infected cells is SERP-1, which
is encoded by the leporipoxvirus myxoma virus (Nash
et al., 1997). Thus, SERP-1 is the only viral serpin
that qualifies as a virokine, and is likely targeted to
inhibit extracellular (or cell surface) proteinases.
Structure
SERP-1 contains the consensus domain structural
features of eukaryotic serpins, including the reactive
center loop bearing an unusual P1±P10 pairing of
Arg±Asn (see Figure 2).
Main activities and
pathophysiological roles
Like all serpins, SERP-1 forms 1:1 inhibitory com-
plexes with serine proteinases. In the case of SERP-1,
1430 Grant McFadden and Richard Moyer

the list of inhibitable proteinases includes tissue postinfection and remain at high levels throughout
plasminogen activator, urokinase, and plasmin (Nash the viral replication cycle. Like all poxviruses, the
et al., 1997, 1998). These inhibitory studies were con- transcriptional regulation is under virus control in
ducted in vitro with human proteinases, indicating cells that support the full replication cycle. Thus,
that SERP-1 target proteinases are highly conserved SERP-1 expression is relatively independent of the
in evolution. A reactive center loop mutant of SERP- type of infected cell.
1 in which the P1±P10 site has been mutated to Ala±
Ala (SAA: see Figure 2) does not bind or inhibit any
of the normal proteinase substrates for SERP-1,
indicating that the viral protein functions as a true PROTEIN
secreted serpin and is classified as an enzymatically
active virokine. Accession numbers
g332305
GENE AND GENE REGULATION
Sequence
Accession numbers
See Figure 1.
M35233

Chromosome location Description of protein

The SERP-1 gene is present as a single copy in the
genome of malignant rabbit fibroma virus (which can
be regarded as a myxoma virus substrain), and as two
copies in the terminal inverted repeat sequences of
myxoma virus, strain Lausanne (Upton et al., 1990).
Regulatory sites and corresponding
transcription factors
SERP-1 is expressed as a classic late poxvirus gene,
and as such is transcribed after DNA replication has
commenced and late transcription factors have been
synthesized (Macen et al., 1993). The SERP-1 pro-
moter sequence includes the consensus motifs of all
late poxvirus genes (Moss, 1996). SERP-1 mRNA
species are first detected at approximately 6 hours
SERP-1 is a classic eukaryotic serpin, and is expressed
as a 55±60 kDa secreted glycoprotein. Although the
protein has not been crystallized, alignment studies
indicate an unusually extended C-terminus, and the
absence of a predicted D-helix, when compared with
other nonviral serpins (Lomas et al., 1993). Despite
the potential absence of the D-helix, modeling studies
predict that the ordering and orientation of the classic
serpin protein domains remain similar to other
serpins, such as ovalbumin and antichymotrypsin
(Lomas et al., 1993).
Important homologies
SERP-1 possesses sequence motifs common to all
serpins, but the closest cellular homologs include anti-

Figure 1 Protein sequence of SERP-1.

MKYLVLVLCL TSCACRDIGL WTFRYVYNES DNVVFSPYGL TSALSVLRIA

AGGNTKREID VPESVVEDSD AFLALRELFV DASVPLRPEF TAFFSSRFNT
SVQRVTENSE NVKDVINSYV KDKTGGDVPR VLDASLDRDT KMLLLSSVRM
KTSWRIVFDP SFTTDQPFYS GNVTYKVRMM NKIDTLKTET FTLRNVGYSV
TELPYKRRQT AMLLVVPDDL GEIVRALDLS LVRFWIRNMR KDVCQVVMPK
FSVESVLDLR DALQRLGVRD AFDPSRADFG QASPSNDLYV TKVLQTSKIE
ADERGTTASS DTAITLIPRN ALTAIVANKP FMFLIYHKPT TTVLFMGTIT
KGEKVIYDTE GRDDVVSSV

Figure 2 A schematic representation of the SERP-1
protein structure indicating the positions of the
predicted  helices that form the serpin structural
fold. Helix a (hA) is the signal sequence responsible for
SERP-1 secretion and is not present in the mature
protein. Helix D (hD), normally present in serpins, is
missing in SERP-1, although this does not appear to
be essential for inhibitory function. The reactive center
loop (RCL) interacts with the active site of the
proteinase and contains the critical P1±P10 residues.
The stable inhibited serpin±enzyme complex is be-
lieved to be an acyl-enzyme linked covalently at the
P1 residue. The RCL sequence of SERP-1 and the
engineered noninhibitory mutant (SAA) are also
shown.
trypsin, antichymotrypsin, and plasminogen activator
inhibitor 1. The specificity of serpins is in part defined
by the amino acid residues at the P1±P10 site, which in
the case of SERP-1 is a unique pairing (Arg±Asn) not
found in any other known serpin (Figure 2).
Posttranslational modifications
Polyclonal antisera against SERP-1 expressed from
myxoma virus reveal a heterogeneous 55±60 kDa
species which can be digested with N-glycosidase F to
the predicted size of the amino acid sequence, indi-
cating that the major posttranslational modification
is N-linked glycosylation. N-Linked sugar residues
are not believed to affect the serpin function of
SERP-1 in terms of reaction parameters in vitro, but
could affect protein half-life or tissue localization
in vivo.
RECEPTOR UTILIZATION
The `true' biological serine proteinases that bind and
mediate the anti-inflammatory properties of SERP-1
in infected tissues are still speculative, but candi-
date proteinases include a variety of soluble or
SERP-1, a Secreted Poxviral Serpin 1431
membrane-associated enzymes. This subject is still
under active investigation.
IN VITRO ACTIVITIES
In vitro findings
A variety of catalytic parameters, including apparent
inhibition constraints, apparent second-order rate
constant for inhibition, and the measured rate con-
stant for dissociation of the inhibited complex, were
determined for SERP-1 against a spectrum of host
serine proteinases in vitro (Lomas et al., 1993; Nash
et al., 1998). The most effectively inhibited substrates
for SERP-1 in vitro were plasmin, tissue plasminogen
activator, urokinase, and thrombin.
IN VIVO BIOLOGICAL
ACTIVITIES OF LIGANDS IN
ANIMAL MODELS
Normal physiological roles
The role of SERP-1 in vivo has been investigated by
targeted disruption of the gene, followed by analysis
of the knockout virus infection in susceptible rabbits
(Upton et al., 1990; Macen et al., 1993). Unlike the
parental myxoma virus, which is uniformly fatal to
the European rabbit (Oryctolagus cuniculus), the
SERP-1-minus virus was severely attenuated in vivo
despite the fact that it replicated normally in tissue
culture (Macen et al., 1993). Histological analysis of
infected lesions indicated that in the absence of
SERP-1 expression a more effective inflammatory cell
response resulted in a more rapid clearance of the
infection (Macen et al., 1993). Thus, the biological
role of SERP-1 can be operationally defined as anti-
inflammatory, even though the target serine protei-
nase(s) remains speculative.
Species differences
All in vitro inhibition assays of SERP-1 protein have
been conducted with human serine proteinases, sug-
gesting a broad species range for its inhibitory func-
tions. However, a precise evaluation of the inhibition
1432 Grant McFadden and Richard Moyer
constants with a single class of serine proteinase from
multiple vertebrates has not been conducted.
Pharmacological effects
SERP-1 protein expressed from myxoma, or vaccinia
virus vectors that overexpress SERP-1, or CHO-
transfected cells, has been purified and tested as an
anti-inflammatory reagent in two models of inflam-
mation: balloon-induced atherosclerosis and antigen-
induced arthritis (Lucas et al., 1996; Maksymowych
et al., 1996). In the coronary restenosis model, single
infusions of purified SERP-1 protein in the picogram
Lucas, A., Liu, L.-Y., Macen, J., Nash, P., Dai, E., Stewart, M.,
Graham, K., Etches, W., Boshkov, L., Nation, P. N.,
Humen, D., Hobman, M. L., and McFadden, G. (1996).
Virus-encoded serine proteinase inhibitor SERP-1 inhibits
atherosclerotic plaque development after balloon angioplasty.
Circulation 94, 2890±2900.
Macen, J. L., Upton, C., Nation, N., and McFadden, G. (1993).
SERP-1, a serine proteinase inhibitor encoded by myxoma
virus, is a secreted glycoprotein that interferes with inflamma-
tion. Virology 195, 348±363.
Maksymowych, W. P., Nation, N., Nash, P. D., Macen, J.,
Lucas, A., McFadden, G., and Russell, A. S. (1996).
Amelioration of antigen-induced arthritis in rabbits treated
with a secreted viral serine proteinase inhibitor. J. Rheumatol.
23, 878±882.
Messud-Petit, F., Gelfi, J., Delverdier, M., Amardeilh, M.-F.,
Py, R., Sutter, G., and Bertagnoli, S. (1998). SERP-2, an inhi-
dose range at the time of balloon injury were able to
block the influx of macrophages into the injured vas-
culature and thus reduce the resultant development
of atherosclerotic plaque (Lucas et al., 1996). Similar
anti-inflammatory properties of SERP-1 protein were
noted after intra-articular injection in a rabbit model
of collagen-induced arthritis (Maksymowych et al.,
1996). In each case, the therapeutic dosage of SERP-1
protein was in the picogram to nanogram range,
which is lower than other known antiinflammatory
reagents but closer to the natural levels of secreted
SERP-1 expression during virus infection.
References
Lomas, D. A., Evans, D. L., Upton, C., McFadden, G., and
Carrell, R. W. (1993). Inhibition of plasmin, urokinase, tissue
plasminogen activator, and C1S by a myxoma virus serine pro-
teinase inhibitor. J. Biol. Chem. 268, 516±521.
bitor of the interleukin-1-converting enzyme, is critical in the
pathobiology of myxoma virus. J. Virol. 72, 7830±7839.
Moss, B. (1996). In ``Virology'' (ed B. N. Fields, D. M. Knipe, and
P. M. Howley), Replication of poxviruses, pp. 2637±2671.
Lippincott Raven Press, New York.
Nash, P., Lucas, A., and McFadden, G. (1997). In ``Chemistry and
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glycoprotein that inhibits the inflammatory response to myx-
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Nash, P., Whitty, A., Handwerker, J., Macen, J., and McFadden, G.
(1998). Inhibitory specificity of the anti-inflammatory myxoma
virus serpin, Serp-1. J. Biol. Chem. 273, 20982±20991.
Turner, P. C., Musy, P. W., and Moyer, R. W. (1995). In
``Viroceptors, Virokines and Related Immune Modulators
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(1990). Myxoma virus and malignant rabbit fibroma virus
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