Viral tissue tropism in a susceptible host is often ... (LZ), occurring immediately after the fusion domain ... domain (CRD) consisting of 8 cysteine residues in the.
Microbiol. Immunol., 39(1), 1-9, 1995
Minireview
Virus Activation by Host Proteinases. Role in the Spread of Infection, Tissue Tropism and Pathogenicity Yoshiyuki
A Pivotal
Nagai
Department of Viral Infection, Institute of Medical Science, University of Tokyo, Shirokanedai 4-6-1, Minato-ku, Tokyo 108, Japan Received
September
Key words:
26, 1994
Cleavage-activation,
Envelope
glycoprotein,
Cellular
Viral tissue tropism in a susceptible host is often determined by virus-receptor interactions. Nevertheless, closely related viruses utilizing the same receptor molecules can display striking differences in tropism, or a virus can cause a localized infection despite the widespread occurrence of the receptor. Virus-receptor interactions cannot explain these events. Instead, the cellular proteinases required for the activation of viral fusion glycoproteins play a pivotal role. This proteinase-dependent mechanism of viral tropism was first demonstrated with Newcastle disease virus (NDV), an avian paramyxovirus, which uses ubiquitous sialic acids in complex sugars as the receptor but shows striking strain-dependent differences in tropism and virulence (35), and this mechanism is now applicable to many other virus-host systems (32, 33). of
The fusion (F) glycoprotein of NDV and other paramyxoviruses induces fusion between the viral envelope and host cell membrane, and hence enables the viral genome to enter the target cells. An essential step in F protein biosynthesis is the posttranslational endoproteolysis of the inactive precursor F0 glycoprotein by host cell proteinases (14, 35, 43). The precursor F0 is synthesized as a single chain molecule (Fig. Abbreviations:
FX,
immunodeficiency PC, proprotein respiratory
blood
virus convertase;
syncytial
clotting
factor
X; HIV-1,
type 1; NDV, Newcastle
virus;
PIV3,
parainfluenza
VAP, virus-activating
disease
Viral
pathogenicity
1). The signal peptide (SP) at the amino terminus directs the nascent polypeptide into the secretory pathway and is then removed from the nascent chain. A hydrophobic region near the carboxyl terminus (TMD) stops the translocation process and anchors the molecule to the lipid bilayer. The F0 is cleaved at the carboxyl side of a particular arginine to yield the twochain molecule with the aminoterminal F2 and the carboxy-terminalF1subunits, which remain bound together by a disulfide bond. The cleavage results in exposure of a long (about 25 amino acids) hydrophobic fusion-inducing domain (FID) at the amino terminus of the transmembrane F1 subunit (7). Thus, the overall structural features of fusion glycoprotein include three hydrophobic domains: the signal peptide, fusion domain and membrane anchor domain.Three other important structural domains are predicted (Fig. 1). Two of these are the heptad repeat (HR) and the leucine zipper (LZ), occurring immediately after the fusion domain and before the transmembrane domain, respectively (5, 6), and are thought to be important for oligomerization to form a tetramer, the mature form of the glycoprotein spike. The third one is the cysteine-rich domain (CRD) consisting of 8 cysteine residues in the middle of the F1 subunit. Their precise conservation in the primary sequence among paramyxovirus F proteins and the viral behavior to specificmonoclonal antibodies have suggested functional and antigenic importance of this domain (38, 52). We have recently shown with the mature F glycoprotein of Sendai virus, a murine paramyxovirus, that the cysteine residues all participate in disulfide-bridges, forming a bunched structure including two tandem loops, and we also were able to identify the cysteine residues for the intermolecular bridge between the F2 and F, subunits
1. Introduction
2. Structure, Function and Cleavage-Activation Paramyxovirus Fusion Glycoprotein
proteinase,
human virus;
virus
3; RSV , proteinase .
1
2
Y. NAGAI
3. Cleavage-Site-Motif Determines Availability of Activating Proteinases, Hence Viral Tissue Tropism and Pathogenicity
place in the Golgi apparatus (31, 36), more specifically in the trans Golgi network, while the latter is an extracellular event, as described later. The amino acid sequence at the cleavage site appears to be the most important determinant for the different cleavability of the glycoproteins. The readily cleavable Fos usually have a pair or a cluster of basic residues at this position whereas a single arginine is found with these of limited cleavability(Table 1) (53, 54). Obviously, the coincidence among cleavage site motifs, tissue specificity of proteinases for activation and the mode of virus spreading is also seen for other paramyxoviruses (Table 1). The Sendai virus receptor is ubiquitous sialic acids; however, the virus targets only the respiratory tract. Apparently, a specific trypsin-like proteinase recognizes the single-arginine motif in F0of Sendai virus, facilitates cleavage and allows spread of the virus, ultimately to cause pneumonia (49). The absence of significant virus growth in other organs is probably due to the absence of specific virus-activatingproteinases. In contrast, mumps virus, also utilizing ubiquitous sialic acids as the receptor, causes systemic, multiorgan tropic infection because of its highly cleavable oligobasic F0 motif. Measles virus and others exhibit the same pattern of high cleavability of F0and broad tissue tropism as that of mumps virus. However, respiratory syncytial virus (RSV) and parainfluenzatype 3 (PIV3), whose F0precursors also possess an oligobasic cleavage motif, generally cause localized infection of the respiratory tract. In immunocompromised infants, however, RSV was multiorgan-tropic, and in cyclophosphamidetreated animals, the virus spread outside the respiratory tract and into other organs whereas Sendai virus remained localized (3, 18). It thus appears likely that RSV is potentially pantropic, although immune response localizes the infection. PIV3 has a greater predilection to cause pneumonia than PIV1, which has a single arginine cleavage motif, and even induces a significant level of viremia in humans, an indication of systemic infection (11, 17). The consensus sequence RXK/RR at the cleavage site appears to be the minimumrequirement for recognitionby the ubiquitous proteinases.
(a) Paramyxoviruses The F0of virulent NDV strains is cleaved by a proteinase(s) ubiquitously present throughout the body and the infection is consequently pantropic or systemic, while avirulent strains undergo F0cleavage only in a few limited tissue types expressing a specific protease(s), hence causing an infection localized to particular organs such as the respiratory and alimentary tracts (34, 35, 37). Cleavage of the former type takes
(b) Other VirusFamilies The cleavability of the glycoprotein HA of influenza A virus in orthomyxoviridae determines whether a virus strain has a wide spectrum of target tissues (as virulent avian influenza strains have) or a narrow one (as mammalian and avirulent avian influenza strains have). The cleavage sites of highly cleavable HAs generally consist of multiple basic residues, whereas those of HAs of restricted cleavability consist of single
A
B
Fig.
1.
(A)
Precursor-product
coprotein.
For
teine
residues.
lines
and
those of
age
site.
HAI
and
gp
not
influenza
are
and
relationship see
Disulfide
products
41
details,
the
text.
bridges
defined
determined A HA2
noncovalently
virus are
of
Vertical
by HA
are
dashed and
HIV-1
disulfide-linked,
NDV
bars
fusion
connected lines.
gly-
represent
(B)
gp160. •¥, whereas
cysby
solid
Cleavage cleavgp120
associated.
(Fig. 1A) (16). Many enveloped animal viruses in other diverse taxonomic families were then found to share essentially the same mechanism of cleavage activation with the paramyxovirus family for expressing fusion activity and infectivity. Biologically active cleavage products are illustrated with the two wellstudied examples, influenza A virus hemagglutinin (HA) and the Env protein (gp160) of human immunodeficiencyvirus type 1 (HIV-1) (Fig. 1B).
MINIREVIEW
Table
1. Cleavage
site sequence
and cleavability
arginine residue (Table 1). The consensus RXK/RR cleavage motif is widely found in the highly cleavable group, and its requirement for ubiquitous cleavage has been confirmed by numerous site-directed mutagenesis studies (25). Thus, influenza A virus presents the same pattern of tropism and pathogenicity as described for NDV. One unique feature of an influenza HA (H5) is that a sugar chain attached to a site close to the oligobasic cleavage site sterically interferes with the action of ubiquitous processing enzymes (Table 1, ref. 20). Thus, the point mutation leading to the loss of the glycosylation site greatly increases the viral pathogenicity and was the cause of the 1983 outbreak (20).
3
of viral glycoprotein
precursors
In virtually all cases of the other enveloped viruses, an oligobasic cleavage site exists in the precursor glycoproteins, which are therefore activated in a wide variety of cells in culture and possibly in vivo (32, 33). Among these is the gp160 of HIV-1 in retroviridae (Table 1), whose cleavage to gp120 and gp41 at REKR site is necessary for viral fusion activity and infectivity (27). Although receptors and other host molecules are important for determining HIV-1 tropism, high cleavability of the gp160 is apparently a precondition for the virus to spread efficiently within and between target organs. Various structural features have been revealed by cleavage-site-directed mutagen-
Y. NAGAI
4
esis of HIV-1 gp160 but the RXK/RR motif again seems to be important as the minimum requirement for recognition by the ubiquitously occurring proteinases (4). 4. Virus-Activating Sight
Proteinases
Are Coming into
in
the
middle
phase
Although ovo
the
has
role
not
the
suggesting
tion
the
that
restricted
the
blood-clotting
to
cascade
reac-
side
where
X
(a) Blood-Clotting Factor Xa (FXa) Avirulent NDV, Sendai virus and mammalian and avirulent avian influenza A virus are thought to replicate well in the allantoic and amniotic sacs of the chicken embryo because of the availability of a VAP in the fluids filling these sacs. By using allantoic and amniotic fluids as starting materials, this VAP has been isolated and purified to homogeneity (8, 10). The VAP activates the viruses by cleaving their F0 or HA at a specific single arginine site. The VAP is a calciumdependent serine proteinase consisting of two subunits: a catalytic 33-kDa chain and a 23-kDa chain required for calcium binding. The amino-terminal amino acid sequence of each subunit is highly homologous to that of the activated form (FXa) of human or bovine blood-clotting factor X (FX), a member of the vitamin K-dependent serine proteinases. The identity of the VAP with FXa has been established by comparing the primary structure, deduced from the cloned cDNA sequence encoding the VAP, with those of human and bovine FX (46) and by amino acid sequencing of the FXa isolated from chicken plasma (8). FX is generally synthesized in the liver and circulates as one of the plasma proteinase zymogens. It was therefore an unexpected discovery that not only the liver but also a variety of other chicken embryo tissues, including the allantoic and amniotic cells as well as the kidney, intestine and spleen, express the specific mRNA and the translation product (39). Also surprise is the finding that active FXa is naturally present in the allantoic and amniotic fluids, since it is usually generated only
eny
with
site
also
be
trypsin
activated
or
for
acids
and
factor-like kDa
Ca2+
the
first
domains
in
cleavage
(Table are of
are
susceptible
by
required
than
that
the
of
appears
enzyme
on
the ƒÁ-carboxyglu-
two
amino
It will
action
of
the
1).
FXa
FXa
to
which
cleaved
lower
of
the
the com-
HAs,
sac
bound of
explain
they
concentration
via
often
activation
is much
P3
viral
E/QXR,
different
efficiency
to
bilayer
could
amount
vitro
the
influenza
allantoic
carat
as
virus
some
The
high
the
embryo
This
whether
in
This
attributable
lipid
tamic
the
enzymes.
(8).
be
the
in
learn
activation
trypsin
of
However,
seen in
to
other
virus
(8).
are
with
charge
for
The
replaceable
chicken
of
FXa.
negative
1).
FXa
of
generalized
T (Table
ovo
site
importance
the
poorly in
Interestingly,
site,
of
interesting
FXa
to
S or
sequences
are
in
only
cleavage
be
the
(21).
cleavage
specificity
pared
the
an
FXa
substrate to
than
recognition
is G,
is
activation the
appears
rather
similar
type
which
natural
activated
a
supports
this
chloro-
cleavage
suggesting
glycoproteins have
(E)
is
FXa-
macromolecular
mimics
the
chain
substrate
higher
block CMK
(Q),
bonyl for
can
acid
glutamine
with
further
EGR-CMK),
prothrombin,
glutamic
virus
contact
of
a
observa-
glutamyl-glycyl-arginine
peptidyl
bovine
the Sendai
finding
in
evidence
as
direct
Indeed,
(dansyl
cells,
This
activator
from
well
activation
event.
ketone
as
This
dansylated
penetrates
P3
(39).
fide
established,
in
cleavage
extracellular
a bona
come
NDV
surfaces
fluids
methyl
has
of
tissue
as
fully
this
that
inhibitor,
FXa
been
spread
notion
(8).
of
yet
strongly
containing
Although proteinase-mediated virus activation and its importance for the determination of virus tropism and pathogenicity were already recognized in the mid1970s, it is only recently that the candidate proteinases involved have been identified. A virus-activating proteinase (VAP) catalyzing cleavage at a single arginine site was first isolated from the chicken embryo. Subsequently, a different VAP of the same category has been isolated from the rat lung. The oligobasic RXK/ RR motif is shared by many cellular proproteins (15), and the recent progress in identifying their processing enzymes has greatly helped to characterize this type of VAP.
of
tion.
epidermal
growth
terminus
of
the
23-
chain.
(b)
Tryptase Rats
Tryptase
well
bronchial
nine
and HA,
cleavage
both
to
lung,
resulting
tality
rate
enzyme
(51).
and
its
Thus,
is
in
tryptase for
the
pathogenicity
concentration
required remarkably
of
for high
(at
the
the
for virus
in-
lungs,
lesions
Clara
is
lung.
Sendai
virus
least
1 ƒÊg
this prog-
and
anti-
spreading
lung
rat
and
a single-argi-
in and
the mor-
probably
dissemination in
in
purified Fo
required
virus
a reduction
responsible
not
cells
The
Sendai
lumen blocks
Clara
feature is
virus.
proteinase
virus
activates
the
enzyme in
(24).
rats
which
Clara in
this
of
Calcium
Tryptase
extracellularly
from
Sendai
of
Sendai
serine
secreted
activates
motif.
activation.
serum
and
epithelium
virus
to
arginine-specific in
cleaves
fluenza
vitro
mice
is an
exclusively
enzyme
virus
as
Clara
located the
Clara
as
the
of The
Sendai enzyme
activation ml-1;
ref.
in 24),
MINIREVIEW
when
compared
ref.
8).
with
Such
occur
a high
normally
that
following
are
modulated
viruses
as
to
tion
of
type
tryptase
suppresses
the
Bacterial
of
influenza
cescens
of
can
are
of
or
at
been
related
to
these
mating
di-
proteinimporfactor
physiological influenza
of
Subsequently,
PC2
and
proprotein
fpslfms
these
region
a variety
involved
in
secretory
of
proprotein
pathway. and are
tissues
PC3 localized
is
is
and
cell
processing On
the
other
restricted to
secretory
the and
to
and
organi-
subtilisins in
including
PC1)
a
pro-ƒ¿ RR
homologues
(PC
denotes
a unique
Furin, is
that
cell
yeast
the at
constitute (2).
(fur),
endoproteinase
the
identified, called
subtilisins
upstream
localized
KEX2 been
of
marker
in
prokaryotic
(formerly
convertase);
mammalian
field
sequence
the
been have
genetic
processes
both
have PC3
this
(a
toxin
several
eukaryotes
in
KEX2
pro-killer
with
The
always
proteinase
shares
frequently
involved
which
the
which
therefore
A landmark
cerevisiae, and
are
prohormones.
proteinases
function)
homologies
higher
They
as
motifs
has
the
discovery
killer
and
zational
PC2
motif
the
certain
and
motifs
for.
the
factor sites
in
cleave
Hageman
of
pro-
Endoproteinase
and
Saccharomyces
ed
to
plasma
well
cleavage
proproteins
searched was
of
appear
and
in
The
suggested
as
be mar-
48).
cleavage
bacterial
dibasic
interest
biology
furin,
should
replication (1,
activating results
with
Serratia
virus
activation
cellular
great
(19).
it
bacteria
through
devel-
proteinases
and
single-arginine
by
the infection
context,
cleavage-activation
processing
ly
these
a Subtilisin-Like
in
OKR
and
26).
Furin,
long
infec-
Clara
promote
symptoms
by the
Oligobasic
of
Clara
virus
produce
influenza
Earlier
for
to
this
severer
activation
found
the
(23).
aureus
plasminogen
(1,
Sendai
often
In
indirectly
factors
pul-
endogenous
tryptase
combined
they
promote
zymogens.
HAs
believed
and
with
or
(FXII)
by
surfactant
after
produced
tance
the
progeny that
is an
of
Staphylococcus
HAs
ase
secreted
that
to
seems
conditions of
found
cells,
and
release
producing
teinases
(d)
Clara
it
local
now
is also
categories.
that
rectly
was
ml-1;
appear
but
activation
alveolar
pneumonia
various
mice,
It
production
viruses,
noted
not
infection,
the
bacteria
opment
does
ng
Proteinases
Certain
the
2
30
surface
the
Clara.
stimulates
of
virus
which
as
(about
bronchial
facilitate
tryptase
well
inhibitor
(c)
the
surfactant,
cells
FXa
concentration
on Sendai
by
monary
chicken
fami-
encoded Golgi
by
membrane-
ubiquitously lines in
express-
and the
hand,
is possibly constitutive
expression
neuroendocrine vesicles
the
of cells.
and
consid-
5
ered to be responsible for processing in the regulated secretory pathway. Several lymphoid cell lines including NALM-6 exhibited a reduced cleaving activity even for F0of virulent NDV (33). Most of these cell lines were later found to express little endogenous furin at the transcriptional level (42). F0cleavage in NALM-6 cells is stimulated significantly by coexpressing KEX2 in a recombinant vaccinia virus (33). Similar coexpression studies have further revealed that furin is fully capable of cleaving F0 while PC2 and PC3 are entirely incapable or only partially capable of cleavage, and that the cleavage by furin results in enhancement of viral infectivity (9). Neither furin nor PC2 and PC3 can attack the single-arginine site of avirulent strains (9). Coexpression systems, however, often result in the expression in large excess beyond the basal level, perhaps giving rise to an unphysiological status. On the other hand, normal cells, which are fully capable of cleaving NDV F0 and other substrates at the RXK/RR site, express furin usually at a low level. Thus, the data obtained by coexpression systems should be carefully interpreted. LoVo cells, a cell line from a human colon carcinoma, are unable to process several cellular proproteins with the RXK/RR cleavage-motif, and were found to be defective in furin (29, 47). The normal human fur gene encodes a polypeptide of 794 amino acids which consists of the unique structural and functional domains, including the signal peptide, propeptide, subtilisin-like catalytic domain, homo B domain, cysteinerich domain and transmembrane domain, in this order from the amino terminus. Several independent furin cDNA clones isolated from LoVo cells unexceptionally revealed a one-nucleotide deletion at positions 1,283 to 1,286 (numbered according to ref. 55), where four successive T residues are present in the wild-type cDNA; only three T residues were found in the LoVo cDNAs (47). This deletion of one T residue was deduced to cause a frameshift at amino acid position 479, followed by an aberrant termination in the homo B domain of the furin polypeptide. The requirement of an entire homo B domain for processing activity was already demonstrated by carboxy-terminal truncation analysis (13). LoVo cells were thus expected to complement the previous results obtained by those coexpression studies, and were now found to be unable to cleave and activate the F0precursor of a virulent strain NDV (40). Furthermore, constitutive expression of the intact human furin has rendered LoVo cells fully capable of F0processing (manuscript in preparation). Several influenza HAs with an oligobasic cleavage site behave quite similarly to the NDV F0 in LoVo cells
6
Y. NAGAI
(manuscript in preparation). Thus, these studies using LoVo cells greatly substantiated the role of furin in viral glycoprotein cleavage and activation. A mutant line of Chinese hamster cells which is defective in NDV F0 processing was described, and this genetic lesion was also complemented by expressing mouse furin (28). A biochemical study was attempted to isolate an endoproteinase that activates the influenzafowl plaque virus (FPV) HA from Madin-Darby bovine kidney (MDBK) cells and was able to concentrate the enzyme sufficiently to establish its identity with furin using a specific antiserum (45). It has also been shown that HA cleavage increases in cells where the HA and furin are coexpressed by vaccinia virus vectors. Furthermore, cleavage site-specific decanoyl-REKR-CMK, which is able to penetrate cells, inhibits intracellular cleavage-activation of HA (45). A very similar pattern of increased glycoprotein cleavage by coexpressed furin and/or activation inhibition by decanoyl-REKRCMK has been demonstrated for PIV3 F0 (41) and HIV-1gp160 (12, 30). Recently,Siezen et al (44) presented a model for the three-dimensional structure of the catalytic domain of human furin and its interaction with model substrates. This model predicts distinct pockets for S1, S2 and S4 subsites of the catalytic site, a strong predominance of negatively charged residues in these subsites (Fig. 2), hence their electrostatic interactions with the corresponding positively charged P1, P2 and P4 residues of substrates for specific interaction. Exactly the same charge complementarityis seen between these subsites and corresponding residues of the F0 of virulent NDV and HIV-1 gp160 (Fig. 2). The model further predicts that the S3, 55 and S6 subsites, also negatively charged (Fig. 2), may further contribute to the high substrate specificity of furin (44). In this context, it is noteworthy that the positive P5 and the neutral P3 residues of the F0 are respectively quite fit and at least not disturbing the interactionwith furin, while the negative charge at P3 of gp160 apparently repells the S3 subsite, although the neutral P5 does not appear to be an obstacle (Fig. 2). These features strongly support the highly specific recognition and cleavage of the Fo by furin but may somewhat underestimate the affinity of the gp160 to this enzyme (see below). In addition, HIV-1 gp160 appears to be unique in that there is a cluster of basic residues three residues upstream from the authentic cleavage site (Fig. 2) and that site-directed mutagenesis of these basic residues to neutral residues abolished cleavage-activation (4). The negatively charged P3 of FPV (Table1) is likewise an obstacle in the interaction, but three successive positively charged
Fig. 2. Amino acid residues in subsites constituting the catalytic domain of human furin (44) and the cleavage site sequences of viral substrates. Dashes indicate identity with the above sequence. Negatively charged residues are shown by outlined letters. Positively charged residues in viral sequences are boxed.
residues may facilitate the interaction. Almost complete resistance of avirulent NDV F0 to furin (Fig. 2, also see ref.9) substantiates the importance of basic residues at P2 and perhaps at P5 for recognition by furin. A similar modeling study with trypsin predicted presence of no acidic residues but neutral ones for Si, S2 and S4 subsite (unpublished data). Thus, trypsin has a much broader specificity than furin and is able to cleave the F0 of both virulent and avirulent NDV strains (Fig. 2, also see refs. 34, 35, 40). (e) Is Furin the Only Ubiquitous VAP? As described above, there appears to be increasing evidence supporting the concept that furin is responsible for recognition and cleavage of the RXK/RRmotif. However, the conclusion has mainly come from the observation that furin coexpressed by an appropriate vector promoted cleavage-activation of the respective substrates in normal cells with a basal level of endogenous processing activity. The peptidyl CMKs used are not strictly enzyme-specific but can inactivate any enzyme that recognizes and cleaves the related sequences. Besides, the furin-substrate modeling described above suggested that microheterogeneity around and in the cleavage site sequence including the residue at P3 may affect the affinity to furin. LoVo cells defective in furin and incapable of cleaving the F0 of virulent NDV were recently found to be able to process the gp160 of HIV-1with kinetics comparable to that of normal cells, express the glycoprotein in a biologically active, fusogenic form on their surface and produce fully infectious progeny (40). Another observation, which also does not appear to indicate the requirement of furin for HIV-1 gp160 processing, is that a T-cell line, Molt 4, is fully susceptible to HIV-1, although it expresses little or only a marginal level of furin mRNA and does not efficiently process the F0 of virulent NDV (42). These recent
MINIREVIEW
studies would raise a further need to search for and identify the proteinases involved in HIV-1 gp160 processing rather than supporting the notion that furin is responsible. In this context, the Ca2+-independent26kDa proteinase is of considerable interest because it has been isolated from Molt 4 cells and appears to correctly process the gp160 at least in vitro (22), and because the intracellular gp160 processing appears to occur in low Ca2+milieu where NDV F0processing is inhibited(manuscript in preparation). PACE4 and PC6 are also the members of mammalian subtilisins and their involvement in gp160 processing has been suggested because of their broad tissue distribution, although their expression, just like furin, was found to be very low in Molt 4 cells (42). However, in view of the fact that LoVo furin is defective in the presumptive regulatory domains such as the homo B and the cysteine-rich domains but does retain the intact catalytic domain, the possibility has not been ruled out that gp160 cleavage does not require such regulatory domains at all and therefore occurs in LoVo cells, whereas these domains are absolutely necessary for NDV F0 processing. 5. Outlook Virus spread and tissue tropism depend strongly on the proper match between the cleavability of the viral glycoproteinby an endoproteinase and the availability of the proteinase in the host. The structure of the cleavage site is of prime importance for determining whether the virus is activated in a wide variety of tissues or in particular tissue types. With its ability to recognize the single arginine cleavage site, FXa became the first VAP to be identified, followed by tryptase Clara. The VAPs in this category thus differ depending on the host and/or tissue type. It would be interesting to identify influenza-virus-activatingproteinases in the human respiratory tract. Other plasma proteinases as well as coinfecting bacterial proteinases could be involved. Furthermore, many other proteinases are also recruited to the inflammatoryregion late in infection, which likely act as a VAP,but it would be important to identify the endogenous VAP triggering the initial rounds of virus replication. The ubiquitously occurring VAP that recognizes the oligobasic motif has been identified as furin, but the question remains whether it is the only proteinase of this type. In this context, identification of VAP for HIV-1gp160 is of particular importance. Although our current knowledgeof proteinase-mediated virus activation and its significance for viral pathogenicity is still incomplete, it has already stimulated
7
a variety of new approaches to virus disease control. For instance, the therapeutic potential of specific proteinase inhibitors is being tested, with peptidyl CMKs specific for cleavage site sequences taking the lead (8, 12, 41, 45). Avirulent NDVstrains with a single-arginine motif for proteinaserecognition have already been used routinely for protection of birds against the virulent viruses with a highly cleavable oligobasic motif. This highlights the potential of attenuated live vaccines made by cleavage site-directed mutagenesis. For example, if the oligobasic cleavage sites can be made monobasic, measles virus and mumps virus could be attenuated for use as live vaccines. Human influenza A virus is similar to murine Sendai virus in that both possess a single-arginine motif and target the respiratory tract. In view of the potential as a live vaccine of proteinase-activationmutants of Sendai virus that have a cleavage site susceptible to enzymes other than arginine-specific enzymes (50), it would be worth engineering similar mutants of influenza virus. VAPs undoubtedly are not solely involved in virus activation. So, what is the physiological significance of ectopic FX/FXa expression in the chicken embryo? What physiological roles does tryptase Clara play in the rat lung? Is furin the only processing proteinase in the constitutive secretory pathway in higher eukaryotes? These are new issues raised by the research on VAPsand are well worthy of investigation. I thank uscript eling
K. Kakuta
and A. Mitsuzawa
and Y. Takeuchi of proteinase-substrate
ported by research Science and Culture
for providing
for preparing information
interactions.
grants from the and the Ministry
My
the man-
on the modwork
was
sup-
Ministry of Education, of Health and Welfare,
Japan.
References
1) Akaike, T., Molla, A., Ando, M., Araki, S., and Maeda, H. 1989. Molecular mechanism of complex infection by bacteria and virus analyzed by a model using serratial proteinase and influenza virus in mice. J. Virol. 63: 22522259. 2) Barr, P.J. 1991. Mammalian subtilisins: the long-sought dibasic processing endoproteases. Cell 66: 1-3. 3) Blandford, G. 1975. Studies on the immune response and pathogenesis of Sendai virus infection of mice. III. The effects of cyclophosphamide. Immunology 28: 871-883. 4) Bosch, V., and Pawlita, M. 1990. Mutational analysis of the human immunodeficiency virus type 1 env gene product proteolytic cleavage site. J. Virol. 64: 2337-2344. 5) Buckland, R., and Wild, F. 1989. Leucine zipper motif extends. Nature 338: 547. 6) Chambers, P., Pringle, C.R., and Easton, A.J. 1990. Heptad repeat sequences are located adjacent to hydropho-
8
Y. NAGAI
bic
regions
Gen. 7)
in
Virol.
Gething,
the
White,
of
the
B.,
J.M.,
nant
Natl.
M.,
homologous
and
glycoprotein.
Waterfield, of
J.
Sci.
T.,
blood
viral
tropism
in
B.,
Ohnishi,
T.,
Y.
chick
precursor
22)
2737-2740.
Inocencio,
1990.
clotting
analysis
during 75:
An
factor
N.M.,
endoprotease
X
embryo.
21)
1978.
virus:
U.S.A.
Toyoda,
Nagai,
the
M.D.
Sendai
generated
Acad.
and
to
of
fusion
protein
Ogasawara,
Hamaguchi,
virus
sequence
Proc.
Gotoh,
of
fusion
NH2-terminal
activation. 8)
types
3075-3080.
M.J.,
Purification of
several
71:
as
a
EMBO
determi-
J.
9:
4189-
23)
4195. 9)
Gotoh, Nakayama,
K.,
Mammalian vating ity
Barr,
P.J.,
Inocencio,
the
to
G.,
and
fusion PC2
or
Esaki,
Nagai,
proteinases
paramyxovirus
furin/PACE
N.M.,
Thomas,
subtilisin-related of
of
Y.,
in
Y.
J.
1992.
cleavage
glycoprotein:
PC1/PC3.
E.,
acti-
24)
superior-
Virol.
66:
6391-
6397. 10)
Gotoh,
B.,
1992.
Isolation
amniotic
11)
FEBS
Gross,
P.A.,
Rev.
H.-D.,
and
cleavage
13)
with
W.
1992.
of
HIV-1
M.,
growth
of
tural
and
culture
cells.
Hosaka,
M.,
Nakayama, for
K.
and
man.
160
by
spe-
K.
furin,
27)
358-361.
a
cleavage
tissue
Klenk,
furin-mediated
360:
1992.
Kex2 at
like
Arg-X-
28)
296-301. Trypsin
action
culture
cells.
grown
in
on
III.
eggs
the
Struc-
and
tissue
1457-1465. M.,
Kim,
W.-S.,
J.,
Watanabe,
Murakami,
Arg-X-Lys/Arg-Arg by
pathway.
J.
furin
Biol.
T.,
K., motif
catalyzed
secretory
E.,
gp
of
viruses
cleavage
Per-
in
Nakayama,
1973.
Ikemizu,
1991.
precursor
stitutive
in
12:
K.,
of
Nature
111:
M.
Sendai
Virol.
virus
Shaw,
Inhibition
precursor
Nagahama,
Hatsuzawa,
H.,
properties
virus
J.
3
glycoprotein
J. Biochem.
of
1973.
26)
Angliker,
in
Ohuchi,
Sendai
difference
acti-
M.G.M. type
K.,
enzymatic
sites.
Homma,
the
25) Curnen,
ketones.
involved
Lys/Arrg-Arg
15)
V.,
Murakami,
and
Y.
as
894-898.
Garten,
endoprotease
Nagai,
embryo
paramyxovirus
parainfluenza 108:
Bosch,
K.,
for
and
peptidyl-chloromethyl
Molecular
14)
R.H.,
activation
and
chick
274-278.
S.,
Hatsuzawa,
T.,
from
responsible
Dis.
Hallenberger,
Xa
296:
Green,
Resp.
Ogasawara,
factor
Lett.
infection
Am.
cific
F.,
of
endoprotease
vation.
sistent
12)
Yamauchi,
as
within
Chem.
a signal the
266:
con-
12127-
30)
12130. 16)
Iwata,
S.,
Gotoh,
Schmidt,
B.,
ment
of
Sendai 17)
Johnson,
J. Virol.
R.A.,
Immun. Julius, ner, the
J.
Kawaoka, virulence
Nagai,
37:
369-373. Brake,
1984.
Isolation
R.H.
of
1973. of
Suffin,
1982.
S.C.,
Respiratory
Y., of
Blair, of
the
cleaving yeast Naeve, H5N2
1994.
H.,
Assignof
Viremia
during
hamsters.
Proc.
31)
Horswood,
R.L.,
32)
745-748.
G.A.,
A.,
Kido,
glycoprotein
syncytial
cyclophosphamide-treated
D.A.,
M., Y.
fusion
infection
144:
R.M.
in
lysine-arginine
processing 20)
Med.
Suzuki,
3200-3206.
virus
Prince,
Chanock,
infection
19)
Biol.
the
Green, 3
K.,
and in
68:
and type
Exp.
and
M., bridges
D.P.,
Johnson,
Titani,
Hamaguchi,
virus.
Soc.
A.C.,
disulfide
parainfluenza
18)
29)
and
cotton
L.,
C.W.,
R.,
structural
endopeptidase
prepro-ƒ¿-factor.
influenza
Kunisawa,
putative
Cell and viruses
Webster, in
virus
rats.
Infect.
and
Thor-
gene required
37:
for for
1075-1089. R.G.
chickens
1984. associat-
Is
ed with loss of carbohydrate from hemagglutinin? Virology 139: 306-316. Kettner, C., and Shaw, E. 1981. The selective affinity labeling of factor X by peptides of arginine chloromethyl ketone. Thromb. Res. 22: 645-652. Kido, H., Kamoshita, K., Fukutomi, A., and Katunuma, N. 1993. Processing protease for gp160 human immunodeficiency virus type I envelope glycoprotein precursor in human T4 + lymphocytes. J. Biol. Chem. 268: 1340613413. Kido, H., Sakai, K., Kishino, Y., and Tashiro, M. 1993. Pulmonary surfactant is a potential endogenous inhibitor of proteolytic activation of Sendai virus and influenza A virus FEBS Lett. 32: 115-119. Kido, H., Yokogoshi, Y., Sakai, K., Tashiro, M., Kishino, Y., Fukutomi, A., and Katunuma, N. 1992. Isolation and characterization of a novel trypsin-like protease found in rat bronchiolar epithelial Clara cells: a possible activator of the viral fusion glycoprotein. J. Biol. Chem. 267: 13573-13579. Klenk, H.-D., and Garten, W. 1994. Host cell proteases controlling virus pathogenicity. Trends Microbiol. 2: 3943. Lazarowitz, S.G., Goldberg, A.R., and Choppin, P.W. 1973. Proteolytic cleavage by plasmin of the HA polypeptide of influenza virus: host cell activation of serum plasminogen. Virology 56: 172-180. McCune, J.M., Rabin, L.B., Feinberg, M.B., Lieberman, M., Kosek, J.C., Reyes, G.R., and Weissman, I.L. 1988. Endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus. Cell 53: 55-67. Moehring, J.M., Inocencio, N.M., Robertson, B.J., and Moehring, T.J. 1993. Expression of mouse furin in a Chinese hamster cell resistant to pseudomonas exotoxin A and viruses complements the genetic lesion. J. Biol. Chem. 288: 2590-2594. Mondino, A., Giordano, S., and Comoglio, P.M. 1991. Defective posttranslational processing activates the tyrosine kinase encoded by the MET proto-oncogene (hepatocyte growth factor receptor). Mol. Cell. Biol. 11: 60846092. Morikawa, Y., Barsov, E., and Jones, I. 1993. Legitimate and illegitimate cleavage of human immunodeficiency virus glycoproteins by furin. J. Virol. 67: 3601-3604. Morrison, T.G., Ward, L.J., and Semerjian, A. 1989. Intracellular processing of the Newcastle disease virus fusion glycoprotein. J. Virol. 53: 851-857. Nagai, Y. 1993. Protease-dependent virus tropism and
pathogenicity. Trends Microbiol. 1: 81-87. 33) Nagai, Y., Inocencio, N.M., and Gotoh, B. 1991. Paramyxovirus tropism dependent on host proteases activating the viral fusion glycoprotein. Behring Inst. Mit. 89: 3545. 34) Nagai, Y., and Klenk, H.-D. 1977. Activation of precursors to both glycoproteins of Newcastle disease virus by proteolytic cleavage. Virology 77: 125-134. 35) Nagai, Y., Klenk, H.-D., and Rott, R. 1976. Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus. Virology 72:
MINIREVIEW
494-508. 36) Nagai, Y., Ogura, H., and Klenk, H.-D. 1976. Studies on the assembly of the envelope of Newcastle disease virus. Virology 69: 523-538. 37) Nagai, Y., Shimokata, K., Yoshida, T., Hamaguchi, M., Iinuma, M., Maeno, K., Matsumoto, T., Klenk, H.-D., and Rott, R. 1979. The spread of a pathogenic and an apathogenic strain of Newcastle disease virus in the chick embryo as depending on the protease sensitivity of the virus glycoproteins. J. Gen. Virol. 45: 263-272. 38) Neyt, C., Geliebter, J., Slaoui, M., Morales, D., Meulemans, G., and Burny, A. 1989. Mutations located on both Fl and F2 subunits of the Newcastle disease virus fusion
39)
40)
41)
42)
43)
44)
protein confer resistance to neutralization with monoclonal antibodies. J. Virol. 63: 952-954. Ogasawara, T., Gotoh, B., Suzuki, H., Asaka, J., Shimokata, K., Rott, R., and Nagai, Y. 1992. Expression of factor X and its significance for the determination of paramyxovirus tropism in the chick embryo. EMBO J. 11: 467-472. Ohnishi, Y., Shioda, T., Nakayama, K., Iwata, S., Gotoh, B., Hamaguchi, M., and Nagai, Y. 1994. A furin-defective cell line is able to process correctly the gp160 of human immunodeficiency virus type 1. J. Virol. 68: 4075-4079. Ortmann, D., Ohuchi, M., Angliker, H., Shaw, E., Garten, W., and Klenk, H.-D. 1994. Proteolytic cleavage of wild type and mutants of the F protein of human parainfluenza virus type 3 by two subtilisin-like endoproteases, furin and Kex2. J. Virol. 68: 2772-2776. Sakaguchi, T., Fujii, Y., Kiyotani, K., and Yoshida, T. 1994. Correlation of proteolytic cleavage of paramyxovirus F protein precursors with expression of the fur, PACE4 and PC6 genes in mammalian cells. J. Gen. Virol. in press. Scheid, A., and Choppin, P.W. 1974. Identification of biological activities of paramyxovirus glycoproteins. Activation of cell fusion, hemolysis, and infectivity by proteolytic cleavage of an inactive precursor protein of Sendai virus. Virology 57: 475-490. Siezen, R.J., Creemers, J.W.M., and Van De Ven, W.J.M. 1994. Homology modelling of the catalytic domain of human furin. A model for the eukaryotic subtilisin-like
proprotein convertases. Eur. J. Biochem. 222: 255-266. 45) Steineke-Grober, A., Vey, M., Angliker, H., Shaw, E., Thomas, G., Roberts, C., Klenk, H.-D., and Garten, W. 1992. Influenza virus hemagglutinin with multibasic
9
cleavage site is activated by furin, a subtilisin-like endoprotease. EMBO J. 11: 2407-2414. 46) Suzuki, H., Harada, A., Hayashi, Y., Wada, K., Asaka, J., Gotoh, B., Ogasawara, T., and Nagai, Y. 1991. Primary structure of the virus activating protease from chick embryo. Its identity with the blood clotting factor Xa. FEBS Lett. 283: 281-285. 47) Takahashi, S., Kasai, K., Hatsuzawa, K., Kitamura, N., Misumi, Y., Ikehara, Y., Murakami, K., and Nakayama, K. 1993. A mutation of furin causes the lack of precursor-
48)
49)
50)
51)
processing activity in human colon carcinoma LoVo cells. Biochem. Biophys. Res. Commun. 195: 1019-1026. Tashiro, M., Ciborowski, P., Klenk, H.-D., Pulverer, G., and Rott, R. 1987. Role of staphylococcus protease in the development of influenza pneumonia. Nature 325: 536537. Tashiro, M., and Homma, M. 1983. Pneumotropism of Sendai virus in relation to protease mediated activation in mouse lung. Infect. Immun. 39: 879-888. Tashiro, M., and Homma, M. 1985. Protection of mice from wild-type Sendai virus infection by a trypsin-resistant mutant, TR-2. J. Virol. 53: 228-234. Tashiro, M., Yokogoshi, Y., Tobita, K., Seto, J.T., Rott, R., and Kido, H. 1992. Tryptase Clara, an activating protease for Sendai virus in rat lungs, is involved in pneumopatho-
genicity. J. Virol. 66: 7211-7216. 52) Toyoda, T., Gotoh, B., Sakaguchi, T., Kida, H., and Nagai, Y. 1988. Identification of amino acids relevant to three antigenic determinants on the fusion protein of Newcastle disease virus that are involved in fusion inhibition and neutralization. J. Virol. 62: 4427-4430. 53) Toyoda, T., Sakaguchi, T., Hirota, H., Gotoh, B., Kuma, K., Miyata, T., and Nagai, Y. 1989. Newcastle disease virus evolution. II. Lack of gene recombination in generating virulent and avirulent strains. Virology 169: 273-282. 54) Toyoda, T., Sakaguchi, T., Imai, K., Inocencio, N.M., Gotoh, B., Hamaguchi, M., and Nagai, Y. 1987. Structural comparison of the cleavage-activation site of the fusion glycoprotein between virulent and avirulent strains of Newcastle disease virus. Virology 158: 242-247. 55) van den Ouweland, A.M.W., van Duijnhoven, H.L.P., Keizer, G.D., Dorssers, L.C.J., and Van de Ven, W.J.M. 1990. Structural homology between the human fur gene product and the subtilisin-like protease encoded by yeast KEX2. Nucleic Acids Res. 18: 664.
