From the
The replicase ORF1a polyprotein of equine arteritis virus, a
positive-stranded RNA virus, is proteolytically processed into (at
least) six nonstructural proteins (Nsp). A papain-like Cys protease in
Nsp1 and a chymotrypsin-like Ser protease in Nsp4 are involved in this
process. In this paper we demonstrate that the Nsp2/3 junction is not
cleaved by either of these previously described proteases. Comparative
sequence analysis suggested that an additional Cys protease resided in
the N-terminal Nsp2 domain. For equine arteritis virus, this domain was
shown to induce Nsp2/3 cleavage in a trans-cleavage assay.
Processing was abolished when the putative active site residues,
Cys-270 and His-332, were replaced. Other Nsp2 domains and three other
conserved Cys residues were also shown to be essential. The Nsp2 Cys
protease displays sequence similarity with viral papain-like proteases.
However, the presumed catalytic Cys-270 is followed by a conserved Gly
rather than the characteristic Trp. Replacement of Gly-271 by Trp
abolished the Nsp2/3 cleavage. Conservation of a Cys-Gly dipeptide is a
hallmark of viral chymotrypsin-like Cys proteases. Thus, the
arterivirus Nsp2 protease is an unusual Cys protease with amino acid
sequence similarities to both papain-like and chymotrypsin-like
proteases.
Proteolytic processing of viral proteins fulfills a key role in
the life cycle of the majority of
viruses(1, 2, 3) . Positive-stranded RNA viruses
usually synthesize polyproteins composed of structural and/or
replicative subunits. The processing of nonstructural precursors is
almost exclusively conducted by specific virus-encoded proteases. The
majority of these enzymes are assumed to be distant relatives of
papain-like or chymotrypsin-like cellular proteases(4) .
Between positive-stranded RNA viral and cellular papain-like
proteases the only conserved residues appear to be: a catalytic Cys,
followed by a bulky hydrophobic residue (usually Trp), and a more
downstream catalytic His(5) . The distance between Cys and His
tends to be smaller and more variable in viral papain-like proteases.
It remains to be determined whether viral and cellular papain-like
proteases are structurally similar.
Only a fraction of the viral
chymotrypsin-like proteases contains the ``canonical''
catalytic triad of Ser, His, and Asp, which is found in cellular
chymotrypsin-like enzymes(6, 7) . Several viral
chymotrypsin-like proteases employ an active site Cys instead of Ser or
a Glu instead of Asp(8, 9) . It was argued that viral
chymotrypsin-like Cys proteases (also known as picornavirus 3C-like Cys
proteases) might be direct descendants of an ancestral
chymotrypsin-like protease(10, 11) . Recently, the
structures of proteases belonging to the serine (12) and
cysteine (13, 14) branches of the viral
chymotrypsin-like group have been solved. It is now clear that, within
the common framework of the two
Both papain-like and chymotrypsin-like proteases are involved in the
replication cycle of arteriviruses, a newly recognized group of animal
positive-stranded RNA viruses(15) . The genome structure of
three closely related arteriviruses, equine arteritis virus (EAV; Ref.
16),
The pM
Our
computer analysis showed that the arrangement of the conserved Cys-270
and His-332 residues (Fig. 2A) in the N-terminal Nsp2
domain resembles the catalytic dyad of papain-like proteases of
arteriviruses and other positive-stranded RNA viruses. The sequences
surrounding the LDV/PRRSV counterpart of EAV Cys-270 produced a match
with the region around the catalytic Cys-164 of the EAV Nsp1
papain-like protease (PCP; Fig. 2B). A close inspection
of the Cys-270 region revealed that it also resembles the context of
the catalytic nucleophile of chymotrypsin-like Cys proteases (Fig. 2C). Furthermore, it was noticed that His-332 is
the only conserved His residue in the sequences of Nsp2 and Nsp3. This
type of residue fulfills a catalytic role in cysteine, serine, and
metalloproteases(34) . Its presence was an additional indication
for a possible proteolytic function of the Nsp2 N-terminal domain.
Therefore, we speculated that Cys-270 and His-332 form the catalytic
dyad of an unusual type of Cys protease (CP), which mediates the Nsp2/3
cleavage.
Subsequently,
we deleted part of the Nsp2 sequence which separates the putative CP
and its cleavage site (Fig. 3, deletions B and C). Also these deletions inhibited proteolysis, since the
58-kDa C-terminal cleavage product was not produced in detectable
amounts. Even smaller deletions in the hydrophilic, hydrophobic, and
C-terminal Cys-rich Nsp2 regions (Fig. 3, deletions D,
E, and F) were not tolerated, suggesting that an intact
Nsp2 is required for processing of the Nsp2/3 site. Again some of the
constructs yielded some unexplained minor bands (e.g. two
bands in the 55-60-kDa region in the case of deletion B; Fig. 3), which we assume to be unrelated to the processing of the
Nsp2/3 junction.
We have shown that the proteolysis of the Nsp2/3 junction in
the EAV ORF1a protein is not mediated by the previously described Nsp1
and Nsp4 proteases. Instead, our theoretical and experimental data
strongly suggest that Nsp2 contains a novel type of Cys protease, which
cleaves the Nsp2/3 site. A putative catalytic dyad was identified by
comparative sequence analysis and shown to be essential for proteolytic
activity (Fig. 5). The context of Cys-270 resembles that of the
catalytic nucleophiles of known proteases from both the papain-like and
chymotrypsin-like protease families (Fig. 2C and ). Furthermore, it was shown that the Nsp2 CP forms a
distinct domain which can induce cleavage of the Nsp2/3 site in
trans (Fig. 4). Formally, our results do not exclude the
possibility that the Nsp2 N-terminal domain serves as a co-factor for
an unidentified host protease. It should be noticed, however, that to
date many proteolytic cleavages in viral nonstructural polyproteins
were attributed to viral proteases, whereas only two flavivirus
cleavages were shown to be mediated by host
proteases(37, 38) .
Immunofluorescence experiments
with infected cells have revealed that Nsp2 is associated with
intracellular membranes (data not shown). This interaction may be
mediated by the hydrophobic domain of Nsp2. Likewise, the extremely
hydrophobic Nsp3 may be membrane-bound. Both Nsp2 and Nsp3 contain
clusters of conserved Cys residues and remain associated after cleavage
of the Nsp2/3 site(19) . Together with the data from our Nsp2
deletion and site-directed mutagenesis ( Fig. 3and 5), these
observations suggest that the formation of a specific Nsp2-Nsp3
complex, possibly involving host components like membranes, is required
for proteolysis of the Nsp2/3 junction. This could explain the
deleterious effects of our Nsp2 deletions and some amino acid
substitutions. Our failure to demonstrate the Nsp2/3 cleavage in
bacterial cells and reticulocyte lysates
Extensive site-directed mutagenesis of Cys-270 and His-332 showed
that they are indispensable for proteolytic activity. The relative
position of these putative catalytic residues is typical for viral
papain-like proteases. Two papain-like protease domains have been
recognized in the Nsp1 region of arteriviruses (Refs. 20 and 39; Fig. 2B). In LDV and PRRSV both proteases, named
PCP
Still, two important differences
between the arterivirus Nsp2 CP and viral papain-like proteases can be
observed. In the first place, most viral papain-like enzymes cleave
just downstream of the catalytic His residue(5) . In the case of
the EAV Nsp2 CP approximately 500 residues separate the putative
protease and its cleavage site, and this distance may even be up to 450
residues larger in other arteriviruses(19, 39) . Most of
all, however, the conserved Gly-271 which follows catalytic Cys-270 is
a special feature of the Nsp2 CP. In arterivirus Nsp1 PCPs, and other
papain-like proteases, this position is typically occupied by Trp, or
sometimes another bulky amino acid, but never by Gly(5) .
Remarkably, conservation of an active site Cys-Gly dipeptide is a
hallmark of chymotrypsin-like Cys proteases (Fig. 2C),
in which the linear order of catalytic Cys and His is reversed in
comparison with papain-like proteases(4) . When EAV Gly-271 was
replaced by Trp, the Nsp2/3 cleavage was abolished (Fig. 5). This
underlines its importance and indicates that the Trp
The sequence similarities observed
for the arterivirus Nsp2 Cys protease can be analyzed in the context of
the structural properties of the papain-like and chymotrypsin-like
enzymes. The fold and the linear order, context, and type of active
site residues are different for both these groups of cellular
proteases. The only structural resemblance between them, which was
noticed previously by Garavito et al.(41) , is the
apparently similar organization of the catalytic triads of papain and
chymotrypsin, Cys/His/Asn and Ser/His/Asp, respectively. However, these
triads cannot be spatially superimposed, since they are of opposite
hand. The resemblance was therefore concluded to be the result of
convergent evolution(41) .
The recent identification of
different RNA viral Cys proteases has greatly expanded the diversity of
both papain-like and chymotrypsin-like proteases. Similarities within
and between the two groups were highlighted (), and
comprehensive evidence was reported for the common ancestry of a part
of the RNA viral Cys proteases and the cellular and viral
chymotrypsin-like Ser proteases (13, 14, 42). Due to the lack of
structural data, the evolutionary relationships between cellular and
viral papain-like proteases, including the arterivirus Nsp2 CP, are
less clear, but the available amino acid sequence information favors
divergent evolution. On the basis of the analysis of the triplets
encoding a variety of catalytic Ser residues, Brenner has postulated
previously that an ancestral Cys protease may have preceded the
chymotrypsin-like Ser proteases(10) . The analysis of present
day chymotrypsin-like Cys proteases points toward the conserved
Gly-X-Cys-Gly sequence () as the most plausible
context of the catalytic Cys residue in such an ancestral
chymotrypsin-like protease. Remarkably, this characteristic
Gly-X-Cys-Gly sequence is also conserved in the catalytic
center of the arterivirus Nsp2 Cys proteases that belong to the
papain-like group (). Although divergent evolution could
obviously provide the most parsimonious explanation for this similarity
between chymotrypsin-like and Nsp2 Cys proteases, this interpretation
cannot be reconciled with the postulated convergent evolution of the
prototypes of the two families, chymotrypsin and papain(41) .
Future studies of RNA virus proteases, particularly the arterivirus
Nsp2 CP, may resolve this puzzle and increase our insight in a possible
evolutionary relationship between papain-like and chymotrypsin-like
proteases.
We thank Johan den Boon, Leonie van Dinten, and Peter
Bredenbeek for helpful discussions and critical reading of the
manuscript. A. E. G. is most grateful to Michael Rossmann (supported by
a Cooperation in Applied Science and Technology award and a National
Science Foundation grant) for encouragement and support during his work
in Purdue.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-barrel domain fold, the divergent
evolution of catalytic systems and structures of positive-stranded RNA
virus proteases has generated a set of unique proteolytic enzymes.
(
)porcine reproductive and respiratory
syndrome virus (PRRSV; Ref. 17), and lactate dehydrogenase-elevating
virus (LDV; Ref. 18), was determined recently. The 5` three-quarters of
the 12.7-kilobase genome of EAV, the arterivirus prototype, contain two
open reading frames (ORFs), which encode replicative proteins. The
ORF1a product is 1727 amino acids (aa) long. ORF1b is expressed through
ribosomal frameshifting which generates a 3175-aa ORF1ab fusion protein
(16). In infected cells and expression systems, the ORF1a polyprotein
is cleaved at least five times, yielding nonstructural proteins (Nsp) 1
through 6 (Fig. 1; Ref. 19). A papain-like Cys protease (PCP) in
EAV Nsp1 cleaves the Nsp1/2 junction(20) . A chymotrypsin-like
Ser protease was identified in the arterivirus Nsp4 protein (16) and is currently being characterized. In this paper we show
that neither of these proteases mediates the cleavage of the Nsp2/3
site. Instead, a Cys protease in the N-terminal domain of Nsp2 was
shown to be involved in this process. To a certain extent, this new
protease resembles both papain-like and chymotrypsin-like Cys
proteases. In our opinion, this unusual Cys protease occupies a unique
position among the different protease families.
Figure 1:
Expression of EAV ORF1a and ORF1a
deletion mutants using the vaccinia virus/T7 system (33). The upper
part of the figure shows a schematic representation of the
expressed proteins. The EAV PCP and SP domains, corresponding cleavage
sites, and Nsp1-6 are indicated for construct pM1a. Deletions
made in pM1a to obtain other expression vectors are indicated. The
sizes of the full-length expression products were calculated from their
known amino acid sequences. The sizes of the pMX8 and pM cleavage
products are estimates, which are based on the assumption that the
Nsp2/3 cleavage site is located close to residue 825. Expression
products were immunoprecipitated using an anti-Nsp2 serum (19) and
analyzed by SDS-polyacrylamide gel electrophoresis. Lysates from mock- (M) and EAV-infected (E) cells and from
mock-transfected (MT) cells were included as controls. Note
that the vaccinia virus infection induces a set of background bands
(especially in the 30-50-kDa region) which is absent from the M and E lanes. The precipitation of the C-terminal
cleavage products of the pMX8 and pM
proteins by the anti-Nsp2
serum is due to the previously described association between Nsp2 and
Nsp3 (19).
Comparative Sequence Analysis
EAV nucleotide
(nt) and aa numbers refer to previously published
sequences(16) . Pairwise sequence comparisons were performed
using the DotHelix program(21) . Multiple sequence alignments
were produced employing the OPTAL (22) or CLUSTAL V
programs(23) . Different scoring tables, including those of the
PAM (24) and BLOSSUM (25) families, were utilized. Protein
sequences were compared with the nonredundant database maintained by
the National Center for Biotechnological Information and the SwissProt
data base using the BLAST (26) and BLITZ (27) programs,
respectively. Profile analysis (28) was supported by the GCG
package(29) . An improved version of the profile analysis (30) was employed for comparison of profiles with other profiles
and sequences.
Expression Construct pM1a
Recombinant plasmids
were constructed using standard techniques. Expression vector pM1a, a
pBS (Stratagene) derivative, contained the complete
EAV ORF1a sequence downstream of a T7 promoter, a copy of the
encephalomyocarditis virus internal ribosomal entry site, which was
used to enhance translation(31) , and 12 nt encoding the
N-terminal extension Met-Ala-Thr-Thr(19) . The ORF1a sequence in
pM1a was modified to facilitate mutagenesis; translationally silent
mutations were introduced to create unique restriction sites. A
detailed description of pM1a will be published elsewhere. The new
restriction sites used here were NcoI (nt 224, containing the
ORF1a initiation codon) and SphI (nt 1975).
Other Expression Vectors
Constructs pMX6 and pMX8 (Fig. 1) were derived from pM1a by insertion of a termination
codon into SalI (nt 2608) and ApaI (nt 3688)
restriction sites, respectively. To create pM (Fig. 1), two
deletions were made in pM1a. First, a translation initiation codon was
engineered close to the Nsp1/2 border and the Nsp1-encoding sequence
was deleted. The N-terminal sequence of the pM
protein was
Met-Ala-Thr-Thr-Met-Val/Gly-261. The second deletion comprised the
sequence encoding Nsp4 and a part of Nsp5. To this end, SmaI
restriction sites were generated at nt 3415 and nt 4513 and an in-frame SmaI deletion was made. The Nsp2 deletion mutants (Fig. 3) were created by digestion of pairs of unique pM
restriction sites and subsequent religation. The following sites were
used: NcoI (nt 997), HindIII (nt 1501), KpnI
(nt 1802), SphI (nt 1875), ClaI (nt 2296), and SalI (nt 2608). Bicistronic construct pBC (Fig. 4) was
generated by insertion of the following sequence elements into the
pM
HindIII site at nt 1501: a termination codon for the
first cistron, a second encephalomyocarditis virus internal ribosomal
entry site, and an NcoI site containing the initiation codon
for the second cistron.
Figure 3:
Expression of Nsp2 deletion mutants (see
also Fig. 1). Six in-frame deletions (indicated as A through F) were made in the Nsp2-encoding region of construct pM.
The deleted residues are indicated on the left. The hatched box represents the central hydrophobic domain in Nsp2.
Immunoprecipitations were carried out using anti-Nsp2 (
2)
and anti-Nsp5 (
5) sera (19). The position in the gel of
the 58-kDa C-terminal pM
cleavage product, which is indicative of
the Nsp2/3 cleavage, is shown.
Figure 4:
Construction and expression of bicistronic
construct pBC. The putative Nsp2 CP (pBC protease) and its
substrate (pBC precursor) were expressed as two separate
proteins and immunoprecipitated using the anti-Nsp2 (2)
and anti-Nsp5 (
5) sera (19). The results obtained with
pM
and control constructs expressing either the first (pM
X3) or the second (pM
(d261-426))
cistron are shown. The N- and C-terminal pBC cleavage products, which
are indicative of processing of the Nsp2/3 site in the precursor, are
indicated. Replacement of Cys-270 (C270
S) and His-332 (H332
Y) and two in-frame Nsp2 deletions (d427-794 and d427-691) abolished
cleavage. IRES, internal ribosomal entry
site.
Mutagenesis
Nucleotide changes were introduced
using oligonucleotide-directed mutagenesis as described by Kunkel et al.(32) . After complete sequence analysis,
restriction fragments carrying mutations were transferred back to pM1a
or pM1a-derived vectors.
Expression and Protein Analysis
Transient T7
expression using vaccinia virus recombinant vTF7-3(33) , RK-13
cells, and cationic liposomes was carried out as described(19) .
Proteins were labeled from 4 to 7 h postinfection using
[S]methionine. The
2 and
5 antisera,
and the methods for cell lysis, immunoprecipitation, and
SDS-polyacrylamide gel electrophoresis have been described
previously(19) .
Processing of the EAV Nsp2/3 Site Is Not Mediated by
the Nsp1 Papain-like Protease or the Nsp4 Chymotrypsin-like
Protease
When full-length EAV ORF1a construct pM1a was
expressed, the 61-kDa Nsp2 protein was detected (Fig. 1),
indicating that the Nsp2/3 junction was cleaved efficiently. The
slightly smaller size (58-kDa; Fig. 1) of the pMX6 product
indicated that the Nsp2/3 cleavage site is located approximately 30 aa
downstream of Val-795. Cleavage at this site was observed with
truncated constructs pM and pMX8 (Fig. 1). The 120-kDa
pM
product lacked both the Nsp1 and Nsp4 protease domains,
indicating that the Nsp2/3 site was cleaved by a third protease. The
Nsp56 region could be excluded as the possible location of this new
protease, since it was absent from the pMX8 product.
construct was used for a more detailed experimental analysis of the
processing of the Nsp2/3 site (see below). Its N-terminal cleavage
product (62 kDa) consists of Nsp2 and a six-residue N-terminal
extension. Its C-terminal cleavage product, which was estimated at 58
kDa but consistently migrated in the 50-kDa region of our gels, was
comprised of Nsp3 and the C-terminal 297 residues of the ORF1a protein.
Computer-assisted Identification of a Novel Type of
Cysteine Protease in Nsp2
With a few exceptions,
positive-stranded RNA viral nonstructural polyproteins are processed by
virus-encoded proteases. Hence, we assumed that the protease which
cleaves the EAV Nsp2/3 site resided in Nsp2 or Nsp3. Comparison of
arterivirus Nsp2 and Nsp3 sequences with sequence data bases did not
produce any significant matches. Assuming that the unknown protease
could be conserved among arteriviruses, we scanned the EAV, LDV, and
PRRSV sequences for conserved residues that might be involved in
proteolytic catalysis (Ser, Cys, His, Asp, Glu, or Asp/Glu).
Figure 2:
Delineation and comparison of the
arterivirus Nsp2 cysteine protease (CP) domain. A,
alignment of the N-terminal Nsp2 domain of the arteriviruses LDV,
PRRSV, and EAV. asterisk, putative catalytic residues; +,
residues which were mutated (Fig. 5). B, dot plot comparison
of the N-terminal regions of EAV and LDV/PRRSV ORF1a proteins. An
LDV/PRRSV alignment profile was made and used for comparison in the
Proplot program (30) using the Blossum 62 table, a 21-aa window, and a
0.05 cut-off level. Processing schemes of EAV (left) and
LDV/PRRSV (bottom) are shown along the axes. The location of
protease domains and selected (catalytic) residues are shown at the
opposite axes. The inactivated EAV PCP* domain (39) contains a Lys
at the position of the catalytic Cys. The importance of the various PCP
Cys and His residues has been documented elsewhere (20, 39). Matches
revealing similarity between presumed orthologous or paralogous domains
are indicated by fat diagonals. C, comparison of the context
of the putative Nsp2 CP active site residues (asterisk) with
that of catalytic residues in arterivirus Nsp1 papain-like proteases
and a number of viral chymotrypsin-like (picornavirus 3C-like) Cys
proteases. Abbreviations: BaYMV, barley yellow mosaic virus; FCV, feline calicivirus; RHDV, rabbit hemorrhagic
disease virus (42). Swissprot (SW) or GenBank
(GB) data base accession numbers are
shown.
The Entire Nsp2 Protein Appears to Be Essential for
Proteolytic Activity
Four domains could be discriminated in
arterivirus Nsp2 proteins. The N- and C-terminal regions (about 100 and
200 aa, respectively) are highly conserved and rich in Cys residues. A
130-aa hydrophobic domain precedes the conserved C-terminal region. The
remaining part of Nsp2 is hydrophilic and extremely variable, both in
size (160-600 aa) and in sequence(18, 19) . To
test the involvement of the putative Nsp2 CP domain in the Nsp2/3
cleavage, we deleted the sequence encoding the Nsp2 N-terminal region
from construct pM (Fig. 3, deletion A). As
expected, processing of the Nsp2/3 site was completely abolished. In
addition to the pM
precursor protein, an indistinct band in the
90-kDa region was observed. Similar bands were also seen when some of
the other pM
deletion mutants were expressed. Their origin is
unclear, although there appears to be a correlation between their size
and the size of the corresponding precursor protein.
The N-terminal Nsp2 Domain Can Induce the Nsp2/3 Cleavage
in trans
To test trans-cleavage activity, the putative
Nsp2 CP and the rest of the pM protein were separately expressed
from bicistronic construct pBC (Fig. 4). The product of the first
cistron, containing the N-terminal 165 aa of Nsp2, was seen as a double
band of approximately 18 kDa. This doublet is explained by the presence
of two translation initiation codons, separated by 9 nt, at the 5` end
of the ORF(35) . A substantial part of the pBC substrate encoded
by the second cistron was cleaved, apparently at the authentic Nsp2/3
site (Fig. 4). This showed that the N-terminal Nsp2 domain can
induce processing in trans. When Cys-270 or His-332 were
mutated, processing was completely abolished. The same result was
obtained when the central and/or C-terminal domains of Nsp2 were
deleted from the substrate (Fig. 4).
Probing the Putative Protease Domain by Site-directed
Mutagenesis
To characterize the putative CP in more detail, 30
substitutions at 12 different positions in the Nsp2 N-terminal region
were tested (Fig. 5). Substitution of Cys-270 and His-332, the
putative catalytic residues, abolished proteolytic activity. This was
also the case when three other conserved Cys residues (aa 319, 349, and
354; Fig. 2A) were replaced. Previously, similar results
were obtained during a mutagenesis study of conserved Cys residues of
another Cys protease, the poliovirus 2A protein(36) . Even the
mutagenesis of nonconserved Cys residues in EAV Nsp2 was not without
consequences: although a Cys-344 Ala substitution was tolerated,
the Cys-344
His mutant was severely impaired. Conversely, a His,
but not an Ala, substitution was tolerated at the position of Cys-356 (Fig. 5). Conservative replacements within a cluster of acidic
residues (aa positions 291 and 295-297) did not influence
proteolysis. Finally, a Gly-271
Trp substitution completely
disabled the protease. When tested in bicistronic construct pBC, a
selection of these mutations produced results identical to those
obtained with the monocistronic pM
(data not shown).
Figure 5:
Site-directed mutagenesis of the putative
Nsp2 CP (see also Fig. 2A). Amino acid substitutions,
indicated in single-letter codes, were introduced into reporter
construct pM and tested by expression in the vaccinia/T7 system
(33). The position in the gel of the pM
precursor (P) and
the N-terminal (N) and C-terminal (C) cleavage
products is indicated.
(
)may be
attributed to the absence of certain host components. Apparently, a
complex with a cleavable Nsp2/3 site can be formed when the Nsp2 CP
domain is expressed separately and the remaining part of Nsp23 is
intact (Fig. 4). It is also remarkable that the previously
described insertion of a 10-kDa heterologous sequence into Nsp2 (at aa
427) did not affect cleavage at the Nsp2/3 site(19) .
and PCP
, are cis-acting Cys proteases which
produce Nsp1
and Nsp1
(39). The PRRSV/LDV PCP
domain and
Nsp1
/2 cleavage correspond to the EAV Nsp1 PCP and Nsp1/2
cleavage, respectively. The EAV counterpart of LDV/PRRSV PCP
(PCP
*; Fig. 2B) has lost the catalytic
Cys(39) . The Nsp1 PCPs are clearly more diverged than the Nsp2
CPs (Fig. 2B), but a number of similarities can be
detected and the Nsp2 CP appears to be more closely related to the
viral papain-like protease group than to other currently known groups
of proteases. Thus, the Nsp2 CP is likely to be another descendant of
an ancestral papain-like protease.
Gly
replacement is probably coupled to additional substitutions.
Chymotrypsin-like proteases contain a catalytic Asp/Glu in the region
separating the active site His and Ser/Cys residues. In EAV Nsp2, the
region between Cys-270 and His-332 contains a conserved Asp, but
mutagenesis of this and other acidic residues did not affect the Nsp2/3
cleavage (Fig. 5). However, this result may be inconclusive,
since mutagenesis proved to be an inappropriate tool for the
identification of a catalytic Asp in at least one viral
protease(40) . Together, the characteristics described above
distinguish the Nsp2 CP from previously described papain-like and
chymotrypsin-like Cys proteases.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.