JOURNAL OF VIROLOGY, Oct. 2004, p. 10765–10775
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Vol. 78, No. 19
Temporal Modulation of an Autoprotease Is Crucial for Replication
and Pathogenicity of an RNA Virus
T. Lackner,1A. Mu ¨ller,1A. Pankraz,1P. Becher,1H.-J. Thiel,1A. E. Gorbalenya,2*
and N. Tautz1*
Institut fu ¨r Virologie (Fachbereich Veterina ¨rmedizin), Justus-Liebig-Universita ¨t Giessen, Giessen, Germany;1
and Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands2
Received 9 March 2004/Accepted 24 May 2004
Pestiviruses belong to the family Flaviviridae, and their genome is a single-stranded RNA of positive polarity
encoding one large polyprotein which is further processed into mature proteins. Noncytopathogenic (noncp)
strains of the pestivirus bovine viral diarrhea virus (BVDV) can establish persistent infection. In persistently
infected animals, noncp BVDVs occasionally acquire mutations in viral nonstructural protein 2 (NS2) that give
rise to cytopathogenic (cp) BVDV variants, and, eventually, lead to the onset of lethal disease. A molecular
marker of cp BVDV infection is a high-level expression of the replicative NS3 protease/helicase that together
with NS2 is derived from NS2-3. Here, we present evidence for NS2-3 autoprocessing by a newly identified
cysteine protease in NS2 that is distantly related to the NS2-3 autoprotease of hepatitis C and GB viruses. The
vital role of this autoprotease in BVDV infection was established, implying an essential function for NS3 in
pestiviral RNA replication which cannot be supplied by its NS2-3 precursor. Accordingly, and contrary to a
current paradigm, we detected almost complete cleavage of NS2-3 in noncp BVDV at early hours of infection.
At 6 to 9 h postinfection, NS2-3 autoprocessing diminished to barely detectable levels for noncp BVDV but
decreased only moderately for cp BVDV. Viral RNA synthesis rates strictly correlated with different NS3 levels
in noncp and cp BVDV-infected cells, implicating the NS2 autoprotease in RNA replication control. The
biotype-specific modulation of NS2-3 autoprocessing indicates a crucial role of the NS2 autoprotease in the
pathogenicity of BVDV.
Pestiviruses are animal pathogens that are recognized as a
separate genus of the family Flaviviridae, which also includes
the genera Flavivirus and Hepacivirus (hepatitis C viruses
[HCV]), as well as the unassigned GB viruses (32). Pestiviruses
are widely used as a surrogate model for studying HCV, which
grows poorly in available cell culture systems. Persistent HCV
infections are a major cause of liver cirrhosis and hepatocel-
lular carcinoma in humans worldwide.
The pestiviral genome is a positive-stranded RNA of 12.3
kb. It is translated into a large polyprotein, which is cotrans-
lationally and posttranslationally processed by viral and cellu-
lar proteases. The order of proteins in the polyprotein is NH2-
COOH. The autoprotease Nprogenerates its C terminus and
the N terminus of the downstream core protein C. The pro-
teolytic releases of the structural glycoproteins Erns(RNase
secreted), E1, E2, and p7 are mediated by cellular signal pep-
tidases. The nonstructural protein 4A (NS4A)-dependent chy-
motrypsin-like serine protease in NS3 mediates processing in
the NS region downstream of NS3 (32). The mechanism of
NS2-3 cleavage was hitherto unknown and is the subject of this
study (see below).
The pestivirus bovine viral diarrhea virus (BVDV) can es-
tablish lifelong persistent infections in animals, which become
the primary sources for the horizontal spread of the virus (3).
One prerequisite of viral persistence is a diaplacental infection
of a bovine fetus in conjunction with an acquired immunotol-
erance against the infecting virus. BVDV strains that cause
persistent infections are noncytopathogenic (noncp) in cell cul-
ture. During persistence, noncp BVDV strains occasionally
mutate into cytopathogenic (cp) BVDV strains, and together
the two strains trigger the development of lethal mucosal dis-
ease in the infected animals (37). In contrast to their noncp
parents, the cp BVDV strains are not able to establish persis-
tent infection. Accordingly, BVDV pathogenesis as well as
spread of the virus is determined by the viral biotype, cp or
The molecular mechanism of biotype control of BVDV is
not yet understood, although the level of NS3 accumulation,
involving NS2-3 cleavage in many BVDV isolates, seems to be
of crucial importance (37). In noncp BVDV-infected cells, only
uncleaved NS2-3 has been detected so far (37, 41). In contrast,
all cp BVDV strains, which are derived from noncp parents
through a mutation(s) in various regions of the viral genome,
efficiently express NS3 (32, 37). For BVDV strain CP7, its cp
biotype and associated efficient cleavage of NS2-3 were linked
to the cp-specific insertion of a unique 9-amino-acid (9-aa)
peptide into NS2 (36). When this insertion was reconstructed
in the noncp BVDV background, it was sufficient to induce
efficient NS2-3 processing (51). This processing does not in-
volve proteolytic activity of the NS3 serine protease (28, 37,
51), and accordingly, the NS2-3 cleavage site differs signifi-
cantly from sites processed by the NS3 protease (28, 32, 35).
* Corresponding author. Mailing address for N. Tautz: Institut fu ¨r
Virologie (FB Veterina ¨rmedizin), Justus-Liebig-Universita ¨t Giessen,
Frankfurter Strasse 107, 35392 Giessen, Germany. Phone: 49 641 99 3
83 94. Fax: 49 641 99 3 83 59. E-mail: Norbert.Tautz@vetmed.uni
-giessen.de. Mailing address for A. E. Gorbalenya: Department of
Medical Microbiology, Leiden University Medical Center, P.O. Box
9600, E4-P, 2300 RC Leiden, The Netherlands. Phone: 31 71 5 26 16
52. Fax: 31 71 5 26 67 61. E-mail: firstname.lastname@example.org.
NS2-3 processing in viruses of two other genera of the family
Flaviviridae is based on different mechanisms. In the genus
Flavivirus, the NS2B-dependent serine protease in NS3 medi-
ates NS2-3 processing. In contrast, a viral cysteine autopro-
tease residing in NS2 and the N-terminal part of NS3 mediates
NS2-3 cleavage of hepaciviruses (32). The hepaciviral NS2
seems to provide a cysteine-dependent proteolytic activity
while the NS3 N-terminal domain, which encompasses an es-
sential noncatalytic Zn-binding (ZnB) site, plays an accessory
role (17, 19, 25). This role does not involve NS3-associated
proteolytic activity, and accordingly, the hepaciviral NS2-3
cleavage site does not match the consensus of the NS3-pro-
cessed cleavage sites (25).
In this report, combining the results of bioinformatics, mo-
lecular genetics, and biochemical analyses of BVDV, we pres-
ent evidence that NS2 is an autoprotease. A model for pesti-
virus NS2-3 processing, resembling the mechanism employed
by hepaciviruses, consistently explains the available and newly
obtained data. We unravel modulation of NS2-3 processing
over the course of BVDV infection with a profound biotype-
specific amplitude and correlation with RNA synthesis. The
NS2 protease is further shown to be crucial for both noncp and
cp BVDV biotypes.
MATERIALS AND METHODS
Bioinformatics analysis. Protein sequences were retrieved from GenBank.
Amino acid sequence alignments were generated with ClustalX, version 1.81
(52), and MACAW (48) programs assisted by Blosum position-based weight
matrices (24) and were processed for presentation with GeneDoc (38).
Cells and viruses. Madin-Darby bovine kidney (MDBK) cells and BHK-21
cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10%
fetal calf serum. Cells were maintained at 37°C and 5% CO2. Vaccinia virus
modified virus Ankara (MVA)-T7pol (49) was generously provided by G. Sutter
(GSF, Oberschlei?heim, Germany). BVDV strains CP7 and NCP7 were de-
scribed previously (11).
BVDV infection. Cells were infected with BVDV at a multiplicity of infection
(MOI) of 10 for 1 h. For metabolic labeling, cells were incubated for 30 min with
DMEM without cysteine and methionine (label medium) at 37°C prior to the
addition of 1 ml of label medium containing 280 ?Ci of [35S]methionine-cysteine
([35S]-ProMix; Amersham Biosciences, Freiburg, Germany) to a 2-cm dish with
106cells; protein expression was allowed to proceed for 1 h at 37°C.
In vitro transcription and electroporation. The infectious cDNA clones of
BVDV CP7 (pCP7-5A) (4) and NCP7 (pNCP7-5A) (5) have been described
previously. After linearization of 3 ?g of plasmid DNA with SmaI at the 3? end
of the cDNA, RNA was transcribed with the MAXIscript SP6 kit (Ambion,
Huntingdon, United Kingdom) without DNase digestion according to the proto-
col of the manufacturer. All transcripts were prepared in parallel, and the amount of
RNA was estimated by agarose gel electrophoresis. Similar amounts of each
RNA (about one-third of each transcript) were used to electroporate one-third
of the MDBK cells from a confluent 10-cm dish as previously described (50).
Expression plasmids. pCITE (Novagen, Madison, Wis.) encompasses the in-
ternal ribosomal entry site of encephalomyocarditis virus downstream of the T7
RNA polymerase promoter. pC/E2-4A and pN/E2-4A have been described pre-
viously (45). Mutations were introduced by the QuikChange method (Stratagene,
Heidelberg, Germany). The following constructs are based on pCITE and code
for the indicated amino acids of BVDV CP7; the amino acid positions refer to
those of BVDV strain SD-1 (13): for pC/E2-NS3/1645GST, amino acids (aa) 693
to 1645, followed by glutathione S-transferase (GST); for pflagNS2-3/1645GST,
pflagNS2-3/1599GST, pflagNS2-3/1596GST, and pflagNS2-3/1595GST, aa 1137
to 1645, 1137 to 1599, 1137 to 1596, and 1137 to 1595, respectively, preceded by
the peptide MDYKDDDDKL (including the flag epitope) and followed by GST;
for p1210/NS2-3/1645GST and p1272/NS2-3/1645GST, aa 1210 to 1645 and 1272
to 1645, respectively, followed by GST.
Transient expression with the T7-vaccinia virus system. BHK-21 cells (106
cells in a six-well dish) were infected with MVA-T7pol at an MOI of 5 in medium
lacking fetal calf serum for 1 h at 37°C. For transfection of plasmid DNA (2 ?g),
Superfect was applied (QIAGEN, Hilden, Germany).
Radioactive labeling of proteins in BHK-21 cells. At 2 h posttransfection of
plasmid DNA, cells were incubated for 30 min in label medium (lacking cysteine
and methionine) at 37°C. After addition of 1 ml of label medium containing 70
?Ci of [35S]methionine-cysteine ([35S]-ProMix; Amersham Biosciences) to a
2-cm dish with 106cells, protein expression was allowed to proceed for 4 h at
37°C. Cells were lysed in RIPA buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl,
1% [vol/vol] NP-40, 1% [wt/vol] deoxycholate, 0.1% [wt/vol] sodium dodecyl
sulfate [SDS], and 0.5 mM PefablocSC [Merck, Darmstadt, Germany]).
kirchen, Germany) and RIPA buffer were used for RIP (45).
Radiosequencing. A pCITE-based plasmid, which encodes the flag epitope
NS2, and 7 aa of NS3 of BVDV strain CP7 (aa 1137 to 1596) followed by the
peptide Met-Leu-Thr-Met-Ala-Met and GST, was used for MVA-T7pol-based
expression. For protein expression, 106BHK-21 cells were used and labeled with
500 ?Ci of35S-Met (Amersham Biosciences) for 4 h; the cell lysate was pro-
cessed by RIP with anti-GST monoclonal antibody (MAb). Following SDS-
polyacrylamide gel electrophoresis (PAGE) and transfer onto a Sequi-Blot poly-
vinylidene difluoride membrane (Bio-Rad, Munich, Germany), the protein was
subjected to automated Edman degradation in an Applied Biosystems, Inc.,
model 473A protein sequencer, and the obtained fractions were analyzed with a
SDS-PAGE and immunoblotting. Proteins were separated in polyacrylamide-
Tricine gels (8, 10, or 12% polyacrylamide) (47). After SDS-PAGE, proteins
were transferred onto a nitrocellulose membrane (Optitran BA-S83 reinforced
NC; Schleicher & Schuell, Du ¨ren, Germany); the membrane was blocked with
3% (wt/vol) dried skim milk in phosphate-buffered saline with 0.05% (vol/vol)
Tween 20. For antigen detection, peroxidase-coupled species-specific secondary
antibodies and Renaissance Western Blot Chemiluminescence Reagent Plus
(NEN Life Sciences, Boston, Mass.) were applied.
Quantification of NS2-3 cleavage efficiencies. Radioactivity of proteins in the
dried SDS-PAGE gels was determined by phosphorimaging. Cleavage efficiency
was calculated as the quotient of the signals of free NS3 and total NS3 (free NS3
signals plus the NS2-3 signal, considering also the label in NS2); for evaluation
of the protease mutants, the cleavage rate of wild-type (wt) CP7 was set to 100%
(see Fig. 3).
RNA preparation, gel electrophoresis, and Northern blotting. Total RNA
from MDBK cells was prepared with a NucleoSpin RNA II kit (Macherey &
Nagel, Du ¨ren, Germany). For Northern blot analyses, 5 ?g of each RNA was
glyoxylated, separated on 1% agarose gels containing 3.7% formaldehyde, and
transferred to Duralon-UV membranes (Stratagene). For hybridization, an
[?-32P]dCTP-labeled cDNA probe encompassing nucleotides 5171 to 5888 of
BVDV CP7 (51) was used. The probe, derived from a bovine cDNA encoding
glyceraldehyde 3-phosphate dehydrogenase (GAPDH), had a length of about
300 bp and was the kind gift of C. Grassmann (Institut fu ¨r Virologie, Giessen,
Germany). Probes were labeled with a nick translation kit (Amersham Bio-
sciences). Further details have been described previously (51).
Antibodies and antisera. The antiflag tag and anti-GST MAbs were purchased
from Sigma-Aldrich. For the detection of NS3, mouse MAb 8.12.7 (10) was used.
Species-specific secondary antibodies were purchased from Dianova (Hamburg,
Metabolic labeling of viral RNA. A total of 106MDBK cells in a 2-cm dish
were infected with BVDV CP7 or NCP7 at an MOI of 10. Prior to metabolic
labeling for 4 h with 300 ?Ci of [32P]orthophosphate (Amersham Biosciences),
cells were incubated for 30 min with phosphate-free DMEM (Sigma) containing
2 ?g of dactinomycin (Sigma-Aldrich)/ml to inhibit RNA synthesis. Total cellular
RNA was purified, and 5 ?g of each RNA was separated by denaturing agarose
gel electrophoresis. A full-length RNA of BVDV CP7 transcribed in the pres-
ence of [32P]UTP served as a size marker (data not shown). The gels were dried
on Duralon-UV membranes (Stratagene) and analyzed by autoradiography and
Virus titration and immunofluorescence. End point titration was done with
four replicates on MDBK cells, and the 50% tissue culture infective dose (TCID50)
was determined (5). The intracellular synthesis of virus-specific proteins postin-
fection or posttransfection was monitored by indirect immunofluorescence (IF)
analysis with MAb 8.12.7 directed against NS3 of BVDV (10) and a secondary
cyanogen-3-labeled antibody as described previously (45).
Bioinformatics analysis predicts pestiviral NS2 to be a cys-
teine autoprotease distantly related to hepaciviral NS2-3 au-
toprotease. To gain a first insight into the mechanism of NS2-3
10766LACKNER ET AL. J. VIROL.
cleavage, pestiviral sequences were examined for elements as-
sociated with NS2-3 processing in the two other Flaviviridae
genera (see the introduction). Our bioinformatics Flaviviridae-
wide analysis of NS2-3 proteins predicts a multidomain orga-
nization for pestiviral NS2; the C-terminal-most domain of
NS2 was found to be distantly related to NS2 of hepaciviruses
and GB viruses (Fig. 1 and data not shown). Strikingly, the
intergenus conservation in NS2 includes three residues, His,
Glu, and Cys (Fig. 1); these residues are essential for NS2-3
autoprocessing in hepaciviruses and were implicated in catal-
ysis of this cleavage (17, 19, 25). They correspond to H1447,
E1462, and C1512 of noncp BVDV (Fig. 1). In addition, one
Pro, one Ala, and three Gly residues are also conserved. The
same linear arrangement of the catalytic triad was previously
found in picornaviral cysteine proteases with chymotrypsin-like
folds, which have otherwise no statistically significant similarity
with NS2 (17).
Upstream and downstream of the predicted catalytic C1512
residue, two unique sequence blocks are evident in pestiviral
NS2. The upstream insertion of 30 aa includes five Cys and one
His that are conserved among all known pestivirus isolates;
these residues are organized in a linear fashion, suggesting the
presence of a mononuclear ZnB site (12). Accordingly, two
amino acid pairs may coordinate a Zn2?ion: candidate mem-
bers for the N-terminal pair are H1483 or C1484, and C1487,
and C1490, while C1500 and C1503 may form the C-terminal
pair (Fig. 1). This putative NS2 ZnB site of pestiviruses might
take over the role of the NS3 ZnB site unique to hepaciviruses
and essential for their NS2-3 autoprocessing (17, 19, 25). The
downstream additional sequence block in pestiviral NS2 has a
length of 41 aa and is enriched in hydrophilic residues. Finally,
a striking conservation was observed between pestiviruses, he-
paciviruses, and GB viruses in the region adjacent to and
encompassing the C-terminal residues of NS2 and the first two
residues of NS3 (Fig. 1, blocks A and B).
We concluded from this analysis that pestiviruses may em-
ploy a variant of the mechanism of NS2-3 autoprocessing
conserved among hepaciviruses. Our model predicts NS2-3
cleavage by an NS2-associated NS3-independent cysteine au-
toprotease in pestiviruses.
BVDV CP7 NS2-3 processing proceeds at the conserved
1589Arg-Gly1590 bond and does not depend on NS3. The
model presented above was tested experimentally with the
E2-NS4A polyprotein region encoded by cp BVDV strain CP7
(see the introduction). Our attempts to express this fragment
in Escherichia coli failed, likely due to the toxicity of the
highly hydrophobic NS2 (data not shown). Therefore, we
switched to transient expression of this polyprotein fragment
with the T7-vaccinia virus, which has already been proven to be
well suited (51). For cp BVDV strain Oregon, the NS2-3 cleav-
age was previously shown to proceed at the conserved 1589Arg-
Gly1590 junction (Fig. 1) (28). By N-terminal sequencing of
FIG. 1. The NS2 protease domain is conserved between hepaciviruses and pestiviruses. Fragments of polyproteins (locations are indicated in
parentheses) including the C-terminal part of NS2 and five N-terminal amino acid residues of NS3 of a representative set of pestiviruses,
hepaciviruses, and GB virus were aligned (see Materials and Methods). The alignment was subsequently manually adjusted to maximize similarity
(gaps are indicated by dashes). The Gibbs sampler (29) of the MACAW workbench (48) was used to assess the similarity between pestivirus BVDV
CP7 and three hepaciviruses that share ?30% identical residues. Blocks A (1.4e?08) and B (5.1e?04) were statistically significant with the NS2-3
protein and the interblock A-NS3-catalytic-Ser searching spaces, respectively. Blocks H, E, and C are named after respective (putative) catalytic
residues of NS2 (His, Glu, and Cys) and were recognized in hepaciviruses and, subsequently, in pestiviruses with the catalytic residues of HCV NS2
as anchors. Note that a region separating blocks C and A hosts cellular insertions of variable sizes in some cp BVDV isolates (37) (not shown)
supporting this alignment. The position of the NS2-3 cleavage site is indicated below the alignment in yellow. Red characters, (putative) catalytic
His, Glu, and Cys residues; blue background, putative Zn2?-coordinating residues of the ZnB site; black background, invariant residues; dark gray
background, residues conserved to 100% (see similarity groups) or invariant residues in 80% of the positions; light gray background, residues
conserved in not less than 60% of the positions. Amino acid similarity group members: D, N, Q, and E; K, R, and H; F, Y, and W; A and G; S
and T; A, C, L, I, V, M, F, and Y. The experimental data (see Results) did not support the putative functions indicated for E1461 (red) and H1483
and C1484 (blue background). Virus names and respective NCBI protein identification numbers are as follows: BVDV-CP7, BVDV strain CP7
(1518836); BDV, border disease virus X818 (20198946); CSFV-E, classical swine fever virus strain Eystrup (12657942); GBV-A, GB virus A
(1096574); GBV-B, GB virus B (9628102); GBV-CG, GB virus C-hepatitis G virus (4426796); HCV-J6, HCV isolate HC-J6 (221651); HCV-J8,
HCV isolate HC-J8 (221609); HCV-H, HCV isolate H (329738). Numbers in parentheses in the alignment of BVDV-CP7 correspond to amino
acid sequences of BVDV strain SD-1 (289507).
VOL. 78, 2004 PROTEASE IN CONTROL OF REPLICATION AND DISEASE10767
NS3, we determined that BVDV CP7 NS2-3 processing occurs
at the same position in our surrogate system (Fig. 2A). When
either P1 or P1? residues were replaced by Pro, NS2-3 cleavage
was abolished (Fig. 2C).
To determine a minimal, processing-competent part of
NS2-3, a series of NS2-3 derivatives truncated from either the
N or C terminus and fused with GST at the C terminus were
tested (Fig. 2B). The NS2-3/NS3 derivatives were isolated by
RIP with a GST-specific antibody and analyzed by SDS-PAGE
(Fig. 2C). According to this analysis, NS2 followed by seven (or
more) residues of NS3 was efficiently cleaved, while a construct
encompassing 6 aa of NS3 showed no processing (Fig. 2C). The
N-terminal truncation of NS2 by 73 aa significantly reduced
NS2-3 processing, and a deletion of the N-terminal 135 aa
abolished NS2-3 cleavage (Fig. 2C). These results extend and
refine previous analyses with cp BVDV strain Oregon (28).
According to our findings, only a minimal part of NS3 imme-
diately adjacent to the cleavage site and virtually the entire
NS2, including its hydrophobic part, are important for NS2-3
cleavage. These observations are in full agreement with the
alignment model (Fig. 1).
The predicted active-site residues of the putative NS2 pro-
tease are essential for NS2-3 autoprocessing of cp BVDV CP7
in a surrogate system. Next, we determined the effect of single-
amino-acid substitutions in NS2 on NS2-3 cleavage efficiency.
Nine putative active-site or ZnB residues plus 12 other (con-
trol) Cys, His, and Glu residues were replaced with different
residues including Ala at each tested position, generating 64
mutants in total (Fig. 3). At 11 positions, the effect of the Ala
substitution on NS2-3 cleavage was small or negligible (?75%
of wt activity was retained); at 3 positions, one Glu and two Cys
residues, the effect was moderate (at least 10% of wt activity
was retained), and at 7 positions that included three Cys, one
His, and three Glu residues the effect was most prominent
(?10% of wt or no activity). These effects were further quan-
titatively verified in three independent analyses of 19 mutants
123456789 10 11 12 13 14 15 16 17
FIG. 2. Determinants of NS2-3 processing. (A) Radiosequencing.
The diagram depicts the amount of radioactivity released by each cycle
of Edman degradation of the partially purified NS3/1596-Met-GST.
The amino acid sequence encoded by flagNS2-3/1596-Met-GST in the
region of the NS2-3 cleavage site was aligned to the fractions with
respect to the methionine residues. The cDNA construct used for
protein expression is shown above the diagram; the amino acid se-
quence downstream of aa 1589 is shown above pflagNS2-3/1596-Met-
GST (the number corresponds to amino acid sequences in BVDV
strain SD-1); the N-terminal 7 aa of NS3 are followed by a sequence
that includes three methionine residues. (B) Scheme of the expression
constructs. Bars symbolize proteins; numbers below the bars indicate
the amino acid positions in the BVDV polyprotein (the numbers
correspond to amino acids in BVDV strain SD-1): 1137, N terminus of
NS2; 1590, N terminus of NS3. Truncated proteins are indicated by
asterisks. S, signal peptide preceding E2; flag, flag epitope. (C) NS2-3
cleavage studied by transient expression in metabolically labeled
BHK-21 cells; the transfected plasmids are indicated above the lanes.
For RIP, a GST-specific antibody was applied, and precipitated pro-
teins were analyzed by SDS-PAGE and autoradiography. Arrows in-
dicate the positions of NS2-3/1645GST, NS3/1645GST, NS3/1599GST,
NS3*= 10 aa
NS3*= 7 aa
NS3*= 6 aa
10768LACKNER ET AL. J. VIROL.
with replacements at 11 selected positions, including putative
catalytic residues His1447 and Cys1512, two Glu residues,
and candidate His or Cys residues of the predicted ZnB site
(Fig. 3C). Upon expression of E2-NS4A with several muta-
tions (H1447A, H1447C, H1447D, H1447E, H1447R, and
H1447Y; E1462A and E1462C; C1487A; C1512A, C1512E,
C1512H, C1512T, and C1512Y), no NS3 could be detected; for
all C1490 mutants, very inefficient NS2-3 cleavage was ob-
served. Importantly, the predicted catalytic Cys1512 partially
tolerated only a Ser substitution out of six replacements tested
(Fig. 3); similar observations were reported for the Cys nucleo-
phile of proven proteases (23, 30).
This mutagenesis analysis supports the relationship between
hepaciviral and pestiviral NS2 (Fig. 1). It refines the pestiviral
NS2 protease model and confirms the pivotal, putative cata-
lytic role for His1447 and Cys1512 in the protease, and it favors
FIG. 3. Effect of single-amino-acid exchanges in NS2 of cp BVDV strain CP7 on NS2-3 cleavage efficiency. Wt i? indicates wt CP7 with the
cp-specific insertion deleted. The numbers indicate amino acid residues based on those of strain BVDV SD-1. (A) RIP analysis. Metabolically
labeled NS2-3 was isolated from BHK-21 cells by RIP, application of an NS3-specific MAb, and further analysis by SDS-PAGE and phospho-
rimaging. Cleavage of CP7 wt NS2-3 was set at 100%. The order of the six blocks is indicated by numbers. (B) Summary of the results presented
in panel A. Shown is the NS2 amino acid sequence, followed by seven amino acids of NS3; the NS3-derived amino acids are underlined. The
indicated NS2-3 cleavage efficiency is based on a single- or triple-quantified experiment. Color code for cleavage (percentage of wt CP7): red, no
NS3 visible; orange, ?10%; yellow, 10 to 75%; green, ?75%. The 9-aa insertion is indicated by italics. Putative functions of residues according
to the experimental results are indicated by colors and shading of residue numbers: red characters on black background, catalytic; white characters
on blue background, coordination of Zn2?ion. (C) NS2-3 cleavage efficiency, based on the results of three quantified experiments. Error bars reach
from the lowest to the highest values measured. (D) Quantification of NS2-3 cleavage efficiency based one or three (see panel C legend)
VOL. 78, 2004PROTEASE IN CONTROL OF REPLICATION AND DISEASE10769
Cys1487, Cys1490, Cys1500, and Cys1503 as the most plausible
candidates for coordination of a Zn2?ion in the putative ZnB
site (Fig. 3).
The NS2 protease is vital for RNA replication of BVDV CP7.
To determine the significance of the NS2 protease for pestivi-
ral replication, three mutations at the catalytic His1447 and
Cys1512 residues, two mutations at Cys residues located either
in the putative ZnB site (Cys1503) or its vicinity (Cys1484), and
a mutation at the P1 position of the NS2-3 cleavage site
(Arg1589) were introduced into the BVDV CP7 genome. The
effects of these mutations were monitored by IF analysis with
an MAb specific for NS3 (also recognizing the NS3 moiety in
NS2-3) at 24, 48 and 72 h postelectroporation (p.e.) of MDBK
cells with cDNA transcripts. According to previous studies, the
IF-mediated detection of NS3 is indicative of viral RNA rep-
lication (8, 34, 36). In addition, development of a cytopathic
effect was monitored, and the titer of infectious progeny virus
was determined in the cell supernatants harvested at 48 h p.e.
Most cells electroporated with CP7 wt RNA were positive
for viral antigen at 24 h p.e. (Fig. 4). The cytopathic effect
became clearly visible at 48 h p.e. (data not shown). The virus
titer reached 1.1 ? 106TCID50per ml in the cell supernatant
at 48 h p.e. In contrast, no signs of virus replication were
observed upon electroporation of transcripts encompassing ac-
tive-site mutations C1512A and H1447A (no NS2-3 cleavage
in the T7-vaccinia system) and C1512S (26% of wt NS2-3
cleavage) (Fig. 4 and data not shown).
All three mutants carrying replacements of residues other
than the catalytic ones were replication competent, although
the negative effect of the mutations on viral replication varied
widely and was proportional to the reduction of NS2-3 cleav-
age observed in the T7-vaccinia system. Almost all cells elec-
troporated with the C1484A transcript (91% of wt NS2-3 cleav-
age in vitro) were positive by the IF test at 24 h p.e. (Fig. 4),
and virus progeny yielded 2.5 ? 106TCID50/ml at 48 h p.e. In
one of three independent experiments with C1503A transcripts
(45% of wt NS2-3 cleavage), few antigen-positive cells were
observed at 24 h p.e. (data not shown). Interestingly, even the
cleavage-impaired R1589P mutant proved to be quasi-infec-
tious, using this term as defined by Gmyl et al. (16), as NS3 was
detected in 20 to 40 cells per dish at 24 h p.e. (data not shown).
In all three cDNA clones derived from independent reverse
transcription-PCR analyses of the latter cells, a reversion of
the mutated residue aa 1589 back to the wt Arg codon was
observed. A possible contamination by wt CP7 virus was ruled
out by the presence of a silent genetic marker in the revertant
that was originally introduced along with the R1589P mutation
(data not shown).
These results show that the predicted NS2 protease is es-
sential for RNA replication of cp BVDV CP7 and support the
catalytic role of residues His1447 and Cys1512. Moreover, this
experiment strongly implied that uncleaved NS2-3 cannot
functionally replace its cleavage products (see Discussion).
Evidence for the NS2 protease activity in noncp BVDV
NCP7. Cleavage of noncp BVDV NS2-3 has not yet been
observed. Our model (Fig. 1), however, suggested that the NS2
autoprotease may be active in all pestiviruses, including noncp
BVDV. Since this protease is vital for BVDV CP7, its inacti-
vation is expected to be deleterious for BVDV NCP7 as well.
To verify the essentiality of the NS2 protease in the repli-
cation of noncp BVDV, Ala replacements of the catalytic
His1447 and Cys1512 were introduced into the cDNA of
BVDV NCP7 and mutated, and wt RNA transcripts were
electroporated into MDBK cells. Upon electroporation of wt
RNA, the IF assay indicated viral replication at 24, 48, and 72 h
p.e. (Fig. 4 and data not shown) and virus progeny with a titer
of 1.6 ? 105TCID50/ml were detected in the supernatant at
48 h p.e. In contrast, in cells electroporated with the mutated
NCP7 transcripts no viral replication was detected at 24, 48,
and 72 h p.e. (Fig. 4 and data not shown); no virus progeny
were detected in the cell supernatants throughout the obser-
vation period (data not shown). These data strongly suggest
that the NS2 protease is also essential for noncp BVDV.
NS2-3 autoprocessing in BVDV-infected cells is temporally
downregulated with a biotype-specific amplitude. The findings
depicted above implicated an essential role of NS2-3 cleavage
in the replication of noncp BVDV and challenged a present
paradigm, according to which these viruses express no NS3 (37,
41). These observations prompted us to reevaluate the gener-
ation of NS3 in noncp BVDV-infected cells, including, in con-
trast to previous studies, that in the early hours postinfection
To investigate the kinetics of NS2-3 and NS3 appearance,
cells infected by either BVDV CP7 or NCP7 with an MOI of
10 were metabolically labeled for 1-h periods from 5 to 10 h p.i.
and, in addition, from 24 to 25 h p.i. To determine the NS3/
(NS3 ? NS2-3) ratio, both proteins were precipitated from the
cell lysates by RIP with an NS3-specific MAb and further
analyzed by SDS-PAGE followed by phosphorimaging (Fig.
5A). Remarkably, NS3 but almost no NS2-3 was detected in
CP7-infected cells and, for the first time, in NCP7-infected
CP7 24 h p.e.
NCP7 24 h p.e.
FIG. 4. IF analysis of MDBK cells 24 h p.e. with RNA transcribed
from full-length cDNA clones of BVDV strains CP7 and NCP7. The wt
and mutants indicated above the individual pictures were character-
ized. A representative part of each dish is shown; primary magnifica-
10770LACKNER ET AL.J. VIROL.
cells labeled from 5 to 6 h p.i. The combined amounts of NS3
plus NS2-3 steadily increased throughout infection with bio-
type-specific cleavage kinetics of the precursor. In NCP7-in-
fected cells, the rate of NS2-3 cleavage decreased sharply from
highly efficient (96% at 5 to 6 h p.i.) to barely detectable (from
4% to below the limit of detection at 8 to 9 and 24 to 25 h p.i.,
respectively). In contrast for cp BVDV CP7, only a moderate
decrease in the efficiency of NS2-3 processing from about 95%
at 5 to 6 h p.i. to a stable high level of about 70% at 8 to 9 or
24 to 25 h p.i., respectively, was observed (Fig. 5A). This
analysis identified a 3-h window between 6 and 9 h p.i. as the
critical phase of infection when NS2-3 autoprocessing effi-
ciency decreases; most importantly, downregulation of NS2-3
cleavage occurs with a biotype-specific amplitude.
To determine the effect of the observed differences in kinet-
ics of NS2-3 processing on accumulation of NS2-3 and NS3,
cells harvested at different times postinfection were subjected
to Western blot analysis. With CP7-infected cells, NS3 but no
NS2-3 was detected throughout the first 6 h p.i. (Fig. 5B). The
absolute amount of NS3 increased constantly through the ob-
servation period (48 h p.i.); after 8 h p.i., NS2-3 was always de-
tected as well. The NS3/NS2-3 ratio slightly changed through-
out the entire period in favor of uncleaved NS2-3 (Fig. 5B and
data not shown).
In NCP7-infected cells, NS3 is more prevalent than NS2-3 at
6 h p.i. (Fig. 5B). However, the NS3/NS2-3 ratio is sharply
reversed at 8 h p.i., and the prevalence of NS2-3 over NS3
increased further at later time points. To confirm the presence
of NS3 beyond 12 h p.i., we analyzed approximately 10 times
more cell lysate per sample by longer-run gels (Fig. 5B, com-
pare the middle and bottom panels).
The results of the Western blot analysis supplement the
kinetic data obtained for NS2-3 processing and imply that the
NS3 turnover rate is slower than its synthesis rate. Collectively,
these analyses reveal the presence of NS3 in both cp and noncp
BVDV-infected cells and identify a temporal and biotype-spe-
cific downregulation of NS2-3 autoprocessing.
Synthesis of viral RNA is correlated with quantity of NS3 in
cp and noncp BVDV-infected cells. The vital character of NS2-
3 processing and the detection of NS3 in cells infected with cp
and noncp strains of BVDV suggest that reproduction of this
virus requires functions provided by the cleavage product(s) of
NS2-3, which are not supplied by this precursor. Viral RNA
replication requires NS3 but neither NS2 nor NS2-3 (7). The
profound and biotype-specific variation of the NS3 levels in the
course of infection observed in our experiments therefore sug-
gested that viral RNA synthesis may be modulated accordingly.
To verify this hypothesis, viral RNA synthesis was charac-
terized in MDBK cells infected at an MOI of 10 with either
CP7 or NCP7. Prior to metabolic labeling of the cells with
[32P]orthophosphate for 4-h periods starting at 7, 24, and 48 h
p.i., cellular RNA synthesis was inhibited by the addition of
dactinomycin. Total intracellular RNA was prepared and an-
alyzed by denaturing agarose gel electrophoresis, followed by
phosphorimaging and autoradiography (Fig. 6A). Similar
RNA synthesis rates were observed for CP7 and NCP7 be-
tween 7 and 11 h p.i. At the two later time points, RNA
synthesis steadily declined to become barely detectable in
NCP7-infected cells but increased in CP7-infected cells. Thus,
biotype-specific RNA synthesis correlates with the dynamics of
NS3 accumulation (Fig. 5B and 6A).
Furthermore, we investigated the viral RNA levels in in-
fected cells by Northern blotting. Total intracellular RNA was
prepared from cells harvested at 12, 24, 36, and 48 h p.i. with
BVDV strain CP7 or NCP7 at an MOI of 10. These RNAs
were hybridized in the Northern blot analysis against32P-la-
beled probes specific for BVDV RNA or bovine GAPDH
5-6h 6-7h7-8h 8-9h
NS3 / (NS3 + NS2-3) (%)
FIG. 5. NS2-3 cleavage in cells infected with noncp BVDV NCP7
and cp BVDV CP7. NS3 and NS2-3 are marked with arrows. (A) RIP
analysis of MDBK cells infected with strain NCP7 (left) or CP7 (right)
after metabolic labeling with [35S]methionine-cysteine for the indicat-
ed time periods p.i. The diagram above indicates the NS2-3 cleavage
rate as [NS3/(NS3 ? NS2-3)], with values in percentages; values were
obtained by phosphorimager analysis. (B) Western blot analysis of
MDBK cells infected with strain CP7 (top panel) or NCP7 (middle and
bottom panels) with an NS3/NS2-3-specific MAb. The lysate separated
in each lane represents about 5 ? 104cells (top and middle) or 5 ? 105
cells (bottom). Lysates were prepared at the indicated time points p.i.
VOL. 78, 2004PROTEASE IN CONTROL OF REPLICATION AND DISEASE10771
mRNA, the latter serving as a loading control (Fig. 6B). Re-
sults of three independent experiments were quantified by
phosphorimaging. Throughout the time period observed, the
amount of intracellular viral RNA was found to be significantly
(approximately 3 to 13 times) lower in the NCP7-infected cells
than in CP7-infected cells (Fig. 6B). A three to fourfold in-
crease in the amount of intracellular viral RNA was observed
for the viruses of both biotypes between 12 to 24 h p.i. In
NCP7-infected cells, the amount of intracellular viral RNA
showed no further significant increase over the next 12 h and
even decreased slightly thereafter. In contrast, in CP7-infected
cells the amount of viral RNA further accumulated by a factor
of about 3 between 24 and 48 h p.i. The largest difference in the
amounts of intracellular viral RNA (about 13 times) between
CP7- and NCP7-infected cells was observed at 48 h p.i. (Fig.
6B). This difference is apparently not reflected in the produc-
tion of progeny virus (Fig. 7) which suggests that the RNA
amount is not the limiting factor for virus production.
In this report, we provide evidence for the NS2 autoprotease
responsible for NS2-3 cleavage in BVDV and, by implication,
other pestiviruses. Our analysis revealed biotype-specific, tem-
poral modulation of NS2-3 autoprocessing identifying the NS2
autoprotease as a key factor in the control of the pathogenicity
Modular relationship of autoproteases in pestiviral NS2
and hepaciviral NS2-3. The identification of the long-elusive
protease responsible for NS2-3 cleavage was a crucial step
toward unraveling the unprecedented modulation of proteo-
lytic processing of BVDV replicase. Our bioinformatics-led
mutagenesis analysis, which involved the characterization of 64
mutants in vitro and a dozen selected mutants in vivo, identi-
fied a cysteine autoprotease in the C-terminal domain of NS2.
This protease is homologous to the proteolytic domain of the
NS2-3 autoprotease of hepaciviruses. We confirmed that, like
the catalytic Cys and His residues of the HCV NS2-3 protease
(27), their counterparts in the BVDV NS2 are indeed essential
both for the viability of viral biotypes and for NS2-3 processing
Most interesting are the observations of the contrasting phe-
notypes of the active-site mutant C1512S, which is partially
active in vitro but is lethal in vivo, and of the cleavage site
mutant R1589P, which shows no detectable cleavage in vitro
but is quasiinfectious in vivo. The former phenotype implies
that the NS2-3 autoprocessing, like autocleavages in other
virus systems (9, 22), may represent an essential rate-limiting
aspect of RNA synthesis. Specifically, the mutant NS2 protease
with the Ser nucleophile may function with slowed kinetics,
severely affecting a downstream process(es) like membrane
insertion and/or association, which is known to depend on
NS2-3 cleavage in the HCV system (46). Consequently, these
effects may interfere with the formation of a functional repli-
cation complex and block RNA synthesis and the generation of
a (pseudo)revertant(s) entirely. In contrast, a much stronger
inhibitory effect of the cleavage site mutation R1589P on
NS2-3 cleavage may not prevent the otherwise-intact enzy-
matic domain from correctly processing the NS2-3 junctions in
a few molecules with proper kinetics, resulting in the formation
FIG. 6. Intracellular accumulation of viral RNA. (A) Analysis of
viral RNA synthesis in cells infected with BVDV strain NCP7 or CP7.
Metabolically labeled RNA was purified and separated by denaturing
agarose gel electrophoresis. Labeling periods in hours are specified
below the lanes. The position of a BVDV full-length RNA transcript is
indicated by an arrow. (B) (Top) Graph depicting the relative amounts
of viral RNA. The amount of BVDV CP7 RNA measured at 48 h p.i.
was set at 100%; bovine GAPDH RNA served to standardize the RNA
amounts. Values were obtained by phosphorimager analysis. Error
bars reach from the lowest to the highest value measured in three in-
dependent experiments. (Bottom) Northern blot analysis. Total RNA
was prepared from MDBK cells infected with BVDV strains NCP7 or
CP7 at the indicated time points p.i. Upon separation by denaturing
agarose gel electrophoresis, RNA was blotted onto a membrane and
hybridized in parallel against probes specific for BVDV or GAPDH.
The entire experiment was done in triplicate, and the data shown is
representative of all experiments.
12h 24h36h 48h
FIG. 7. Growth kinetics for BVDV strains CP7 and NCP7. The
graph shows mean values derived from three independent experi-
ments. MDBK cells were infected at an MOI of 10. Culture superna-
tants were harvested at the indicated times p.i. Virus titers are given as
log 50% tissue culture infective doses per milliliter. For cDNA-derived
viruses, see previously published results (6).
10772LACKNER ET AL. J. VIROL.
of functional replication complexes. The few complexes pro-
duced in this way may be sufficient for the generation of viable
revertants (quasiinfectious virus).
Although the mutagenesis analysis supports the bioinfor-
matics-based NS2 model, the identity of a possible third cata-
lytic residue in the NS2 protease remains unresolved. NS2-3
processing in vitro is mildly sensitive to mutations of the
Glu1462 residue and tolerates a range of replacements of
Glu1461, which is absolutely conserved in NS2 of pestiviruses,
hepaciviruses, and GB viruses (Fig. 1). Because of overall poor
conservation, we cannot rule out that the generated alignment
for the region around Glu1461-Glu1462 will be revised. The
role of the Glu1461 counterpart in the HCV NS2-3 autopro-
tease has also not been firmly established, and future studies
must address these issues.
The conservation of the NS2 protease domain in pestiviruses
and hepaciviruses was an unexpected observation, since the
other essential part of the NS2-3 autoprotease, which resides in
the N-terminal part of NS3 and encompasses ?180 aa in HCV
(18, 24), was reduced to no more than 7 aa in BVDV (19, 25).
The dependence of the HCV NS2-3 autoprotease on NS3 was
previously linked to a noncatalytic ZnB site naturally engi-
neered on the two-beta-barrel chymotrypsin-like fold (17).
This ZnB site is located at the side opposite to the active-site
cleft of the NS3 serine protease (26, 33). This site may be
regarded as a peculiar variant of a Zn finger otherwise found
only in 2A cysteine proteases of enteroviruses or rhinoviruses
(40, 54) but not in the NS3 protease of pestiviruses. Strikingly,
our experimentally backed NS2 alignment features a putative
ZnB site, which is critical for NS2-3 autoprocessing and viral
replication (Fig. 8), as part of a unique insertion in the pesti-
viral protease domain.
To fully understand its function, the NS2 ZnB site must be
characterized structurally and biochemically. In the meantime,
the genus-specific structural requirements of the NS2-3 cleav-
age in pestiviruses and hepaciviruses could be reconciled as
variations of a common mechanism. We suggest that NS2-3
autoprocessing is mediated by a protease associated with the
NS2 domain and is assisted by a Zn2?-dependent structure
supplied by either NS3 in hepaciviruses or NS2 in pestiviruses.
In contrast, flaviviruses employ NS2B to assist the serine pro-
tease in NS3 in cleaving the NS2-3 junction (Fig. 8) (32). These
genus-based variations in NS2-3 autoprocessing seem sensible
from the evolutionary perspective, as hepaciviruses and pesti-
viruses form sister phylogenetic lineages within the Flaviviri-
dae. This similarity in the mechanism of NS2-3 autoprocessing
between BVDV and HCV further strengthens the validity of
pestiviruses as a model system to study HCV replication.
Despite continuous efforts, evidence of NS2-3 processing in
reticulocyte lysate, in E. coli, and in trans, which was reported
for HCV (19, 39, 44), could not be demonstrated for BVDV
CP7 or NCP7 (N. Tautz and A. E. Gorbalenya, unpublished
data). Aside from purely technical issues, these differences may
be genuine and may involve the hydrophobic N-terminal re-
gion of NS2, which is only moderately important for NS2-3
cleavage of HCV (39) but is an essential part of the BVDV
NS2 protease (Fig. 2C).
Modulation of NS2-3 cleavage efficiency. It was a paradigm
that NS2-3 is cleaved only in cp but not in noncp BVDV-
infected cells (10, 37). We were able to demonstrate efficient
NS2-3 cleavage in noncp BVDV-infected cells at early time
points not included in previous analyses. The observed pro-
found downregulation of the NS2-3 cleavage as early as 9 h p.i.
implies that between 6 and 9 h p.i., either a potent activator of
the autoprocessing is depleted or an inhibitor of this process is
induced or produced.
The CP7-specific insertion in the central region of NS2 ob-
viously interferes with the downregulation of the autoprocess-
ing. Nevertheless, we also observed a downregulation of cleav-
age in CP7-infected cells between 6 and 9 h p.i., although the
resulting cleavage level of about 67% [NS3/(NS3 ? NS2-3)]
was still high. Interestingly, upon transient expression in BHK-
21 cells, e.g., in the context of the E2-NS4A polyprotein frag-
ment, cleavage of noncp BVDV-derived NS2-3 was not de-
tected (36, 51; this report). Under the same conditions, NS2-3
of BVDV CP7 is cleaved, while not fully approximating the
level observed in CP7-infected MDBK cells at 9 h p.i. and later.
In this context, it is important to note that BHK-21 cells are
not natural host cells for BVDV and do not support efficient
RNA replication of noncp BVDV even though cell lines can be
selected that allow a low-level replication (20). Less-efficient
NS2-3 processing under the conditions used for transient ex-
pression may thus originate from a shortage of virus- or cell-
derived cofactors or the presence of an inhibitor(s). Further-
more, the available data suggest that the 9-aa insertion in NS2
of CP7 switches on NS2-3 autoprocessing and makes it less
sensitive to hypothetical cofactors present in BVDV-infected
With respect to potential cofactors of the pestiviral NS2-3
processing, the identification of a cellular J-domain protein,
which interacts with pestiviral NS2 and is capable of promoting
NS2-3 cleavage in noncp BVDV-infected cells, may be relevant
(45); a possible role of this protein in modulating the NS2
protease is currently under investigation (N. Tautz, unpub-
lished data). Regardless of the identity of the factor, it is
evident that BVDV NS2-3 autoprocessing is sensitive to the
FIG. 8. NS2-3 cleavage in three genera of the family Flaviviridae.
Polyprotein fragments encompassing NS2-3 of the three Flaviviridae
genera are depicted schematically. The locations of the helicase and
protease domains are indicated; (putative) ZnB motifs (Zn2?) are
shaded in gray. The positions of the minimal protease domains capable
of cleaving the NS2-3 junction in vitro are shown below the polypro-
teins by filled ovals (catalytic domains) and rectangles (essential ac-
cessory domains). Curved arrows point to the cleavage sites processed
by the respective protease.
VOL. 78, 2004 PROTEASE IN CONTROL OF REPLICATION AND DISEASE10773
cellular environment and may be host restricted. The underly-
ing mechanism is an important subject for future studies.
Regulatory effects of NS2-3 autoprocessing in the pestiviral
life cycle. Proteolytic processing of viral polyproteins is a major
mechanism regulating the replication of positive-strand RNA
viruses. For picornaviruses or the alphavirus Sindbis virus, a
pivotal role of differential cleavages or a temporal regulation of
certain processing steps on diverse aspects of the viral replica-
tion cycle has been established (21, 31, 43).
The temporal modulation of NS2-3 cleavage, especially in
noncp BVDV-infected cells, provides a mechanism for regu-
lating the ratio between NS2-3 and its processing products
during the course of infection. Intriguingly, when compared to
noncp BVDV strains, the high levels of NS3 expressed by cp
BVDV correlate with an enhanced accumulation of intracel-
lular viral RNA (6, 34, 53). This is in line with our observation
that the intracellular concentration of NS3 strongly correlates
with the efficiency of RNA replication (Fig. 5 and 6). NS3,
which has protease, helicase, and NTPase activities, was pre-
viously shown to be essential for the replication of subgenomic
pestiviral RNAs (replicons) encoding viral proteins NS3 to
NS5B (7). Since these RNAs replicated efficiently, NS2 and
NS2-3 do not play essential roles in positive- or negative-strand
RNA synthesis (7). Our finding that NS3 cannot be function-
ally replaced by uncleaved NS2-3, as well as the strict correla-
tion between NS3 level and efficiency of RNA replication, thus
strongly suggest that NS3 but not NS2-3 is an essential com-
ponent of the viral replication complex, further implicating the
NS2 protease in replication control.
While cp BVDV strain CP7 generates NS3 by cleavage of
NS2-3, the majority of cp BVDV strains express NS3 from a
duplicated genomic region (37). In the respective polyproteins,
cellular sequences like ubiquitin are often located directly up-
stream of NS3. They promote the generation of the authentic
N terminus of NS3 by cellular proteases. It is not known
whether these viruses also require an active NS2 protease for
their replication. After infection with these cp BVDV strains,
there is efficient generation of NS3 throughout infection, viral
RNA accumulates to high concentrations, and the infected
cells undergo apoptosis. Accordingly, efficient generation of
NS3 generally correlates with an upregulated synthesis of viral
RNA and the cp biotype of the virus. This is in line with our
data that show that the concentration of NS3 appears to limit
RNA replication of noncp BVDV, which may be crucial for its
A correlation between increased viral RNA synthesis and
viral cytopathogenicity was also described for the picornavirus
hepatitis A virus (references 18 and 55 and references therein)
and the alphavirus Sindbis virus (1, 14, 15). For natural isolates
of BVDV, enhanced viral RNA synthesis was described only
for cp strains (6, 34, 53); the only exception to this trend is a cp
BVDV-derived noncp virus isolated under selection in tissue
culture (42). Although this virus showed high levels of NS3
expression and RNA replication, it exhibited the noncp phe-
notype. This last observation indicates that the mechanism of
cytopathogenicity in vitro is complex and needs further study.
With respect to the viral life cycle, effective processing of
NS2-3 shortly after infection appears to be required for effi-
cient RNA replication. At about 8 to 10 h p.i., the production
and secretion of newly generated infectious BVDV progeny
begins (data not shown). At that time, NS2-3 cleavage effi-
ciency drops sharply, leading to the accumulation of unproc-
essed NS2-3. It has been observed that uncleaved NS2-3 is es-
sential for the generation of infectious progeny virus (2; Tautz,
unpublished). Accordingly, temporal regulation of NS2-3 auto-
processing is most likely necessary for the switch between the
phase of highly active viral RNA replication and virus mor-
NS2-3 cleavage and outcome of infection. BVDV represents
a model system for persistent infections in mammals. noncp
BVDV strains have a high prevalence in cattle and persist in a
low percentage of cattle worldwide, accompanied by continu-
ous shedding of infectious virus (3). In contrast, cp BVDV
strains are unable to establish persistent infections and are
therefore almost exclusively isolated from sporadic cases of
animals with mucosal disease. In cattle persistently infected
with noncp BVDV, the emergence of a cp BVDV variant, like
BVDV CP7 analyzed in detail in this study, leads to the onset
of lethal disease. The viral biotype is thus a crucial determinant
for the pathogenicity of BVDV and the spread of the virus in
its host population. In this context, our study strongly suggests
that the observed temporal modulation of NS2-3 autoprocess-
ing may be central for the adaptation of BVDV to its animal
host. Restriction of this process, as shown here for cp BVDV
CP7, is correlated with viral cytopathogenicity and the conver-
sion of persistent infection into lethal disease. The results
obtained in the present study therefore contribute to the un-
derstanding of the molecular basis for viral persistence and
progression to disease.
We thank S. Jacobi for excellent technical assistance. We are grate-
ful to M. Ziess and C. Birghan for their contributions in the initial
phase of the project and thank J. Ziehbur (Institut fu ¨r Virologie und
Immunologie, Universita ¨t Wu ¨rzburg) for his support in the radiose-
quencing of proteins. A.E.G. gives special thanks to The Netherlands
Organization for Scientific Research (NWO) for support at the initial
stage of the project and Willy Spaan (Leiden University) for encour-
This study was supported by SFB 535 Invasionsmechanismen und
Replikationsstrategien von Krankheitserregern (T.L.) and Gradui-
ertenkolleg 455 Molekulare Veterina ¨rmedizin (A.M.) of the Deutsche
1. Agapov, E. V., I. Frolov, B. D. Lindenbach, B. M. Pragai, S. Schlesinger, and
C. M. Rice. 1998. Noncytopathic Sindbis virus RNA vectors for heterologous
gene expression. Proc. Natl. Acad. Sci. USA 95:12989–12994.
2. Agapov, E. V., C. L. Murray, I. Frolov, L. Qu, T. M. Myers, and C. M. Rice.
2004. Uncleaved NS2–3 is required for production of infectious bovine viral
diarrhea virus. J. Virol. 78:2414–2425.
3. Baker, J. C. 1987. Bovine viral diarrhea virus: a review. J. Am. Vet. Med.
4. Baroth, M., M. Orlich, H.-J. Thiel, and P. Becher. 2000. Insertion of cellular
NEDD8 coding sequences in a pestivirus. Virology 278:456–466.
5. Becher, P., M. Orlich, and H.-J. Thiel. 2000. Mutations in the 5? nontrans-
lated region of bovine viral diarrhea virus result in altered growth charac-
teristics. J. Virol. 74:7884–7894.
6. Becher, P., M. Orlich, and H.-J. Thiel. 2001. RNA recombination between
persisting pestivirus and a vaccine strain: generation of cytopathogenic virus
and induction of lethal disease. J. Virol. 75:6256–6264.
7. Behrens, S.-E., C. W. Grassmann, H.-J. Thiel, G. Meyers, and N. Tautz.
1998. Characterization of an autonomous subgenomic pestivirus RNA rep-
licon. J. Virol. 72:2364–2372.
8. Behrens, S.-E., L. Tomei, and R. de Francesco. 1996. Identification and
properties of the RNA-dependent RNA polymerase of hepatitis C virus.
EMBO J. 15:12–22.
9. Birghan, C., E. Mundt, and A. E. Gorbalenya. 2000. A non-canonical lon
10774 LACKNER ET AL.J. VIROL.
proteinase lacking the ATPase domain employs the Ser-Lys catalytic dyad to Download full-text
exercise broad control over the life cycle of a double-stranded RNA virus.
EMBO J. 19:114–123.
10. Corapi, W. V., R. O. Donis, and E. J. Dubovi. 1990. Characterization of a
panel of monoclonal antibodies and their use in the study of the antigenic
diversity of bovine viral diarrhea virus. Am. J. Vet. Res. 51:1388–1394.
11. Corapi, W. V., R. O. Donis, and E. J. Dubovi. 1988. Monoclonal antibody
analyses of cytopathic and noncytopathic viruses from fatal bovine viral
diarrhea infections. J. Virol. 62:2823–2827.
12. de Moerlooze, L., M. Desport, A. Renard, C. Lecomte, J. Brownlie, and J. A.
Martial. 1990. The coding region for the 54-kDa protein of several pestivi-
ruses lacks host insertions but reveals a “zinc finger-like” domain. Virology
13. Deng, R., and K. V. Brock. 1992. Molecular cloning and nucleotide sequence
of a pestivirus genome, noncytopathogenic bovine viral diarrhea virus strain
SD-1. Virology 191:867–879.
14. Dryga, S. A., O. A. Dryga, and S. Schlesinger. 1997. Identification of muta-
tions in a Sindbis virus variant able to establish persistent infection in BHK
cells: the importance of a mutation in the nsP2 gene. Virology 228:74–83.
15. Frolov, I., E. Agapov, T. A. Hoffman, Jr., B. M. Pragai, M. Lippa, S.
Schlesinger, and C. M. Rice. 1999. Selection of RNA replicons capable of
persistent noncytopathic replication in mammalian cells. J. Virol. 73:3854–
16. Gmyl, A. P., E. V. Pilipenko, S. V. Maslova, G. A. Belov, and V. I. Agol. 1993.
Functional and genetic plasticities of the poliovirus genome: quasi-infectious
RNAs modified in the 5?-untranslated region yield a variety of pseudorever-
tants. J. Virol. 67:6309–6316.
17. Gorbalenya, A. E., and E. J. Snijder. 1996. Viral cysteine proteinases. Per-
spect. Drug Discov. Des. 6:64–86.
18. Gosert, R., D. Egger, and K. Bienz. 2000. A cytopathic and a cell culture
adapted hepatitis A virus strain differ in cell killing but not in intracellular
membrane rearrangements. Virology 266:157–169.
19. Grakoui, A., D. W. McCourt, C. Wychowski, S. M. Feinstone, and C. Rice.
1993. A second hepatitis C virus-encoded proteinase. Proc. Natl. Acad. Sci.
20. Grassmann, C. W., O. Isken, N. Tautz, and S. E. Behrens. 2001. Genetic
analysis of the pestivirus nonstructural coding region: defects in the NS5A
unit can be complemented in trans. J. Virol. 75:7791–7802.
21. Griffin, D. E. 2001. Alphaviruses, p. 917–962. In D. M. Knipe and P. M.
Howley (ed.), Fields virology, 4th ed., vol. 1. Lippincott-Raven Publishers,
22. Hahn, C. S., and J. H. Strauss. 1990. Site-directed mutagenesis of the
proposed catalytic amino acids of the Sindbis virus capsid protein autopro-
tease. J. Virol. 64:3069–3073.
23. Hellen, C. U. T., M. Fa ¨cke, H.-G. Kra ¨usslich, C.-K. Lee, and E. Wimmer.
1991. Characterization of poliovirus 2A protease by mutational analysis:
residues required for autocatalytic activity are essential for induction of
cleavage of eucaryotic initiation factor 4F polypeptide p220. J. Virol. 65:
24. Henikoff, S., and J. G. Henikoff. 1994. Position-based sequence weights. J.
Mol. Biol. 243:574–578.
25. Hijikata, M., H. Mizushima, T. Akagi, S. Mori, N. Kakiuchi, N. Kato, T.
Tanaka, K. Kimura, and K. Shimotohno. 1993. Two distinct proteinase
activities required for the processing of a putative nonstructural precursor
protein of hepatitis C virus. J. Virol. 67:4665–4675.
26. Kim, J. L., K. A. Morgenstern, C. Lin, T. Fox, M. D. Dwyer, J. A. Landro,
S. P. Chambers, W. Markland, C. A. Lepre, E. T. O’Malley, S. L. Harbeson,
C. M. Rice, M. A. Murcko, P. R. Caron, and J. A. Thomson. 1996. Crystal
structure of the hepatitis C virus NS3 protease domain complexed with a
synthetic NS4A cofactor peptide. Cell 87:343–355.
27. Kolykhalov, A. A., K. Mihalik, S. M. Feinstone, and C. M. Rice. 2000.
Hepatitis C virus-encoded enzymatic activities and conserved RNA elements
in the 3? nontranslated region are essential for virus replication in vivo.
J. Virol. 74:2046–2051.
28. Ku ¨mmerer, B., D. Stoll, and G. Meyers. 1998. Bovine viral diarrhea virus
strain Oregon: a novel mechanism for processing of NS2–3 based on point
mutations. J. Virol. 72:4127–4138.
29. Lawrence, C. E., S. F. Altschul, M. S. Boguski, J. S. Liu, A. F. Neuwald, and
J. C. Wootton. 1993. Detecting subtle sequence signals: a Gibbs sampling
strategy for multiple alignment. Science 262:208–214.
30. Lawson, M. A., and B. L. Semler. 1991. Poliovirus thiol proteinase 3C can
utilize a serine nucleophile within the putative catalytic triad. Proc. Natl.
Acad. Sci. USA 63:5013–5022.
31. Lemm, J. A., T. Ru ¨menapf, E. G. Strauss, J. H. Strauss, and C. M. Rice.
1994. Polypeptide requirements for assembly of functional Sindbis virus
replication complexes: a model for the temporal regulation of minus- and
plus-strand RNA synthesis. EMBO J. 13:2925–2934.
32. Lindenbach, B. D., and C. M. Rice. 2001. Flaviviridae: the viruses and their
replication, p. 991–1042. In D. M. Knipe and P. M. Howley (ed.), Fields
virology, 4th ed., vol. 1. Lippincott-Raven Publishers, Philadelphia, Pa.
33. Love, R. A., H. E. Parge, J. A. Wickersham, Z. Hostomsky, N. Habuka, E. W.
Moomaw, T. Adachi, and Z. Hostomska. 1996. The crystal structure of
hepatitis C virus NS3 proteinase reveals a trypsin-like fold and a structural
zinc binding site. Cell 87:331–342.
34. Mendez, E., N. Ruggli, M. S. Collett, and C. M. Rice. 1998. Infectious bovine
viral diarrhea virus (strain NADL) RNA from stable cDNA clones: a cellular
insert determines NS3 production and viral cytopathogenicity. J. Virol. 72:
35. Meyers, G., D. Stoll, and M. Gunn. 1998. Insertion of a sequence encoding
light chain 3 of microtubule-associated proteins 1A and 1B in a pestivirus
genome: connection with virus cytopathogenicity and induction of lethal
disease in cattle. J. Virol. 72:4139–4148.
36. Meyers, G., N. Tautz, P. Becher, H.-J. Thiel, and B. Ku ¨mmerer. 1996.
Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea
viruses from cDNA constructs. J. Virol. 70:8606–8613.
37. Meyers, G., and H.-J. Thiel. 1996. Molecular characterization of pestiviruses.
Adv. Virus Res. 47:53–117.
38. Nicholas, K. B., N. H. B. J. Nicholas, and D. W. Deerfield. 1997. GeneDoc:
analysis and visualization of genetic variation. EMBNET News 4:1–4.
39. Pallaoro, M., A. Lahm, G. Biasiol, M. Brunetti, C. Nardella, L. Orsatti, F.
Bonelli, S. Orru, F. Narjes, and C. Steinkuhler. 2001. Characterization of the
hepatitis C virus NS2/3 processing reaction by using a purified precursor
protein. J. Virol. 75:9939–9946.
40. Petersen, J. F., M. M. Cherney, H. D. Liebig, T. Skern, E. Kuechler, and
M. N. James. 1999. The structure of the 2A proteinase from a common cold
virus: a proteinase responsible for the shut-off of host-cell protein synthesis.
EMBO J. 18:5463–5475.
41. Pocock, D. H., C. J. Howard, M. C. Clarke, and J. Brownlie. 1987. Variation
in the intracellular polypeptide profiles from different isolates of bovine viral
diarrhea virus. Arch. Virol. 94:43–53.
42. Qu, L., L. K. McMullan, and C. M. Rice. 2001. Isolation and characterization
of noncytopathic pestivirus mutants reveals a role for nonstructural protein
NS4B in viral cytopathogenicity. J. Virol. 75:10651–10662.
43. Racaniello, V. R. 2001. Picornaviridae: the viruses and their replication, p.
685–722. In D. M. Knipe and P. M. Howley (ed.), Fields virology, 4th ed., vol.
1. Lippincott-Raven Publishers, Philadelphia, Pa.
44. Reed, K. E., A. Grakoui, and C. M. Rice. 1995. Hepatitis C virus-encoded
NS2–3 protease: cleavage-site mutagenesis and requirements for bimolecu-
lar cleavage. J. Virol. 69:4127–4136.
45. Rinck, G., C. Birghan, T. Harada, G. Meyers, H.-J. Thiel, and N. Tautz.
2001. A cellular J-domain protein modulates polyprotein processing and
cytopathogenicity of a pestivirus. J. Virol. 75:9470–9482.
46. Santolini, E., L. Pacini, C. Fipaldini, G. Migliaccio, and N. La Monica. 1995.
The NS2 protein of hepatitis C virus is a transmembrane polypeptide. J. Vi-
47. Scha ¨gger, H., and G. von Jagow. 1987. Tricine-sodium dodecyl sulfate-
polyacrylamide gel electrophoresis for the separation of proteins in the range
from 1 to 100 kDa. Anal. Biochem. 166:368–379.
48. Schuler, G. D., S. F. Altschul, and D. J. Lipman. 1991. A workbench for
multiple alignment construction and analysis. Proteins 9:180–190.
49. Sutter, G., M. Ohlmann, and V. Erfle. 1995. Non-replicating vaccinia vector
efficiently expresses bacteriophage T7 RNA polymerase. FEBS Lett. 371:9–
50. Tautz, N., T. Harada, A. Kaiser, G. Rinck, S. E. Behrens, and H.-J. Thiel.
1999. Establishment and characterization of cytopathogenic and noncyto-
pathogenic pestivirus replicons. J. Virol. 73:9422–9432.
51. Tautz, N., G. Meyers, R. Stark, E. J. Dubovi, and H.-J. Thiel. 1996. Cyto-
pathogenicity of a pestivirus correlated with a 27-nucleotide insertion. J. Vi-
52. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G.
Higgins. 1997. The CLUSTAL_X Windows interface: flexible strategies for
multiple sequence alignment aided by quality analysis tools. Nucleic Acids
53. Vassilev, V. B., and R. O. Donis. 2000. Bovine viral diarrhea virus induced
apoptosis correlates with increased intracellular viral RNA accumulation.
Virus Res. 69:95–107.
54. Yu, S. F., and R. E. Lloyd. 1992. Characterization of the roles of conserved
cysteine and histidine residues in poliovirus 2A protease. Virology 186:725–
55. Zhang, H., S. F. Chao, L. H. Ping, K. Grace, B. Clarke, and S. M. Lemon.
1995. An infectious cDNA clone of a cytopathic hepatitis A virus: genomic
regions associated with rapid replication and cytopathic effect. Virology
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