JOURNAL OF VIROLOGY, Dec. 2009, p. 12415–12423
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 23
A Single Point Mutation in Nonstructural Protein NS2 of Bovine Viral
Diarrhea Virus Results in Temperature-Sensitive Attenuation of
Alexander Pankraz,1§ Simone Preis,1Heinz-Ju ¨rgen Thiel,1Andreas Gallei,1† and Paul Becher1,2*
Institute of Virology, Justus-Liebig University, D-35392 Giessen, Germany,1and Institute of Virology, Department of
Infectious Diseases, University of Veterinary Medicine, D-30559 Hannover, Germany2
Received 17 July 2009/Accepted 15 September 2009
For Bovine viral diarrhea virus (BVDV), the type species of the genus Pestivirus in the family Flaviviridae,
cytopathogenic (cp) and noncytopathogenic (ncp) viruses are distinguished according to their effect on cultured
cells. It has been established that cytopathogenicity of BVDV correlates with efficient production of viral
nonstructural protein NS3 and with enhanced viral RNA synthesis. Here, we describe generation and char-
acterization of a temperature-sensitive (ts) mutant of cp BVDV strain CP7, termed TS2.7. Infection of bovine
cells with TS2.7 and the parent CP7 at 33°C resulted in efficient viral replication and a cytopathic effect. In
contrast, the ability of TS2.7 to cause cytopathogenicity at 39.5°C was drastically reduced despite production
of high titers of infectious virus. Further experiments, including nucleotide sequencing of the TS2.7 genome
and reverse genetics, showed that a Y1338H substitution at residue 193 of NS2 resulted in the temperature-
dependent attenuation of cytopathogenicity despite high levels of infectious virus production. Interestingly,
TS2.7 and the reconstructed mutant CP7-Y1338H produced NS3 in addition to NS2-3 throughout infection.
Compared to the parent CP7, NS2-3 processing was slightly decreased at both temperatures. Quantification of
viral RNAs that were accumulated at 10 h postinfection demonstrated that attenuation of the cytopathogenicity
of the ts mutants at 39.5°C correlated with reduced amounts of viral RNA, while the efficiency of viral RNA
synthesis at 33°C was not affected. Taken together, the results of this study show that a mutation in BVDV NS2
attenuates viral RNA replication and suppresses viral cytopathogenicity at high temperature without altering
NS3 expression and infectious virus production in a temperature-dependent manner.
The pestiviruses Bovine viral diarrhea virus-1 (BVDV-1),
BVDV-2, Classical swine fever virus (CSFV), and Border disease
virus (BDV) are causative agents of economically important
livestock diseases. Together with the genera Flavivirus, includ-
ing several important human pathogens like Dengue fever virus,
West Nile virus, Yellow fever virus, and Tick-borne encephalitis
virus, and Hepacivirus (human Hepatitis C virus [HCV]), the
genus Pestivirus constitutes the family Flaviviridae (8, 20). All
members of this family are enveloped viruses with a single-
stranded positive-sense RNA genome encompassing one large
open reading frame (ORF) flanked by 5? and 3? nontranslated
regions (NTR) (see references 8 and 28 for reviews). The ORF
encodes a polyprotein which is co- and posttranslationally pro-
cessed into the mature viral proteins by viral and cellular pro-
teases. For BVDV, the RNA genome is about 12.3 kb in length
and encodes a polyprotein of about 3,900 amino acids. The first
third of the ORF encodes a nonstructural (NS) autoprotease
and four structural proteins, while the remaining part of the
genome encodes NS proteins which share many common char-
acteristics and functions with the corresponding NS proteins
encoded by the HCV genome (8, 28). NS2 of BVDV repre-
sents a cysteine autoprotease which is distantly related to the
HCV NS2-3 protease (26). NS3, NS4A, NS4B, NS5A, and
NS5B are essential components of the pestivirus replicase (7,
10, 49). NS3 possesses multiple enzymatic activities, namely
serine protease (48, 52, 53), NTPase (46), and helicase activity
(51). NS4A acts as an essential cofactor for the NS3 protein-
ase. NS5B represents the RNA-dependent RNA polymerase
(RdRp) (22, 56). The functions of NS4B and NS5A remain to
be determined. NS5A has been shown to be a phosphorylated
protein that is associated with cellular serine/threonine kinases
According to their effects in tissue culture, two biotypes of
pestiviruses are distinguished: cytopathogenic (cp) and noncy-
topathogenic (ncp) viruses (17, 27). The occurrence of cp
BVDV in cattle persistently infected with ncp BVDV is di-
rectly linked to the induction of lethal mucosal disease in cattle
(12, 13). Previous studies have shown that cp BVDV strains
evolved from ncp BVDV strains by different kinds of muta-
tions. These include RNA recombination with various cellular
mRNAs, resulting in insertions of cellular protein-coding se-
quences into the viral genome, as well as insertions, duplica-
tions, and deletions of viral sequences, and point mutations (1,
2, 9, 24, 33, 36, 37, 42). A common consequence of all these
genetic changes in cp BVDV genomes is the efficient produc-
tion of NS3 at early and late phases of infection. In contrast,
NS3 cannot be detected in cells at late time points after infec-
tion with ncp BVDV. An additional major difference is that the
* Corresponding author. Mailing address: Institute of Virology, De-
partment of Infectious Diseases, University of Veterinary Medicine,
Bu ¨nteweg 17, D-30559 Hannover, Germany. Phone: 49 511 953 8452.
Fax: 49 511 953 828452. E-mail: email@example.com.
§ Present address: Biocontrol Ingelheim, Konrad-Adenauer-Strasse
17, D-55218 Ingelheim am Rhein, Germany.
† Present address: BioScreen European Veterinary Disease Man-
agement Center GmbH, Mendelstrasse 11, D-48149 Mu ¨nster, Ger-
?Published ahead of print on 23 September 2009.
cp viruses produce amounts of viral RNA significantly larger
than those of their ncp counterparts (7, 32, 50). While there is
clear evidence that cell death induced by cp BVDV is mediated
by apoptosis, the molecular mechanisms involved in pestiviral
cytopathogenicity are poorly understood. In particular, the role
of NS3 in triggering apoptosis remains unclear. It has been
hypothesized that the NS3 serine proteinase might be involved
in activation of the apoptotic proteolytic cascade (21, 55).
Furthermore, it has been suggested that the NS3-mediated,
enhanced viral RNA synthesis of cp BVDV and subsequently
larger amounts of viral double-stranded RNAs may play a
crucial role in triggering apoptosis (31, 54).
In this study, we describe generation and characterization of
a temperature-sensitive (ts) cp BVDV mutant whose ability to
cause viral cytopathogenicity at high temperature is strongly
attenuated. Our results demonstrate that a single amino acid
substitution in NS2 attenuates BVDV cytopathogenicity at
high temperature without affecting production of infectious
viruses and expression of NS3 in a temperature-dependent
MATERIALS AND METHODS
Cells and viruses. Madin-Darby bovine kidney (MDBK) cells and sheep fetal
thymus cells were obtained from the American Type Culture Collection (Rock-
ville, MD) and the Friedrich Loeffler Institute (Isle of Riems, Germany), respec-
tively. Cells were grown in Dulbecco’s modified Eagle’s medium supplemented
with 10% horse serum. Cells were tested regularly for the absence of pestiviruses
by reverse transcription (RT)-PCR and immunofluorescence (IF) (3). The cyto-
pathogenic BVDV-1 strain CP7 and its isogenic ncp derivative NCP7 were
generated from the authentic full-length infectious BVDV cDNA clones pCP7-
388 and pNCPC-5A, which have been described previously (1, 39). The first
passage after transfection of pCP7-388- and pNCP7-5A-derived RNAs was used
throughout this study.
Chemical mutagenesis and plaque purification of ts mutants. To generate ts
mutants, a set of MDBK cells was infected with BVDV CP7 at a multiplicity of
infection (MOI) of 1 PFU/cell. After 1 h adsorption, media that contained
various concentrations of proflavin (4.0 to 1,000 ?g/ml) were laid over the cells,
and the infected cells were incubated at 37°C for 48 h. To determine the effects
of proflavin on virus replication and survival, progeny viruses from the superna-
tants of infected cells were titrated. The decrease in the yield of infectious virus
was assessed by comparison with the titer produced in untreated cells. The
supernatants from cell cultures that had been treated with 36 ?g/ml proflavin
showed about 2% survival compared to the control. Serial dilutions of this
supernatant were used for infection of MDBK cells. After adsorption for 1 h, the
cells were washed with phosphate-buffered saline (PBS) and overlaid with semi-
solid medium containing 0.6% low-melting-point agarose (Gibco-BRL) and 5%
horse serum. After 3 days of incubation at 33°C, the plaques were circled and the
plates were incubated at 39.5°C for 2 days. Plaques that failed to enlarge at
39.5°C were considered potentially ts and picked for further analysis.
RNA preparation, RT-PCR, and molecular cloning. Total cellular RNA was
prepared using the QIAshredder and RNeasy MinElute cleanup kits (Qiagen).
RT of heat-denatured RNA, PCR, molecular cloning, and nucleotide sequencing
were essentially done as described previously (3). Primers were deduced from the
BVDV CP7 sequence reported previously (5, 34). For determination of the 5?
and 3? terminal sequences of BVDV TS2.7, an RNA ligation method was em-
ployed as described elsewhere (4, 5). The cDNA fragments obtained after RT-
PCR were separated by agarose gel electrophoresis, purified using the Qiaex
DNA purification kit, and cloned into vector pDrive (Qiagen).
Nucleotide sequencing and sequence analysis. Nucleotide sequences were
determined by cycle sequencing using the Thermo Sequenase kit (Amersham
Buchler, Braunschweig, Germany) and dye (IRD 800)-labeled primers (MWG
Biotech). Analysis of sequencing gels was carried out with the DNA sequencer
Li-Cor 4000 L (MWG Biotech). Computer analysis of sequence data was per-
formed using HUSAR (DKFZ, Heidelberg, Germany), which provides the GCG
software package (16). Multiple sequence alignments of the amino acid se-
quences were generated with the program PILEUP.
Construction of BVDV mutant CP7-Y1338H. All nucleotide numberings in-
cluded in this study refer to pCP7-388 (39). For construction of CP7-Y1338H,
the point mutation identified in the genome of ts mutant TS2.7 at position 4394
was introduced into the CP7 cDNA by QuikChange PCR (Stratagene, Heidel-
berg, Germany) using a subclone of pCP7-388, which lacks nucleotides (nt) 5883
(SacI) to 11076 (ClaI) of the CP7 sequence. Finally, the XhoI (nt 222)/AgeI
(5356) fragment carrying the mutation was cloned into pCP7-388 precut with
XhoI and AgeI. The presence of the mutation at position 4394 and absence of
additional mutations were confirmed by nucleotide sequencing of the genomic
region encompassing nt 222 to 5356. Further details of the cloning strategies as
well as primer sequences are available upon request.
In vitro synthesis of RNA. SmaI (12294)-linearized plasmids of full-length
BVDV clones were treated with phenol-chloroform, precipitated with ethanol,
and then used as DNA templates to generate in vitro transcripts using the SP6
RNA polymerase (Takara, Shiga, Japan), as described previously (5). After
transcription, a DNase I (Ambion, Austin, TX) digestion was performed for 15
min at 37°C. Photometric quantification of the transcribed RNA was carried out
by using a GeneQuantII photometer (Pharmacia). The quality and the calculated
amount of RNA were assessed by ethidium bromide staining of samples after
agarose gel electrophoresis. The RNA transcripts used for transfection contained
?80% of full-length RNA.
Transfection of RNA. The confluent MDBK cells from a dish 10 cm in diam-
eter were trypsinized, washed, resuspended in 0.4 ml of PBS without Ca2?and
Mg2?, and mixed with 2 ?g of in vitro-transcribed RNA immediately before the
pulse (950 ?F and 180 V). For electroporation, Gene Pulser II (Bio-Rad, Mu-
nich, Germany) was used. The electroporated cells were resuspended in 6 ml of
medium containing 10% horse serum, and this cell suspension was then distrib-
uted to three wells of a six-well plate immediately posttransfection. Cells were
checked 2 and 3 days posttransfection by light microscopy for the appearance of
a cytopathic effect (CPE) and by IF analysis using monoclonal antibody 8.12.7
(directed against NS3), kindly provided by E. J. Dubovi (Cornell University,
Ithaca, NY) (15).
Plaque assay and immunostaining of infected cells. MDBK cells (2 ? 106)
were infected with 10-fold serial dilutions of infectious supernatants obtained
from the second cell culture passage of BVDV CP7, TS2.7, and CP7-Y1338H.
After adsorption for 1 h, the cells were washed with PBS and overlaid with
semisolid medium containing 0.6% low-melting-point agarose (Gibco-BRL) and
5% horse serum. After 6 or 7 days of incubation at 33°C or 39.5°C, 2% para-
formaldehyde (wt/vol) was used for fixation of the cells. After removal of the
agarose overlays, the cells were washed with PBS, air dried, and subjected to
immunostaining using an anti-BVDV polyclonal antiserum and a peroxidase-
linked goat anti-bovine immunoglobulin antibody (5).
Determination of growth kinetics and indirect IF. MDBK cells (2 ? 106) were
infected with infectious supernatants obtained from the second cell culture
passage of BVDV CP7, NCP7, TS2.7, and CP7-Y1338H at an MOI of 0.1. After
adsorption for 1 hour at 33°C or 39.5°C, the cells were washed six times with PBS,
overlaid with medium containing 10% horse serum, and incubated over a 3-day
period. At the indicated time points, aliquots (200 ?l) of the supernatant were
removed, and the 50% tissue culture infectious dose (TCID50) of progeny virus
was determined. In addition to microscopic examination of a CPE, indirect IF
with monoclonal antibody 8.12.7 directed against NS3 was used to monitor the
presence of viral antigen (15) as previously described (3).
Immunoblot analysis. Sodium dodecyl sulfate-polyacrylamide gel electro-
phoresis, transfer to nitrocellulose, and detection of NS2-3 and NS3 with mono-
clonal antibody 8.12.7 (15) was described previously (2).
Quantitative real-time RT-PCR. For comparative quantification of viral
genomic RNAs, duplicate sets of MDBK cells were infected with transcript-
derived virus of BVDV strains CP7, TS2.7, and CP7-Y1338H at an MOI of 1.0
and incubated at 33°C and 39.5°C. Total cellular RNA was prepared 10 h
postinfection (p.i.). Subsequently, photometric analyses were performed in order
to determine the RNA concentration for each sample; 1 ?l of each sample
(corresponding to 0.45 to 0.68 ?g) was then subjected to real-time RT-PCR
analysis. After RT using the reverse primer pv03R (5?-TCCATGTGCCATGT
ACAGCAG-3?; nt 367 through 387) and Superscript II reverse transcriptase
(Invitrogen, Karlsruhe, Germany), the quantitative real-time PCR was run using
the AbiPrism 7000 sequence detection system (Applied Biosystems, Branchburg,
NJ) in the “absolute quantification” mode and the TaqMan universal PCR
master mix (Applied Biosystems) without MgCl2. For PCR, the BVDV-1-specific
probe pvtaq01 (6-carboxyfluorescein [FAM]-ACAGTCTGATAGGATGCTGC
AGAGGCCC-6-carboxytetramethylrhodamine [TAMRA]; nt 317 through 344)
and the primer pair pv02 (5?-GTGGACGAGGGCATGCC-3?; nt 235 through
251; sense primer)/pv03R were used. Cycling conditions were 1 cycle (2 min at
50°C, 10 min at 95°C) followed by 38 cycles (15 s at 95°C, 1 min at 60°C). To
compare the amounts of viral genomic RNA that accumulated in cells infected
with the BVDV mutant viruses TS2.7 and CP7-Y1338H to those in cells infected
12416 PANKRAZ ET AL.J. VIROL.
by parental BVDV CP7, the formula % RNA ? 1/(1.98??CT) ? 100 was used.
Finally, the results were normalized to the different amounts of input RNA.
Three replicates were assayed for each sample.
Generation of ts BVDV mutant TS2.7. For generation of ts
mutants of BVDV, various concentrations of the chemical
mutagen proflavin were applied to MDBK cells infected with
cp BVDV strain CP7. To minimize initial genetic variation of
the virus population prior to mutagenesis, the first passage of
BVDV strain CP7 derived from the infectious cDNA clone
pCP7-388 was used for infections (39). The percentage of
progeny virus survival was calculated by comparison of mu-
tated progeny virus titers to progeny virus titers without mu-
tagen (data not shown). The yield of infectious virus produced
decreased with increasing amounts of proflavin. It has been
reported that the optimal mutagen concentration for produc-
tion of ts mutants results in a 1 to 10% survival of viral progeny
(41). This condition was met when the BVDV infection was
performed in the presence of 36 ?g proflavin per ml. Using
serial dilutions of the mutagenized virus stock, plaque assays
were performed and incubated at 33°C (chosen as the permis-
sive temperature for this study). After an incubation period of
72 h, the plaques were circled and the plates were placed at
39.5°C (chosen as the restrictive temperature for this study) for
an additional 48 h. One out of about 120 examined plaques
failed to enlarge after transfer to 39.5°C, and thus, the respec-
tive virus represented a potential ts mutant. This plaque was
picked, subjected to two rounds of plaque purification, and
amplified through two passages at the permissive temperature.
Growth characteristics of TS2.7. The temperature sensitivity
of the obtained virus stock, termed TS2.7, was first examined
by comparing its abilities to cause cytopathogenicity at permis-
sive and restrictive temperatures. For this analysis, the virus
stocks of TS2.7 and the parental BVDV strain CP7 were ti-
trated in duplicate 96-well plates and incubated at 33°C and
39.5°C for 3 days. Microscopic examination revealed that
CPEs, including cell lysis, were found after infection with com-
parable dilutions of the unmutagenized CP7 strain at both
temperatures (Fig. 1A). In contrast, the amount of virus re-
quired by TS2.7 to cause cytopathogenicity at the restrictive
temperature was about 1,000-fold more than that required for
induction of cytopathogenicity at the permissive temperature
(Fig. 1A). After examination of CPEs, the 96-well plates were
subjected to IF analysis in order to detect viral antigen. For
CP7, titers determined by IF analysis were identical to titers
obtained by examination of a CPE. Surprisingly, IF analysis of
cells infected with TS2.7 at 39.5°C revealed a titer almost
FIG. 1. (A) Results of virus titrations of supernatants obtained 3 days after infection of MDBK cells with BVDV CP7 and TS2.7 at 33°C.
For titration, cells in duplicate 96-well plates were infected and incubated at 33°C (left) and 39.5°C (right) for 3 days. The titers were first
determined by microscopic examination of the occurrence of a CPE, and then viral antigen was detected via IF analysis. Mean values and ?
standard deviation ranges (error bars) were calculated from two independent experiments, each analyzed by quadruplicate titrations.
(B) Plaque assays of cells infected with CP7 and TS2.7. Infected cells were visualized by immunostaining at 7 and 6 days p.i. at 33°C (top)
and 39.5°C (bottom). (C) Bovine MDBK cells infected with BVDV strains CP7, TS2.7, and NCP7 at an MOI of 1 and incubated at 33°C (top)
and 39.5°C (bottom). The photographs were taken 48 h after infection. As a control, mock-infected cells are included. A strong CPE occurred
after infection of cells with CP7 at both temperatures and with TS2.7 at 33°C. In contrast, the ability of TS2.7 to cause viral cytopathogenicity
was drastically reduced at 39.5°C.
VOL. 83, 2009 BVDV NS2 MUTATION ATTENUATES VIRAL CYTOPATHOGENICITY12417
1,000-fold higher than the one previously determined by ex-
amination of CPE (Fig. 1A). Accordingly, the phenotype of
mutant TS2.7 is characterized by a significant attenuation of
cytopathogenicity at 39.5°C without reducing the yield of in-
fectious virus titers. In contrast, the ability of TS2.7 to cause a
CPE at 33°C is not affected. This phenomenon is also well
documented by plaque assays performed in parallel at both
temperatures. Infection of cells with dilutions of CP7 (10?1to
10?5) at 33°C and 39.5°C and with dilutions of TS2.7 (10?1to
10?5) at 33°C resulted in cell lysis and the formation of plaques
(Fig. 1B; data shown only for dilutions of 10?5). In contrast,
infection with TS2.7 at 39.5°C caused a CPE only when high
virus concentrations (undiluted or dilutions up to 10?2) were
used (data not shown), while higher dilutions of TS2.7 (10?3to
10?5) led to formation of foci (visualized by immunostaining),
without any signs of CPE (Fig. 1B; data shown only for dilu-
tions of 10?5). TS2.7 and its parent produced large plaques at
33°C, while immunostained foci produced by TS2.7 at 39.5°C
were smaller than the plaques produced by CP7. A similar
attenuation of viral cytopathogenicity at 39.5°C was observed
when sheep fetal thymus cells were used for infection with
TS2.7 (data not shown).
To further characterize viral growth properties, MDBK cells
were infected in duplicate six-well plates with BVDV TS2.7,
CP7, and the ncp virus NCP7 at an MOI of 1 and incubated at
33°C and 39.5°C for 48 h. As expected, there was no CPE
detectable after infection of cells with NCP7 at both temper-
atures. Infection with TS2.7 at 33°C caused a strong CPE
indistinguishable from that produced after infection with par-
ent CP7 (Fig. 1C, top). In agreement with the results obtained
from plaque assays, the ability of TS2.7 to cause cytopathoge-
nicity was drastically inhibited at 39.5°C (Fig. 1C, bottom). In
addition, the growth kinetics of TS2.7, CP7, and NCP7 at both
temperatures were determined. Duplicate sets of MDBK cells
were infected at an MOI of 0.1 and incubated in parallel at
33°C and 39.5°C. Virus released into the medium was sampled
for a 3-day period, and the respective virus titers were deter-
mined by IF analysis. Each of the three viruses reached titers
of ?5 ? 106TCID50/ml at 48 h after infection at both tem-
peratures (Fig. 2). With regard to infections at 33°C, the peak
titer of the mutant virus TS2.7 reached at 48 h p.i. was 2.3 ?
107TCID50/ml and thus slightly higher than the peak titer of
CP7 (5.9 ? 106TCID50/ml); the peak titer of NCP7 was 1.8 ?
107TCID50/ml. With respect to infections at 39.5°C, the titers
of TS2.7 reached at 24 h and 36 h were about three- and
four-fold lower, respectively, than the titers of the parental
CP7 strain. Taken together, the growth kinetics of TS2.7, CP7,
and NCP7 were very similar at both temperatures. Similar
results, including the absence of significant (?4-fold) differ-
ences between the titers of T2.7 and CP7, were obtained by two
additional comparative analyses of the growth kinetics of TS2.7
and CP7 (data not shown).
To study possible effects of temperature shifts on the induc-
tion of cytopathogenicity, sets of MDBK cells were infected
with TS2.7 at MOIs of 1.0, 10?1, 10?2, 10?3, and 10?4; incu-
bated at various combinations of temperatures for 7 days; and
examined daily by light microscope. Periods of 6 h and 24 h at
33°C before shifting to 39.5°C did not result in increased cy-
topathogenicity of TS2.7 at 39.5°C (data not shown). Vice
versa, a period of 6 h at 39.5°C before shifting to 33°C did not
attenuate the cytopathogenicity of TS2.7 at 33°C, while incu-
bation for 24 h at 39.5°C before shifting to 33°C led to a
temporal delay of the occurrence of CPEs (data not shown).
Genetic characterization of TS2.7. To determine the genetic
alteration(s) present in the genome of TS2.7, the entire
genomic sequence of TS2.7 was determined and compared to
the sequence of BVDV CP7; RNA from the second tissue
culture passage of TS2.7 was used for the respective analysis.
When differences from the wild-type BVDV CP7 sequence
were detected, the sequencing procedure was repeated and
only the confirmed changes were considered to be mutations
present in the genome of TS2.7. A total of five nucleotide
changes were found in the genomic regions encoding structural
proteins C and E2 as well as NS proteins NS2 and NS5B. Four
of these mutations resulted in changes of the deduced amino
acid sequence. The identified mutations and corresponding
deduced amino acid changes found in the genome of TS2.7 are
detailed in Fig. 3A.
A single nucleotide substitution is responsible for the ts
phenotype of TS2.7. To identify the mutation(s) responsible for
the ts phenotype of TS2.7, each of the four individual substi-
tutions causing amino acid changes was transferred into the
infectious cDNA clone pCP7-388. After in vitro transcription
and transfection of MDBK cells with each of the resulting
RNAs carrying a single substitution and incubation at 33°C for
3 days, infectious cp viruses were recovered. In two indepen-
dent experiments the specific infectivity of the four mutant
RNAs ranged between 4.2 ? 105and 2.0 ? 106PFU/?g RNA
and thus were very similar to that of parent CP7 (6.0 ? 105and
2.0 ? 106PFU/?g RNA). To examine whether one of these
mutations was responsible for the ts phenotype of TS2.7, du-
plicate plaque assays were performed using cells infected with
dilutions of the first passage of the respective viruses and
incubated at 33°C and 39.5°C for 7 days. Like those of the
parental CP7 strain, dilutions (up to 10?5) of the three mutants
with single nucleotide substitutions in the genomic regions
encoding C, E2, and NS5B (Fig. 3A) resulted in cell lysis and
formation of large plaques at both temperatures (data not
shown). Infection of cells with dilutions (up to 10?5) of the
FIG. 2. Growth kinetics of BVDV CP7, NCP7, and TS2.7 at 33°C
(left) and 39.5°C (right). MDBK cells were infected with the indicated
viruses at an MOI of 0.1. The titers of released virus were determined
by IF analysis over a time period of 72 h. Error bars indicate the ?
ranges from quadruplicate titrations.
12418PANKRAZ ET AL.J. VIROL.
mutant CP7-Y1338H carrying the single mutation at nucleo-
tide position 4394 located in the NS2 gene also resulted in cell
lysis and plaque formation at 33°C. However, CP7-Y1338H
was not able to produce plaques at 39.5°C when dilutions
between 10?2and 10?5were used for infection of cells but
resulted in the formation of infected foci (visualized by immu-
noperoxidase staining) without any signs of cytopathogenicity
(Fig. 3B). Accordingly, the phenotype of CP7-Y1338H is very
similar to the ts phenotype of TS2.7. Taken together, the re-
sults of these analyses demonstrated that a single mutation at
position 4394 within the NS2 gene resulted in attenuation of
cytopathogenicity of BVDV CP7 at high temperature.
Growth characteristics of BVDV CP7-Y1338H. To compare
the growth characteristics of the reconstructed ts mutant CP7-
Y1338H and the parental virus strain CP7, growth rates and
virus yields were determined using duplicate sets of MDBK
cells infected at an MOI of 0.1 and incubated at 33°C and
39.5°C. Virus titers were determined for a 3-day period. As
observed for the original TS2.7 mutant (Fig. 2), the growth
kinetics of CP7-Y1338H and CP7 were similar and both viruses
replicated at similar titers at 33°C and 39.5°C (Fig. 4A). Similar
results were obtained by an additional comparative analysis of
the growth kinetics of CP7-Y1338H and CP7 (data not shown).
Taken together, the results show that the Y1338H mutation
had no significant effect on virus yields and growth rates. Fur-
thermore, the results of this analysis showed that attenuation
of cytopathogenicity of CP7-Y1338H at 39.5°C does not cor-
relate with reduced viral growth.
Analysis of viral RNA synthesis and expression of NS2-3
and NS3. It is well known that cp and ncp BVDV strains
significantly differ in viral RNA production. For CP7 and other
cp BVDV strains, amounts of viral RNA accumulated in in-
fected cells were about 5- to 10-fold larger than the viral RNA
amounts produced by their ncp counterparts (7, 32, 50). An-
other striking difference between cp and ncp BVDV concerns
expression of viral NS3. While cp BVDV strains produce NS3
at early and late time points postinfection, for ncp BVDV, NS3
is usually no longer detectable after the first 12 h of infection
(26). It was therefore interesting to investigate whether the
observed attenuation of cytopathogenicity of TS2.7 and CP7-
Y1338H correlates with changes of viral RNA synthesis and/or
expression of NS3.
For analysis of viral RNA synthesis, cells were infected with
BVDV CP7, TS2.7, and CP7-Y1338H at an MOI of 1. To
minimize effects of potential differences in the levels of viral
spread and replication efficiency of the individual viruses, total
cellular RNAs were prepared 10 h p.i.; this time point precedes
the end of the first viral replication cycle (7, 32, 50). The
relative amounts of accumulated viral RNAs were determined
by quantitative real-time RT-PCR analysis. Comparative anal-
ysis revealed that the amounts of viral RNA produced at 33°C
were very similar for CP7, TS2.7, and the reconstructed ts
mutant; significant differences were not observed (Fig. 4B). In
contrast, the amounts of accumulated viral RNA after infec-
tion of cells with TS2.7 and CP7-Y1338H at 39.5°C were about
fivefold lower than the amount produced after infection with
parental BVDV CP7.
To monitor expression of NS3 and NS2-3, sets of cells were
infected with BVDV CP7, NCP7, TS2.7, and CP7-Y1338H at
an MOI of 2 and incubated at 33°C and 39.5°C. Cells were
lysed and harvested at 12 h and 20 h p.i., and aliquots corre-
sponding to 6.0 ? 104cells (collected at 12 h p.i.) and 6.0 ? 103
cells (collected at 20 h p.i.) were used for immunoblot analysis
(Fig. 4C). The results of this analysis revealed that CP7, TS2.7,
and CP7-Y1338H express significant amounts of NS2-3 and
NS3 throughout infection at both temperatures. Compared to
parental CP7, infection with TS2-7 and CP7-Y1338H resulted
in slightly decreased NS2-3 processing. This phenomenon was
observed at both temperatures (Fig. 4C). In contrast, after
infection with NCP7 at 33°C and 39.5°C, expression of detect-
able levels of NS3 was limited to the early phase of infection
(12 h p.i.), while large amounts of NS2-3 were expressed at 12 h
and 20 h p.i. Taken together, the results show that attenuation
of cytopathogenicity of BVDV CP7 at 39.5°C caused by the
Y1338H substitution correlated with reduced viral RNA syn-
FIG. 3. Genetic basis of the ts phenotype of BVDV TS2.7. (A) Genetic characterization of TS2.7. A schematic representation of the genome
organization of the parental BVDV strain CP7 is shown on the top. Nucleotide sequencing of the entire genome of BVDV TS2.7 and subsequent
comparison with the sequence of BVDV CP7 led to identification of five nucleotide changes at positions 1139, 3575, 4394, 10156, and 10542. The
resulting four amino acid changes are indicated on the bottom. The mutation at position 4394 together with the resulting substitution of a tyrosine
(Y) residue by a histidine (H) residue at position 1338 of the CP7 polyprotein is highlighted by a box. (B) The ts phenotype of BVDV mutant
CP7-Y1338 carrying the point mutation at position 4394 in the CP7 genome. Plaque assays of cells infected with BVDV CP7-Y1338H at 33°C
resulted in detection of plaques (top), while infection at 39.5°C led to production of foci (bottom). Plaques and foci were visualized by
immunostaining 7 days after infection of cells.
VOL. 83, 2009 BVDV NS2 MUTATION ATTENUATES VIRAL CYTOPATHOGENICITY12419
thesis but that production of NS3 was not affected in a tem-
The existence of two biotypes represented by cp and ncp
viruses is a particularly interesting feature of pestiviruses. In
this study we describe isolation and characterization of a ts
mutant of cp BVDV strain CP7 whose ability to cause cy-
topathogenicity at 39.5°C is strongly reduced despite effi-
cient viral propagation. This is the first report on a BVDV
mutant which differs from the parental virus by tempera-
ture-dependent attenuation of viral cytopathogenicity. Our
analysis demonstrated that the genetic basis of this unique
phenotype is represented by a single tyrosine-to-histidine
substitution (Y1338H) at residue 193 of NS2. An alignment of
partial NS2 protein sequences of a representative set of pesti-
viruses showed that Y1338 is highly conserved among pestivi-
rus species BVDV-1, BVDV-2, BDV, and the tentative species
“Giraffe” but not among CSFV and a pestivirus isolated from
a pronghorn antelope (Fig. 5).
A common characteristic of cp BVDV strains is the efficient
production of NS3 throughout infection, which represents a
major difference from ncp BVDV (15, 18, 35, 40). Cytopatho-
genic BVDV strains can use various strategies for expression of
NS3, including processing of the precursor protein NS2-3 (1, 9,
24, 32–34, 36, 37, 42, 49). This strategy, which is used by BVDV
CP7 and some other cp BVDV strains, depends on the auto-
proteolytic activity of NS2 (26). Studies of cp BVDV strain
Oregon demonstrated that point mutations within NS2 can
influence processing of NS2-3 (24, 25). Mutants of BVDV
Oregon carrying point mutations in NS2 which significantly
reduced NS2-3 processing were either nonviable or propa-
gated much more slowly and rapidly reverted to viruses,
allowing efficient NS2-3 cleavage. These viruses apparently
caused cytopathogenicity only after reversion (25). The re-
sults of these studies significantly contributed to the conclu-
sion that cytopathogenicity of BVDV correlates with in-
creased NS3 expression and efficient viral replication. It was
therefore expected that attenuation of cytopathogenicity of
TS2.7 correlates with reduced processing of NS2-3 at high
temperature. However, the results of our study showed that,
irrespective of the incubation temperature used, infections
with the original ts mutant TS2.7 and the reconstructed
mutant CP7-Y1338H resulted in expression of substantial
FIG. 4. Characterization of BVDV CP7-Y1338H. (A) Growth kinetics of BVDV CP7 and the genetically engineered ts mutant CP7-Y1338H
at 33°C (left) and 39.5°C (right). MDBK cells were infected at an MOI of 0.1 with supernatants of the second cell culture passages of the indicated
viruses. The titers of released virus were determined by IF analysis over a time period of 72 h. Error bars indicate the ? ranges from quadruplicate
titrations. (B) Relative amounts of accumulated viral genomic RNAs obtained after infection of MDBK cells with BVDV CP7, TS2.7, and
CP7-Y1338H at 33°C (left) and 39.5°C (right). For infection an MOI of 1 was used. Total cellular RNAs were extracted at 10 h p.i. and subjected
to quantitative BVDV-specific real-time RT-PCR. Results are indicated as percentages of the mean value obtained for reference strain CP7
(100%). Data show the means ? standard deviation ranges from two independent experiments, each analyzed by the measurement of triplicates.
(C) Immunoblot. MDBK cells infected with CP7, NCP7, TS2.7, and CP7-Y1338H at 33°C (left side) and 39.5°C (right side) were lysed 12 and 20 h
p.i. (h.p.i.). For infection an MOI of 2 was used. The samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8%
polyacrylamide) under reducing conditions, transferred to nitrocellulose, and incubated with the anti-NS3 monoclonal antibody 8.12.7 (15).
Noninfected (n.i.) cells served as negative controls. The sizes (in kDa) of the molecular mass marker proteins (in thousands) are indicated on the
left. The positions of NS2-3 and NS3 are indicated on the right.
12420 PANKRAZ ET AL. J. VIROL.
amounts of NS3 (Fig. 4C). This observation uncouples cy-
topathogenicity of BVDV from efficient production of NS3
at early and late phases of infection and shows that a single
point mutation in NS2 can suppress cytopathogenicity of
BVDV CP7 at 39.5°C without compromising NS2-3 process-
ing in a temperature-dependent manner.
Another major difference between cp and ncp BVDV con-
cerns the efficiency of viral RNA synthesis. Analyses of several
BVDV pairs, including the isogenic pair consisting of CP7 and
NCP7, have shown that the cp viruses produce amounts of viral
RNA about 5- to 10-fold larger than their ncp counterparts (7,
32, 50). The ability of the ts mutants described here to produce
large amounts of viral RNA at 39.5°C was significantly reduced
(Fig. 4B). Accordingly, attenuation of viral cytopathogenicity
correlated with reduced amounts of accumulated viral RNAs.
This result supports the hypothesis that synthesis of large
amounts of viral RNA, including double-stranded RNA, trig-
gers cp BVDV-induced apoptosis (31, 54).
Apart from the work described here, there is one other
report describing isolation and characterization of several ncp
variants of a cp BVDV strain (NADL) (43). Similar to the
ts mutant TS2.7, these ncp variants expressed substantial
amounts of NS3 and produced slightly elevated accumulation
of NS2-3 relative to that of NS3. However, for these ncp
variants of NADL, attenuation of cytopathogenicity did not
correlate with significantly reduced amounts of viral RNA, as
was observed for TS2.7 and CP7-Y1338H. For all these ncp
variants, a substitution of a tyrosine residue at position 15
of NS4B by a cysteine residue was responsible for the change
of biotype (43). The results of this study with the ncp vari-
ants of BVDV NADL and our study demonstrate that efficient
production of NS3 throughout infection does not inevitably
result in a CPE and that at least two viral NS proteins, NS4B
and NS2, can attenuate viral cytopathogenicity. While NS4B
represents an essential component of the viral RNA replica-
tion complex, NS2 is dispensable for viral RNA replication.
Furthermore, analyses of several naturally occurring and ge-
netically engineered cp BVDV replicon RNAs lacking the NS2
coding region of the viral genome have shown that NS2 is also
not required for induction of viral cytopathogenicity (7, 10, 49).
This suggests that the Y1338H mutation identified in NS2 of
TS2.7 causes induction of an antiapoptotic function of NS2
rather than suppression of a proapoptotic function residing in
NS2. The mechanism by which NS2 of TS2.7 attenuates cyto-
pathogenicity remains to be determined. It can be speculated
that the Y1338H mutation results in a temperature-dependent
alteration of the NS2 structure and that the respective struc-
tural change modulates the interaction of NS2 with viral or
In the field of virology ts mutants have been exploited ex-
tensively to study biological processes. For many plus-strand
RNA viruses, including members of the Togaviridae, Picorna-
viridae, Coronaviridae, and Flaviviridae, a number of ts mutants
have been described (references 11, 14, 19, 23, 30, 38, 41, 45,
and 47 and references therein). To our knowledge, only one
other ts pestivirus (cp BVDV strain RIT 4350) has been re-
ported so far (29). The genome of RIT 4350 harbors two
cellular insertions and a large duplication of viral sequences
(6). While these genomic alterations were shown to be respon-
sible for expression of NS3 and induction of cytopathogenicity,
the molecular basis of the ts phenotype remains unknown (6,
7). In contrast to the yield of the ts mutants described here, the
virus yield obtained after infection of cells with RIT 4350 at
high temperature was significantly reduced by at least two
orders of magnitudes (29). Our own evaluation of the virus
titers produced after infection with RIT 4350 confirmed that its
ts phenotype is characterized by a significant reduction of virus
propagation at nonpermissive temperature (data not shown).
Irrespective of the incubation temperature used, the infectious
virus titers of RIT 4350 determined by plaque assays were
identical to the titers obtained by subsequent IF analysis. Thus,
the phenotype of the ts mutants TS2.7 and CP7-Y1338H is
clearly different from that of BVDV RIT 4350.
In this study a novel ts mutant of a cp BVDV strain exhib-
iting a unique phenotype was isolated and characterized. The
results of our analyses showed that a single point mutation in
NS2 interferes with viral RNA synthesis in a temperature-
dependent manner and attenuates BVDV-induced cytopatho-
genicity despite efficient production of NS3 and high levels of
infectious virus yield. Thus, NS2 can play an unexpected role in
attenuation of BVDV cytopathogenicity. Accordingly, this
study provided further insights into the role of NS proteins
NS2 and NS3 in viral cytopathogenicity. Future experiments
will concentrate on the mechanism by which NS2 of TS2.7
causes the ts attenuation of cytopathogenicity. Such studies will
FIG. 5. Y1338 of NS2 is highly conserved among pestivirus species
BVDV-1, BVDV-2, BDV, and “Giraffe” but not among CSFV and a
pestivirus from a pronghorn antelope. Fragments of polyproteins (of a
representative set of 23 pestiviruses) that include amino acid Y1338
(highlighted by a black box and white letters) were aligned (see Ma-
terials and Methods). The virus strains (and their GenBank accession
numbers) are BVDV-1 strains NADL (M31182), Osloss (M96687),
NCP7 (U63513), SD-1 (M96751), Oregon (AF041040), KS86-1ncp
(AB078950), ZM-95 (AF526381), and ILLC (U86599); BVDV-2
strains 890 (U18059), NY93 (AF502339), p24515 (AY149216), p11Q
(AY149215), 1373 (AF145967), and C413 (AF002227); BDV strains
X818 (AF037405), BD-31 (U70263), and Reindeer-1 (AF144618); pes-
tivirus strain Giraffe-1 (NC_003678); CSFV strains Alfort-T (J04358),
Paderborn (AY072924), C-strain (Z46258), and Brescia (P21530); and
a pestivirus from pronghorn antelope (AAX12371).
VOL. 83, 2009 BVDV NS2 MUTATION ATTENUATES VIRAL CYTOPATHOGENICITY12421
contribute to our understanding of the molecular basis of pes-
We thank M. Orlich for excellent technical assistance in the initial
stage of the project.
This study was supported by Sonderforschungsbereich 535, Invasion
Mechanisms and Replication Strategies of Infectious Agents (project
B8), and grant BE 2333/2-1 from the Deutsche Forschungsgemein-
schaft (DFG). P.B. is supported by a Heisenberg professorship from
the DFG (BE 2333/1-1).
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