Previous Article | Next Article 
J Virol, May 1998, p. 4139-4148, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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
Gregor
Meyers,1,*
Dieter
Stoll,2 and
Michael
Gunn3
Department of Clinical Virology, Federal
Research Centre for Virus Diseases of Animals, D-72001
Tübingen,1 and
Naturwissenshaftliches und Medizinisches Institut an der
Universität Tübingen in Reutlingen, D-72762
Reutlingen,2 Germany, and
Veterinary
Research Laboratory, Abbotstown, Castleknock, Dublin 15, Ireland3
Received 25 November 1997/Accepted 10 February 1998
 |
ABSTRACT |
Pestiviruses represent the first RNA viruses for which
recombination with cellular protein-coding sequences has been reported. As a result of such recombinations cytopathogenic (cp) pestiviruses can
develop from noncytopathogenic (noncp) viruses. In the case of
bovine viral diarrhea virus (BVDV), the generation of cp mutants is
linked to the induction of the lethal syndrome mucosal disease (MD) in
cattle. The cp BVDV JaCP was isolated from an animal which had come
down with MD. The genome of JaCP contains a novel kind of cellular
insertion (LC3*) which is flanked by duplicated pestivirus sequences.
Neither insertion nor duplication is present in the genome of the
accompanying noncp virus JaNCP. As part of the viral polyprotein, the
insertion in the JaCP genome is translated into a polypeptide almost
identical to a fragment of light chain 3, a subunit of the
microtubule-associated proteins 1A and 1B from the rat.
Transient-expression studies revealed that the LC3* sequence is able to
induce an additional cleavage of the viral polyprotein. The respective
cleavage occurs directly downstream of the LC3*-encoded sequence and is
not dependent on the NS3 serine protease. Insertion of LC3* into an
infectious noncp pestivirus cDNA clone without duplicated viral
sequences resulted in recovery of a defective cp virus able to
replicate only in the presence of a noncp helper virus. In contrast,
introduction of both insertion and duplication led to an autonomously
replicating cp virus.
| |
INTRODUCTION |
Economically important diseases of
farm animals such as classical swine fever and bovine viral diarrhea
are caused by pestiviruses, which together with flaviviruses and
hepatitis C virus constitute the family Flaviviridae (for
reviews see references 35 and
45). Pestiviruses have a positive-sense
single-stranded RNA genome of about 12.5 kb, which consists of 5' and
3' noncoding regions flanking a long open reading frame. The genomic
RNA is translated into a polyprotein of approximately 3,900 amino acids
(aa). Processing by host and virus-encoded proteases leads to the
mature virus proteins arranged in the polyprotein in the order
NH2-Npro-C-Erns-E1-E2-p7-NS2-3-NS4A-NS4B-NS5A-NS5B-COOH.
C, Erns, E1, and E2 are present in pestivirus virions,
whereas the other polypeptides represent nonstructural proteins
(4, 6, 11, 36, 40, 41, 46, 50).
The most severe clinical condition resulting from infection with the
pestivirus bovine viral diarrhea virus (BVDV) is called mucosal disease
(MD) (45). The etiology of MD has been elucidated only
recently. Two biotypes of BVDV, cytopathogenic (cp) and
noncytopathogenic (noncp) viruses, are required for induction of
this lethal disease. In a first step, intrauterine infection with
a noncp BVDV that leads to induction of specific immunotolerance and
birth of persistently infected calves has to occur (45).
Such animals frequently go down with MD early in life; the disease is
either induced by superinfection with an antigenically closely related
cp BVDV or by generation of a cp mutant of the persisting noncp virus
(24, 25). This switch from a noncp to a cp phenotype is in
most cases the result of RNA recombination (reviewed in reference
31). Recombination can occur between the noncp BVDV
genome and RNAs of viral or cellular origin (26, 29-31, 34,
42-44). So far, two types of cellular sequences, which code for
ubiquitin or a protein of unknown function, have been identified in
pestivirus RNAs (2, 27, 29-31, 34, 43). We report here the
identification of a third type of cellular insertion and
present an analysis of its effects on polyprotein processing and
virus phenotype.
| |
MATERIALS AND METHODS |
Cells and viruses.
MDBK cells and BVDV isolate NADL were
obtained from the American Type Culture Collection (Rockville, Md.).
BVDV JaCP and JaNCP have been isolated from the serum of a bullock
named Jasper, which developed MD while housed in isolation
(14). The cp virus, JaCP, was cloned twice by plaque
purification, and the noncp virus, JaNCP, was obtained by two rounds of
limiting dilution. MDBK cells were grown in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS)
and nonessential amino acids.
Infection of cells.
Since pestiviruses tend to be associated
with the host cells, suspensions composed of cell culture supernatant
and infected cell lysate were used for infection of culture cells.
Material for infection was prepared by freezing and thawing cultures
48 h postinfection and was stored at 
70°C. If not specified, a
multiplicity of infection of ~0.1 was used. Infection with noncp BVDV
was detected by immunofluorescence with a bovine hyperimmune serum.
Northern (RNA) hybridization.
RNA preparation by the
guanidine isothiocyanate method, glyoxylation of RNA, and
electrophoresis through 1% agarose gels containing formaldehyde were
carried out as described before (30). Following electrophoresis, the RNA was transferred to nylon membranes (Duralon; Stratagene, Heidelberg, Germany) according to standard procedures (37). Hybridization with radioactive probes labeled by nick translation (Nick Translation Kit; Amersham and Buchler, Braunschweig, Germany) at 54°C and posthybridization washes at the same temperature were carried out as described before (30). The cDNA insert
of BVDV clone NCII.1 (29) was used as a probe.
cDNA synthesis, cloning, and nucleotide sequencing.
Establishment of cDNA libraries in lambda ZAPII (Stratagene) was done
as described before (29). Briefly, total RNA of cells infected with JaCP or JaNCP, respectively, was used for cDNA synthesis primed with oligonucleotides Ol-BVDV13, Ol-BVDV14, and Ol-Pes9 (29). Second-strand synthesis and ligation of
EcoRI adaptors was done with the You-Prime cDNA synthesis
kit as suggested by the supplier (Pharmacia, Freiburg, Germany). Size
selection of double-stranded cDNA for molecules larger than 2 kb was
done by preparative agarose gel electrophoresis as previously described (29). Ligation of cDNA fragments with lambda ZAPII DNA,
packaging with Gigapack III-Gold, and plating on Escherichia
coli XL-Blue1 cells were done as recommended by the manufacturer
(Stratagene). Screening was done with the same probe used for Northern
hybridization. Subcloning of cDNA fragments into pBluescript plasmids
by in vivo excision was performed as recommended by the supplier
(Stratagene). Exonuclease III and S1 were used to establish deletion
libraries of cDNA clones (16). Dideoxy sequencing
(38) of double-stranded DNA templates was carried out by
using the T7 sequencing kit (Pharmacia). Computer analysis of sequence
data was performed with the Genetics Computer Group software
(9).
From a variety of cDNA clones derived from RNA of cells infected with
BVDV JaCP, pJaCP/A17 and pJaCP/A19 were chosen for sequencing. The
insert of pJaCP/A17 has a length of about 1.6 kb; the 5' and 3' ends of
the cDNA fragment correspond to positions 7096 and 5808, respectively,
in the published sequence of noncp BVDV SD-1 (8). Clone
pJaCP/A19 contains an insert of about 2.8 kb which starts at position
5815 and ends at position 5808 compared to BVDV SD-1. The sequence data
have been obtained by sequencing both strands of the two clones and
deposited at the EMBL/GenBank data libraries under accession no.
U80885.
Clone pJaNCP35 was isolated from the library established with RNA of
cells infected with BVDV JaNCP. The insert in this plasmid has a length
of 4.6 kb and corresponds to positions 2051 to 6814 of the BVDV genome.
From this cDNA fragment a region of only 0.3 kb, extending from
position 5142 to 5439, was sequenced.
Construction of clones for transient expression and recovery of
infectious virus.
Restriction, subcloning and other standard
procedures were done essentially as described previously
(37). Restriction and modifying enzymes were purchased
from New England Biolabs (Schwalbach, Germany), Pharmacia, and
Boehringer-Mannheim (Mannheim, Germany). Plasmid pACYC177 was
obtained from NEB. To obtain pEx7, a 3.1-kb NcoI/SalI fragment of pA/BVDV (28) was
cloned into pCITE-2A (Angewandte Gentechnologie Systeme GmbH,
Heidelberg, Germany); the cDNA fragment does not contain the
27-nucleotide insertion responsible for NS2-3 cleavage and
cytopathogenicity of BVDV CP7 (42). Plasmids pEx7/JaCP and
pEx7/JaNCP were established by exchanging a 0.3-kb
BamHI/HpaI fragment of pEx7 for equivalent fragments of 0.65 and 0.3 kb from cDNA clones pJaCP/A17 and pJaNCP35, respectively. The former fragment contains the LC3* insertion, whereas
the latter is colinear with the BVDV CP7 sequence. The position of the
BamHI site corresponds to residues 5142 to 5147 of the
BVDV SD-1 genome, and the HpaI site is located at positions 5434 to 5439. Similarly, pEx7/JaCP and pEx7/JaNCP, each with a destroyed NS3 protease, were generated by insertion of the appropriate BamHI/HpaI fragment into a pEx7 homolog in which
the triplet encoding the active-site serine of the NS3 protease had
been exchanged for an alanine codon.
The full-length constructs pA/B-JaCP and pA/B-JaNCP were obtained by
exchanging the
NcoI/
SalI fragment in
pA/BVDV/Ins
for
the corresponding fragments of pEx7/JaCP and
pEx7/JaNCP, respectively.
By starting with pA/B-JaCP, pA/BVDV/Ins
was reconstructed by
deletion of a 0.45-kb
NotI/
SacI fragment together with a 1-kb
SacI fragment and insertion of a 1-kb
NotI/
SacI fragment derived
from pA/BVDV/Ins
.
To obtain full-length clones containing the duplication detected in the
genome of BVDV JaCP, pEx7/JaCP was restricted with
SacI and
PstI. The latter enzyme cuts in the multiple-cloning
site of
the vector. Integration of a
SacI/
BamHI fragment
from
cDNA clone pJaCP/A19 together with the
BamHI/
PstI fragment from
pEx7/JaCP or pEx7/JaNCP
resulted in plasmids pEx7/JaCP/dp or pEx7/JaNCP/dp,
respectively. To
establish the full-length construct pA/B-JaCP/dp
harboring the
JaCP-specific duplication of viral sequences, the
NcoI/
SalI fragment of pEx7/JaCP/dp was used to
replace the corresponding
fragment in pA/BVDV/Ins
. Similarly,
pA/B-JaNCP/dp was constructed
by starting with pEx7/JaNCP/dp and
pA/BVDV/Ins
.
Plasmid pEx7
was generated by cutting pEx7 with
HpaI and
XhoI, followed by end filling with Klenow polymerase and
religation.
The BVDV-derived cDNA fragment of the resulting construct
corresponds
to positions 4648 to 5436 of the BVDV SD-1 genome. The
equivalent
clones containing sequences derived from BVDV JaCP or JaNCP
(pEx7/JaCP
or pEx7/JaNCP
, respectively) were constructed the same
way, starting
with plasmids pEx7/JaCP or pEx7/JaNCP, respectively.
Site-directed mutagenesis.
Mutagenesis according to the
method of Kunkel et al. (21) was done with the Muta-Gene
Phagemid in vitro-mutagenesis kit (Bio-Rad, Munich, Germany)
essentially as recommended by the manufacturer, with the exception that
single strands were produced with the filamentous phage VCSM13
(Stratagene). The presence of the desired mutations was verified by
nucleotide sequencing. For mutagenesis, single-stranded DNA of pEx7 was
produced and annealed with oligonucleotide Ol-C7/S-A and the
mutagenized strand was completed by incubation with T4 DNA polymerase
and T4 ligase in synthesis buffer using the materials supplied with the
mutagenesis kit.
The sequence of the oligonucleotide used for mutagenesis of the
active-site serine codon of the NS3 protease gene to an alanine
codon,
Ol-C7/S-A, was 5'-TGAAGGATGGGCGGGTCTACCCAT-3'.
Transient expression, metabolic labeling, immunoprecipitation,
and SDS-PAGE.
Transient expression of transfected plasmids using
vaccinia virus vTF7-3 (kindly provided by B. Moss
[12]) was done as described before (44),
except that labeling time was reduced to 5 h. BVDV-infected MDBK
cells (1.5 × 106 per 3.5-cm-diameter dish) were
labeled for 8 h with 0.5 mCi/ml of
[35S]methionine-[35S]cysteine (Promix;
Amersham). Nonradioactive cysteine and methionine were absent from the
labeling medium. Cell extracts were prepared under denaturing
conditions (15). Extracts were incubated with 5 µl of
undiluted serum. Precipitates were formed with cross-linked Staphylococcus aureus (18), analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and
processed by fluorography using En3Hance (New England
Nuclear, Boston, Mass.). For analyses of proteins with molecular masses
of up to 60 kDa, Tricine gels prepared according to the method of
Schägger and Jagow (39) were used, while larger proteins were analyzed by electrophoresis performed according to the
method of Doucet and Trifaro (10).
The following antisera were used for detection of BVDV proteins. (i)
Antiserum A3 was generated against a bacterial fusion
protein
encompassing sequences of CSFV Alfort/Tübingen and recognizes
NS2-3 and NS3 (
46). (ii) Antiserum pep6 was raised against a
peptide corresponding to residues 1571 to 1586 of the BVDV CP7
polyprotein and reacts with NS2 and NS2-3 (
42). (iii)
Antiserum
P1 was raised against a bacterial fusion protein containing
sequences
of CSFV Alfort/Tübingen and recognizes both NS4A and
NS4B (
30).
In vitro transcription and RNA transfection.
cDNA constructs
(2 µg) were linearized with SmaI (full-length BVDV clones,
partial cut for pA/B-JaCP/dp and pA/B-JaNCP/dp) or PvuII
(pEx7 and equivalent constructs) and purified by phenol extraction
and ethanol precipitation. Transcription with T7 RNA polymerase (NEB)
was carried out in a total volume of 50 µl of transcription mix (40 mM Tris-HCl, pH 7.5; 6 mM MgCl2; 2 mM spermidine; 10 mM
NaCl; 0.5 mM [each] ATP, GTP, CTP, and UTP; 10 mM dithiothreitol; 100 µg of bovine serum albumin per ml) with 50 U of T7 RNA polymerase in
the presence of 15 U of RNAguard (Pharmacia). After incubation at
37°C for 1 h, the reaction mixture was passed through a Sephadex G-50 spun column (37) and further purified by phenol
extraction and ethanol precipitation.
Transfection was done with a suspension of 3 × 10
6
MDBK cells and about 100 ng of in vitro-transcribed RNA bound to
DEAE-dextran
(Pharmacia). The RNA-DEAE-dextran complex was established
by mixing
RNA dissolved in 100 µl of HBSS (
47) with 100 µl of DEAE-dextran
(1 mg/ml in Hanks balanced salt solution [HBSS])
and incubating
the mixture for 30 min on ice (
32). Pelleted
cells were washed
once with DMEM without FCS, centrifuged, and then
resuspended
in the RNA-DEAE-dextran mixture. After 30 min of
incubation at
37°C, 20 µl of dimethyl sulfoxide was added and the
mixture was
incubated for 2 min at room temperature. After the addition
of
2 ml of HBSS, cells were pelleted and washed once with HBSS and
once
with medium without FCS. Cells were resuspended in DMEM with
FCS and
seeded in a 10.0-cm-diameter dish. At 48 to 72 h posttransfection,
cells were apportioned and seeded as appropriate for subsequent
analyses.
In vitro translation.
Translation of in vitro-transcribed
RNA was done with nuclease-treated rabbit reticulocyte lysate (RRL)
(Promega, Heidelberg, Germany). The reaction was carried out in the
presence or absence of microsomal membranes (1.8 µl; Promega) with
0.5 µg of RNA in a total volume of 25 µl in the presence of
[35S]methionine (Amersham), according to the
manufacturer's suggestions. For analysis of the translation products,
10% of the reaction mix was separated in a 10% acrylamide gel
(39).
N-terminal sequence analysis of radiolabeled NS3.
The NS3
protein used for radiosequencing was transiently expressed from
pEx7/JaCP by using vaccinia virus vTF7-3 (see above). Labeling was
carried out with 500 µCi of [35S]cysteine and about
106 cells in 0.5 µl of labeling medium. The protein was
immunoprecipitated with antiserum A3, separated by SDS-PAGE, and
transferred to an Immobilon polyvinylidene difluoride membrane
(Millipore, Eschborn, Germany). The protein was localized by
autoradiography and subjected to automated Edman degradation.
| |
RESULTS |
Virus isolation and genome analysis.
To investigate putative
virus recombinants involved in the development of MD, a
persistently infected bullock named Jasper was housed under
isolated conditions from the age of 1 year until the clinical
syndrome developed over 2 years later (14). The animal was
euthanatized when in extremis, and cp as well as noncp BVDV was
isolated from serum samples, biologically cloned, and further analyzed.
In a Northern blot hybridization, the RNA of the noncp virus, JaNCP,
comigrated with the genome of the BVDV reference strain, NADL, with a
size of 12.5 kb (5). In contrast, the genomic RNA of JaCP
was found to have a length of more than 14 kb (Fig.
1). This finding is reminiscent of other
cp BVDV isolates, such as CP1 or III-C, which contain ubiquitin-coding
cellular insertions that are flanked by large duplications of viral RNA (29, 31, 34). However, the genome of JaCP did not hybridize to a ubiquitin probe or to the second known cellular insert (cIns) (data not shown).
View larger version (43K):
[in this window]
[in a new window]
|
FIG. 1.
Northern blot with RNA from bovine kidney (MDBK) cells
infected with the indicated viruses. An RNA ladder served as a size
marker (in kilobases). Control, RNA from noninfected MDBK cells.
|
|
After cDNA cloning and sequencing, a large duplication of viral
sequences and a nonviral insertion were identified in the
genome of
JaCP. The 5' part of the determined sequence is colinear
with a noncp
BVDV genome from residue 5815 to 7517. The latter
position
(C
5 in Fig.
2A) is located in
the NS4B gene. Residue
C
5 is followed by a fragment derived
from the NS2 gene, starting
with position 5057 (F") and ending with
position 5152 (position
A). Downstream of position A is located a
nonviral insertion which
is followed by the viral sequence
starting with position B (Fig.
2A). Position B corresponds to
nucleotide 5153 of a BVDV genome
without rearrangement. Taken
together, it can be concluded that
the JaCP genome contains a
duplication of 2,460 nucleotides.
View larger version (47K):
[in this window]
[in a new window]
|
FIG. 2.
(A) Genome organization of BVDV JaCP (bottom) compared
to that of a noncp BVDV (top) and BVDV isolates Osloss, CP1, and III-C,
which contain ubiquitin-coding insertions. For CP1 and III-C,
duplicated viral sequences were found in addition to the cellular
insertion (29, 34). The viral genomes are indicated as bars.
The regions coding for NS2-3 or NS2-3-derived polypeptides are shaded.
Important genomic positions flanking insertions or representing the
start or end of duplicated sequences are labeled with letters as in
reference 31. The genome of BVDV CP1 contains a
duplication of the sequence located between B and C. In the RNAs of
BVDV III-C or JaCP, the regions flanked by F' and C4 or F"
and C5, respectively, are duplicated. In both cases the
duplicated region located closer to the genomic 3' end contains the
cellular insertion. Note that A and B mark the equivalent genomic
position in all five genomes. Hatched bar, ubiquitin-coding insertion
(Ub); cross-hatched bar, insertion homologous to the rat LC3 sequence
(LC3*). (B) Comparison of the sequence published for rat LC3 of MAPs 1A
and 1B (23) with the amino acid sequence encoded by the
nonviral insertion identified in the RNA of JaCP. For JaCP, the
sequence encoded by the insertion is shown in capital letters while the
flanking regions are given in lowercase letters. The translational stop
of rat LC3 is symbolized by an asterisk.
|
|
The genome organization of BVDV JaCP as deduced from these data is very
similar to that proposed for BVDV III-C. Both virus
genomes contain a
cellular insertion integrated into a 5'-terminally
truncated version of
the NS2-3 gene that has been duplicated in
the course of the
recombination. In contrast to BVDV JaCP and
C-III, the genome of cp
BVDV CP1 contains no duplicated NS2 sequences
and the RNA of cp BVDV
Osloss contains a ubiquitin-coding insertion
but no duplication at all
(Fig.
2A). Position B is not only conserved
as an integration site in
the genomes of BVDV JaCP and III-C but
represents the crossing-over
site for all viruses containing ubiquitin-coding
insertions and also
for most pestiviruses with duplicated and
rearranged viral sequences
(
31). Position B is regarded as the
5' end of the NS3 gene.
The nonviral insertion in the JaCP RNA has a length of 348 nucleotides
and shows no homology to ubiquitin genes or cIns, the
other cellular
sequence known to be present in the genomes of
cp pestiviruses. A
search in data libraries revealed 84% identity
between the insertion
and a rat cDNA sequence coding for light
chain 3 (LC3), a subunit of
the neuronal microtubule-associated
proteins (MAPs) 1A and 1B
(
23); the deduced amino acid sequences
exhibit 98%
identity. The JaCP insertion (LC3*) encompasses nucleotides
39 to 386 of the published LC3 sequence. LC3* has been integrated
into the viral
genome in the same reading frame as in the LC3
mRNA. Thus, a viral
polyprotein is expressed that contains aa
5 to 120 of LC3 (Fig.
2B).
Protein analysis.
The genomes of most cp BVDV isolates exhibit
individual rearrangements. In all cases, these rearrangements affect
the genomic region coding for the nonstructural viral protein NS2-3, a
chymotrypsin-like serine protease that also has helicase function
(1, 13, 48, 49). The protease identified in NS2-3 is
responsible for cleavage at its own carboxy terminus and the
nonstructural protein sites 4A/4B, 4B/5A, and 5A/5B (41,
50). In consequence of the rearrangements, processing of cp BVDV
polyproteins yields NS3, which is not present in cells infected with
non-cp viruses. Either NS3 is generated by cleavage of an NS2-3 protein
containing, e.g., a ubiquitin insertion, or it is expressed from
duplicated sequences which again contain an insertion (29-31, 43,
44) (Fig. 2A, BVDV CP1). Also, in the case of BVDV JaCP,
expression of NS3 was observed in addition to that of NS2-3, whereas in
JaNCP-infected cells, only NS2-3 could be detected (data not shown). To
analyze whether LC3* is responsible for the generation of NS3, this
sequence was integrated at the appropriate site into pEx7, a plasmid
that was originally designed for expression of proteins from BVDV CP7. pEx7 codes for aa 1422 to 2447 of the BVDV polyprotein (numbers are
according to the sequence of BVDV SD1 [8]). The
amino-terminally truncated NS2-3 expressed from this construct is not
processed (Fig. 3). To integrate the LC3*
insertion, a BamHI/HpaI fragment from pEx7 was
exchanged for the corresponding fragment from cDNA clone pJaCP/A17. The
resulting construct, pEx7/JaCP encodes a polypeptide that contains the
cellular sequence and in addition exhibits four amino acid exchanges in
the NS2-3 region. As a control, a third construct was assembled from
pEx7 and cDNA clone pJaNCP35; the resulting plasmid, pEx7/JaNCP, lacks
the cellular insert but contains the same mutations as pEx7/JaCP. Thus,
the only difference between the polyproteins encoded by the last two of
these constructs is the absence or presence of the LC3* insertion,
respectively.
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 3.
Immunoprecipitation of proteins transiently expressed
from cDNA constructs pEx7, pEx7/JaNCP, and pEx7/JaCP. The constructs
encompass the genomic region coding for the carboxy-terminal half of
NS2 together with NS3, NS4A, and the amino-terminal half of NS4B
(codons 1422 to 2447 of the open reading frame). The upper panels show
the proteins precipitated with an antiserum directed against
nonstructural protein NS3 (antiserum A3 [46]) analyzed
on a 12% gel according to the method of Doucet and Trifaro
(10) (A) and the polypeptides recognized by an antiserum
directed against NS2 (antiserum pep6 [42]) separated
on a 10% gel according to the method of Schägger and Jagow
(39) (B). Numbers on the left side of each gel indicate the
molecular masses (in kilodaltons) of marker proteins, whereas the
letters and arrows on the right side mark the important bands.
The panels on the bottom show schematic presentations of the
different expression products. NS2*, aminoterminally truncated NS2;
LC3*, insertion derived from the cellular LC3 of MAPs 1A and 1B
control, precipitation of proteins extracted from cells
infected with vaccinia virus vTF7-3 (12).
|
|
The proteins derived from the three constructs were analyzed by
transient expression using vaccinia virus vTF7-3 (
12) and
precipitation with antisera directed against NS2 or NS3, respectively
(
42,
46). For pEx7 and pEx7/JaNCP, further processing of the
amino-terminally truncated NS2-3 protein was not observed; only
one
specific band of ~92 kDa precipitated with the antisera against
NS2
and NS3 (bands B in Fig.
3). In contrast, the same protein
containing
the LC3* polypeptide was efficiently processed to yield
NS3 (band C in
Fig.
3A); only a rather small proportion of the
expressed protein
remained uncleaved (band A in Fig.
3A). Using
the anti-pep6 serum that
is directed against the assumed carboxy-terminal
part of NS2
(
42) the amino-terminal cleavage product of 32 kDa
was
detected in addition to the uncleaved product (bands D and
A,
respectively, in Fig.
3B). The reaction of this cleavage product
with
the anti-pep6 serum, its size, and the fact that the obtained
NS3
protein comigrated with NS3 from cp BVDV isolates (not shown)
strongly
indicate that the 32-kDa protein contains at least the
majority of the
LC3*-encoded polypeptide.
For cp BVDV isolates containing ubiquitin-coding insertions, host
ubiquitin-specific proteases were found to cleave at the
carboxy
terminus of the cellular sequence and thus liberate the
amino terminus
of NS3 (
43). The genomes of the second type of
cp strains
contain duplicated N
pro genes; in these cases, the
autoproteolytic activity of N
pro generates the amino
terminus of NS3. However, for the other cp
BVD viruses, the protease
responsible for the cleavage at the
2-3 site is still not known. In
order to test whether the NS3
serine protease is involved in NS2-3
processing, mutants of pEx7/JaCP
and pEx7/JaNCP were generated that
encode, at a position corresponding
to aa 1752 of the polyprotein, an
alanine instead of a serine;
the latter amino acid represents the
active-site residue of the
NS3 protease (
1,
13,
49). After
expression of these constructs,
release of NS4A (band F in Fig.
4) or the carboxy-terminally truncated
NS4B encoded by the plasmids (band G in Fig.
4) was no longer
observed.
Instead, polypeptides of high molecular weight were
precipitated with
the respective antiserum for these two constructs.
Thus, the NS3
protease activity was indeed destroyed by the mutation.
Accordingly,
only the full-length expression product of ~115 kDa
was detected
after expression of pEx7/JaNCP harboring the protease
mutation (band B
in Fig.
4). However, the LC3*-dependent processing
of NS2-3 still
occurred, since for the mutated pEx7/JaCP both
the amino-terminally
truncated NS2/LC3* and a product of ~97 kDa
composed of NS3, NS4A,
and the truncated NS4B could be detected
in addition to the full-length
expression product (bands C, D,
and A, respectively, in Fig.
4). These
experiments show that the
NS3 serine protease itself is not involved in
the observed NS2-3
cleavage.
View larger version (42K):
[in this window]
[in a new window]
|
FIG. 4.
Immunoprecipitation of proteins transiently expressed
from cDNA constructs pEx7, pEx7/JaNCP, and pEx7/JaCP. In the latest two
of these constructs, the codon coding for the active-site serine of the
NS3 protease (position 1752 of the BVDV polyprotein) was exchanged for
an alanine codon. The panel on the upper-left side shows the results of
immunoprecipitations with the antiserum A3, directed against NS3
(46), analyzed on a 12% gel (10); the other two
panels present 10% gels (39) on which the polypeptides
recognized by the antiserum against NS2 (42) (middle) or the
antiserum P1 (30) (right), directed against NS4A/4B, were
separated. Numbers on the left side of each gel indicate the molecular
masses (in kilodaltons) of marker proteins, whereas the letters and
arrows on the right side mark the important bands. A schematic
presentation of the different expression products is shown in a box
below the gels. NS2*, amino-terminally truncated NS2; NS4B*,
carboxy-terminally shortened NS4B; LC3*, insertion derived from the
cellular LC3 of MAPs 1A and 1B. Control, precipitation of proteins
extracted from cells infected with vaccinia virus vTF7-3
(12).
|
|
In vitro translation studies.
To obtain further information on
the processing of NS2-3 in the presence of the LC3*-encoded sequence,
in vitro translation studies were conducted. The majority of the NS3
coding region was removed from pEx7, pEx7/JaCP, and pEx7/JaNCP by
deletion of the 3'-terminal 2.3 kb of the cDNA inserts, resulting in
constructs pEx7, pEx7/JaCP, and pEx7/JaNCP. Because of the
carboxy-terminal truncation of the encoded proteins, the serine residue
of the active center of the intrinsic serine protease was deleted from NS3. RNA was transcribed from these plasmids after linearization with
PvuII and was translated in RRL either in the absence or presence of canine microsomal membranes. The translation products were
analyzed by SDS-PAGE and subsequent fluorography. Proteins obtained by
transient vaccinia virus expression served as controls. For pEx7 and
pEx7/JaNCP, only one major translation product (29 kDa), which
comigrated with the polypeptide obtained after transient expression,
was detected (bands B in Fig. 5).
However, translation of the RNA transcribed from pEx7/JaCP yielded a
cleavage product of 31 kDa (band C in Fig. 5) in addition to the
full-length product (band A in Fig. 5). The second cleavage product,
representing the truncated NS3 protein of about 14 kDa, could not be
detected, probably because truncated NS3 proteins tend to be unstable
(unpublished observation). The detection of NS2-3 cleavage was not
dependent on the presence of microsomal membranes as observed in the
case of BVDV Oregon, a cp virus without recombination-induced genome alteration (20). In conclusion, the insertion of LC3* leads to expression of NS3 through mediating polyprotein cleavage by a not
yet identified protease that either is contained in the RRL or
represents an intrinsic activity of the expressed viral protein.
Moreover, the experiment showed that major parts of NS3 can be deleted
without deleterious effects on the cleavage.
View larger version (46K):
[in this window]
[in a new window]
|
FIG. 5.
Proteins obtained by in vitro translation of RNA
transcribed from cDNA constructs pEx7, pEx7/JaNCP, and
pEx7/JaCP, separated by 10% SDS-PAGE (39). The
translation was performed either in the absence () or presence (+) of
canine microsomal membranes (MM). Proteins obtained by
immunoprecipitation with the antiserum pep6 serum after T7-driven
(vTF7-3) transient expression of the same constructs in eucaryotic
cells served as controls. Numbers on the left side of the gel indicate
the molecular masses (in kilodaltons) of marker proteins, whereas the
letters and arrows on the right side mark the important bands. A
schematic presentation of the different expression products is shown in
the box below the gel. NS2*, amino-terminally truncated NS2; NS3*,
carboxy-terminally shortened NS3; LC3*, insertion derived from the
cellular LC3 of MAPs 1A and 1B.
|
|
N-terminal sequencing of NS3.
The results of the protein
analyses indicated that the processing of the truncated NS2-3 expressed
from pEx7/JaCP occurred very close to the carboxy-terminal end of the
LC3* encoded insertion. This conclusion is based on the size of the
precipitated proteins and the reactivity of the antiserum that is
directed against aa 1571 to 1586 of the BVDV polyprotein (antiserum
pep-6 [42]). To determine the cleavage site precisely,
NS3 transiently expressed from pEx7/JaCP and labeled with
[35S]cysteine was precipitated with the antiserum A3
serum, run through a polyacrylamide gel, transferred to an Immobilon
membrane, and subjected to 20 cycles of automated Edman degradation.
The radioactivity released in each degradation step was counted and
plotted, resulting in the curve shown in Fig.
6. Two peaks, at positions 5 and 14, were
detected. Within the protein encoded by pEx7/JaCP, such a spacing of
two cysteine residues is found only once, namely, at positions
corresponding to residues 1594 and 1603 of the BVDV polyprotein
(numbers refer to BVDV SD1 [8]). The LC3*-derived sequence is located in the pEx7/JaCP-encoded polypeptide as well as in
the JaCP polyprotein between an arginine and a glycine corresponding to
residues 1589 and 1590 of the BVDV polyprotein without insertion. Thus,
cleavage of the polypeptide expressed from pEx7/JaCP occurs between the
last residue of the LC3*-encoded sequence and the first amino acid of
the viral sequence downstream of the cellular insertion. The amino
terminus of the NS3 protein generated by this processing corresponds to
glycine 1590 of the BVDV polyprotein.
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 6.
N-terminal sequence of NS3 expressed from pEx7/JaCP. The
proteins transiently expressed from the cDNA construct were labeled
with [35S]cysteine and purified by immunoprecipitation
and SDS-PAGE. After transfer of the proteins to an Immobilon membrane,
NS3 was isolated and automated Edman degradation was performed. The
graph shows the counts per minute released per sequencing cycle. Above
the graph, the amino acids determined by sequencing (in boldface type
and underlined) and the flanking residues deduced from the pEx7/JaCP
sequence are shown.
|
|
RNA transfection experiments.
Expression of NS3 by cp BVDV is
correlated with induction of a cytopathic effect (CPE). In several
cases, a direct connection between genome rearrangement, expression of
NS3, and lysis of the infected cells could be demonstrated
(28). To test whether the insertion of LC3* is able to
convert a noncp BVDV into a cp virus, a full-length construct named
pA/B-JaCP was generated by insertion of the
NcoI/SalI insert of pEx7/JaCP into pA/BVDV/Ins cut with the same enzymes (Fig. 7A).
Construct pA/BVDV/Ins represents a cDNA clone from which infectious
noncp BVDV RNA can be transcribed (28). As a control, an
equivalent construct without the LC3* insertion was established by
using a fragment of pEx7/JaNCP; this plasmid was termed pA/B-JaNCP
(Fig. 7A). RNA was transcribed from the two plasmids and used for
transfection of MDBK cells. Transcripts derived from pA/BVDV/Ins or
pA/BVDV served as controls for non-cp and cp genomes, respectively. As
described before (28), transfection of RNA derived from
pA/BVDV resulted in lysis of the transfected cells, whereas a CPE was
not observed in the case of pA/BVDV/Ins (Fig. 7B). Upon transfection
of RNA derived from pA/B-JaNCP and pA/B-JaCP, a CPE could not be
detected (Fig. 7B, upper row). While this finding was expected in the
case of pA/B-JaNCP, it was surprising for the RNA containing the LC3*
insertion. Even more surprisingly, further analyses showed that in the
latter case no infectious virus had been generated. In a Northern blot
with total RNA isolated 72 h posttransfection, BVDV-specific
signals could be detected for cells transfected with transcripts
derived from pA/BVDV, pA/BVDV/Ins, and pA/B7-JaNCP RNA but not for
cells transfected with pA/B-JaCP-derived RNA (Fig. 7C). Similarly,
immunofluorescence analyses allowed the detection of positive cells for
the first three constructs but not for pA/B-JaCP (data not shown).
These results indicated that transcripts derived from pA/B-JaCP were
not infectious.
View larger version (32K):
[in this window]
[in a new window]
View larger version (46K):
[in this window]
[in a new window]
View larger version (73K):
[in this window]
[in a new window]
|
FIG. 7.
Results of transfection of MDBK cells with RNA
transcribed from the full-length cDNA constructs pA/B-JaCP and
pA/B-JaNCP. RNA derived from pA/BVDV and pA/BVDV/Ins
(28) served as controls for cp and noncp viruses,
respectively. (A) Schematic presentation of the protein coding region
of the cDNA constructs. pA/BVDV/Ins was established from pA/BVDV by
deletion of a 27-nucleotide insertion responsible for cytopathogenicity
of the virus recovered from the latter construct (28). The
regions derived from pJaCP/A17 or pJaNCP35 and inserted via
BamHI and HpaI cuts are marked by a thick broken
or a dotted line, respectively. For further information, see the legend
to Fig. 2. Checkered bar, Npro gene; dark gray bar, NS2
gene; light gray bar, NS3 gene; small white bar within the NS2 gene of
pA/BVDV, 27-nucleotide insertion responsible for cytopathogenicity. (B)
Crystal violet staining of cells that were fixed 72 h after
transfection with the RNAs transcribed in vitro from the indicated cDNA
constructs. Cells were washed once with phosphate-buffered saline,
fixed for 10 min with 5% formaldehyde, washed extensively with water,
and stained for 5 min with 1% (wt/vol) crystal violet (in 50%
ethanol). The upper row shows the results of the transfection of
noninfected MDBK cells, whereas the lower row shows dishes into which
were seeded cells that had been infected 24 h before transfection,
with non-cp BVDV NCP1 at a multiplicity of infection of 0.1. (C)
Northern blot with RNA derived from cells at 72 h posttransfection
hybridized with a BVDV-specific probe. Only the result for the
cells which were not infected prior to transfection are shown. RNA
ladder (in kilobases) is on the left side of the gel.
|
|
In order to verify that the latter finding was not due to a deleterious
mutation in the cDNA construct, different experiments
were conducted.
After transient expression in the vTF7-3 vaccinia
virus system,
viral proteins were analyzed by immunoprecipitation,
SDS-PAGE, and
fluorography. The expression of polypeptides corresponding
to all viral
proteins could be demonstrated. No abnormality with
regard to
size or amount of the individual proteins was observed
(data not
shown).
As a second control, a plasmid equivalent to pA/BVDV/Ins
was
reconstructed, starting with the defective clone pA/B-JaCP.
The region
of pA/B-JaCP containing the LC3* insertion was exchanged
for the
corresponding fragment from pA/BVDV/Ins
. After transfection
of RNA
transcribed from the reconstructed plasmid, infectious
non-cp virus was
recovered (not shown). The sequence of the fragments
deleted from
pA/B-JaCP did not exhibit differences with regard
to the expected
sequence. Thus, no obvious mistake present in
pA/B-JaCP was responsible
for the fact that RNA derived from this
plasmid did not yield
infectious virus.
Different cp pestivirus isolates were found to be composed of noncp
helper viruses and cp-defective interfering particle (
31).
In these cases, cell lysis was observed upon transfection of viral
RNA
when the cells were infected with a noncp helper virus prior
to
introduction of the RNA. We therefore investigated whether
RNA derived
from pA/B-JaCP was also able to induce a CPE in the
presence of a
helper virus. Accordingly, the transfection experiments
were repeated
with target cells previously infected with a noncp
BVDV. Whereas the
RNA transcribed from pA/BVDV/Ins
or pA/B-JaNCP
was again not able to
induce a CPE, transfection of the RNA containing
LC3* resulted in cell
lysis (Fig.
7B, lower row). This result
was not dependent on the helper
virus strain, since cells infected
with NCP7 or V(pA/BVDV/Ins
), the
virus derived from pA/BVDV/Ins
,
also developed CPE upon transfection
with the RNA containing LC3*
(data not shown). Taken together, these
results indicate that
the presence of LC3* in pA/B-JaCP leads to
recovery of a defective
cp virus.
In order to demonstrate that an autonomously replicating cp BVDV could
be generated by integration of JaCP cDNA fragments
into pA/BVDV/Ins
,
a construct resembling the JaCP genome was
assembled. A 3-kb
NcoI/
SalI fragment from pA/BVDV/Ins
was
exchanged
for the corresponding 5.5-kb fragment from a cDNA clone,
pEx7/JaCP/dp,
that was assembled from pEx7/JaCP and a
SacI/
BamHI fragment of
cDNA clone pJaCP/A19
containing the duplication. The resulting
construct was termed
pA/B-JaCP/dp (Fig.
8A). As a control, a
second
construct, which contained the duplicated sequence but not the
LC3* insertion, was established (Fig.
8A). For this clone the
NcoI/
SalI fragment from cDNA clone pEx7/JaNCP/dp
was used instead
of the corresponding fragment from pEx7/JaCP/dp. After
transfection
of in vitro-transcribed RNA, cell lysis was observed in
the case
of pA/B-JaCP/dp (Fig.
8B). It therefore can be concluded that
RNA containing sequences from JaCP and CP7 can serve as the genome
of
an autonomously replicating cp BVDV. For RNA derived from
pA/B-JaNCP/dp,
cell lysis could not be observed (Fig.
8B). Moreover,
immunofluorescence
analysis and Northern hybridization indicated that
no viable virus
could be recovered (not shown and Fig.
8C,
respectively). Experiments
with a reconstructed plasmid similar to
pA/BVDV/Ins
, which was
obtained by deletion of a
SacI
fragment containing the duplication,
resulted in recovery of noncp BVDV
(not shown). It is therefore
very likely that a BVDV RNA containing the
JaCP duplication without
the LC3* insertion is defective.
View larger version (27K):
[in this window]
[in a new window]
View larger version (49K):
[in this window]
[in a new window]
View larger version (33K):
[in this window]
[in a new window]
|
FIG. 8.
Results of the transfection of MDBK cells with RNA
transcribed from the full-length cDNA constructs pA/B-JaCP/dp and
pA/B-JaNCP/dp. RNA derived from pA/BVDV/Ins served as a control. (A)
Schematic presentation of the protein coding region of the cDNA
constructs. The regions derived from pJaCP/A17 or pJaCP/A19 are marked
by a thick broken line. Sequences derived from pJaNCP35 are marked by a
dotted line. The positions of restriction sites important for the
cloning procedures are indicated. For further information, see the
legends to Fig. 2 and 7. (B) Crystal violet staining of cells that were
fixed 72 h after transfection with the RNAs transcribed in vitro
from the indicated cDNA constructs. (C) Northern blot with RNA derived
from cells at 72 h posttransfection hybridized with a
BVDV-specific probe. RNA ladder (in kilobases) is on the left side of
the gel.
|
|
| |
DISCUSSION |
Molecular characterization of pestiviruses has revealed that the
genomes of most cp viruses exhibit alterations which are not found in
the RNAs of noncp isolates. Different rearrangements of viral sequences
or insertions of cellular sequences, sometimes accompanied by large
duplications of viral sequences, have been identified (2, 26, 27,
29-31, 34, 42-44). In the past, two types of cellular
insertions, which code for either ubiquitin or part of a cellular
protein of unknown function, termed cIns, were found (2, 26, 29,
31, 34). In this communication, we report on a novel kind of
cellular insertion that shows no homology to the already known cellular
inserts and is derived from an mRNA coding for LC3 of MAPs 1A and 1B.
Cellular LC3 represents a small basic protein which is abundant only in
neurons and which is able to bind microtubules as well as the MAPs 1A
and 1B. The function of LC3 in normal cell life has not yet been
elucidated (23). Interestingly, the polypeptide encoded by
LC3* exhibits only four exchanges with respect to the LC3 sequence from
rat. Since LC3* was most likely derived from a bovine mRNA, it can be
concluded that the LC3 amino acid sequence has been highly conserved
during evolution, indicating an important function of this protein.
The genome of JaCP represents the first BVDV RNA, for which the
presence of an LC3 coding sequence has been identified. In contrast,
insertions coding for ubiquitin have been found in the genomes of
several independent virus isolates (for a review, see reference
31). With regard to the 3' end, all nine
ubiquitin-coding insertions identified so far are found at the same
genomic position, which represents the 5' end of the NS3 gene (position
B in Fig. 2A). In the polyprotein, the presence of ubiquitin induces
cleavage by a ubiquitin-specific cellular protease and thus is
responsible for generation of the amino terminus of NS3. Taking into
account our knowledge about different cp pestiviruses, it can be
concluded that the presence and the location of the cellular sequences
or other rearrangements in the genomes of cp pestiviruses are not random but should be regarded as a result of functional selection. The
genome rearrangement leading to a cp BVDV has to induce processing at
the amino terminus of NS3, thus allowing expression of this protein,
which represents a specific feature of cp BVDV. In accordance with
these considerations, LC3* is located in the JaCP genome at the
conserved position B (Fig. 2A) and is able to mediate NS2-3 cleavage.
It therefore can be hypothesized that the polypeptide encoded by LC3*
either (i) exhibits autoproteolytic activity, (ii) serves as a signal
for a cellular protease, or (iii) induces a defined conformation which
allows NS2-3 cleavage via a usually cryptic mechanism. The N-terminal
sequencing of the NS3 protein expressed from pEx7/JaCP showed that the
LC3*-induced processing occurs just downstream of the cellular
insertion. This is again reminiscent of cp BVDV-encoded proteins
containing ubiquitin. It might turn out in the future that LC3*, like
ubiquitin, serves as a target for a specific cellular protease. In any
case, elucidation of the mechanism of LC3*-induced cleavage of NS2-3
should also shed light on the function of its cellular counterpart.
Processing of NS2-3 induced by the LC3*-encoded polypeptide results in
an NS3 protein starting with a glycine corresponding to residue 1590 in
a BVDV polyprotein without insertion. Cleavage of BVDV proteins
containing ubiquitin most likely leads to NS3 proteins with the same
amino terminus. Interestingly, at least three other types of cp BVDV
express NS3 with the identical amino terminus, although the mechanisms
responsible for generation of this protein apparently are different. In
the case of BVDV Pe515CP, CP6, and DI9, the pestivirus Npro
protease is located upstream of NS3. This protease cleaves
autocatalytically at its own carboxy terminus and thereby
releases NS3 (30, 31, 44). The NS2 expressed by BVDV NADL
contains the cellular clns insertion located 54 residues upstream of
the position where ubiquitin or LC3* is found. The mechanism of NS2-3
processing has not been elucidated for this virus, but preliminary
results obtained by N-terminal sequencing of NS3 indicated cleavage
just upstream of a glycine corresponding to residue 1590 (50). Finally, NS2-3 of BVDV Oregon is cleaved because of
point mutations within NS2, giving rise to NS3 with glycine 1590 as the
amino terminus (20). In vitro, cleavage of NS2-3 of BVDV
Oregon is dependent on microsomal membranes, whereas both the ubiquitin
and the LC3*-induced processing can occur in the absence of membranes.
As there is obviously no common mechanism responsible for this high
conservation of the amino terminus of NS3, the function of this protein
for either cytopathogenicity or viability of the viruses has to be
dependent on the correct N terminus. Future analyses with infectious
BVDV full-length clones and replicons will hopefully help to elucidate this interesting phenomenon.
According to a widely accepted hypothesis, RNA virus genomes recombine
by template switching of the viral RNA-dependent RNA polymerase during
replication (for reviews, see references 3, 17, 19,
and 22). Direct generation of the JaCP genomic RNA requires three consecutive template switches, which is a very improbable event. Alternatively, the JaCP RNA could have been generated
via two independent recombination reactions. In a first step, a
recombination between viral and cellular RNA could yield a virus genome
containing LC3*, integrated between NS2 and NS3, but no duplication.
The resulting genomic RNA could be amplified by replication and then
undergo recombination with the RNA of the original noncp BVDV to
produce the JaCP genome containing LC3* and the duplication of viral
sequences. Selection of the mutant generated in the second step would
be favored if the first virus were somehow defective and the
introduction of the duplication helped to overcome this problem. It has
to be stressed in this context that the virus recovered after
transfection of the RNA transcribed from pA/B-JaCP was defective and
therefore dependent on a helper virus. The in vitro-generated RNA
exactly mimics the product of the hypothetical first recombination,
since it contains LC3* at the correct position of the NS2-3 gene but no
duplication. Since viable viruses could be recovered from pA/B-JaCP/dp,
the corresponding construct containing the duplication, and from
pA/B-JaNCP, which lacks both LC3* and the duplication, the defect of
pA/B-JaCP can hardly be due to the genetic background of our infectious clone. The majority of cp BVDV isolates contain in their genomes duplicated viral sequences encompassing the NS3 gene and sequences located further downstream in the genome. The 3' ends of the
duplications vary between position 7456 and 8788 of a regular genome
(BVDV Pe515 CP and BVDV CP6, respectively [31]). The
prevalence of rearranged genomes with duplications could be explained
by the recombination mechanism. A recombination leading to a
duplication allows flexibility with regard to the last template switch
and should therefore be more likely than a recombination integrating an
additional sequence between two neighboring nucleotides. However, the
findings reported here indicate that at least in some cases the
duplications might be functionally relevant. Further investigations are
necessary to elucidate the role of the duplicated sequences with regard
to viability and cytopathogenicity of the viruses.
In BVDV the ability to express NS3 is directly linked to the cp
phenotype (7, 33). Since in the case of JaCP LC3* is apparently responsible for expression of NS3, the integration of the
cellular sequence has to be regarded as causative for the CPE induced
by this virus; this conclusion is strongly supported by the recovery of
cp viruses upon transfection of the in vitro-transcribed RNA containing
LC3*. JaCP has been isolated from an animal suffering from lethal MD
(14). The generation of a cp virus in an animal persistently
infected with a noncp virus is regarded as crucial for development of
the disease. The homology found for corresponding parts of the
sequences from noncp and cp BVDV isolated from one diseased animal
proved that the cp virus represents a mutant of the persisting noncp
virus (31). Since Jasper had been housed under isolated
conditions a long time before he came down with MD (14) it
is obvious that JaCP has also developed from JaNCP within the
persistently infected animal. Thus, Jasper died most likely as a
consequence of a recombination between the genome of JaNCP and an mRNA
coding for MAP LC3. Molecular analysis of further samples taken just
before the onset of MD and during the clinical disease will help to
improve understanding of the processes leading to this interesting
disease.
| |
ACKNOWLEDGMENTS |
We thank Silke Esslinger and Petra Wulle for excellent technical
assistance and K.-K. Conzelmann and B. M. Kümmerer for
helpful comments on the manuscript.
This work was supported by the Deutsche Forschungsgemeinschaft (grant
DFG Me1367/2-3).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Clinical Virology, Federal Research Centre for Virus Diseases of
Animals, P.O. Box 1149, D-72001 Tübingen, Germany. Phone: 49 7071-967207. Fax: 49 7071-967303. E-mail:
gregor.meyers{at}tue.bfav.de.
| |
REFERENCES |
| 1.
|
Bazan, J. F., and R. J. Fletterick.
1989.
Detection of a trypsin-like serine protease domain in flaviviruses and pestiviruses.
Virology
171:637-639[Medline].
|
| 2.
|
Becher, P.,
G. Meyers,
A. D. Shannon, and H.-J. Thiel.
1996.
Cytopathogenicity of border disease virus is correlated with integration of cellular sequences into the viral genome.
J. Virol.
70:2992-2998[Abstract].
|
| 3.
|
Bujarski, J. J.,
P. D. Nagy, and S. Flasinski.
1994.
Molecular studies of genetic RNA-RNA recombination in brome mosaic virus.
Adv. Virus Res.
43:275-302[Medline].
|
| 4.
|
Collett, M. S.,
R. Larson,
S. K. Belzer, and E. Retzel.
1988.
Proteins encoded by bovine viral diarrhea virus: the genomic organization of a pestivirus.
Virology
165:200-208[Medline].
|
| 5.
|
Collett, M. S.,
R. Larson,
C. Gold,
D. Strick,
D. K. Anderson, and A. F. Purchio.
1988.
Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus.
Virology
165:191-199[Medline].
|
| 6.
|
Collett, M. S.,
M. A. Wiskerchen,
E. Welniak, and S. K. Belzer.
1991.
Bovine viral diarrhea virus genomic organization.
Arch. Virol. Suppl.
3:19-27[Medline].
|
| 7.
|
Corapi, W. V.,
R. O. Donis, and E. J. Dubovi.
1988.
Monoclonal antibody analyses of cytopathic and noncytopathic viruses from fatal bovine viral diarrhea virus infections.
J. Virol.
62:2823-2827[Abstract/Free Full Text].
|
| 8.
|
Deng, R., and K. Brock.
1992.
Molecular cloning and nucleotide sequence of a pestivirus genome, noncytopathogenic bovine viral diarrhea virus strain SD-1.
Virology
191:867-879[Medline].
|
| 9.
|
Devereux, J.,
P. Haeberli, and O. Smithies.
1984.
A comprehensive set of sequence analysis programs for the VAX.
Nucleic Acids Res.
12:387-395.
|
| 10.
|
Doucet, J.-P., and J.-M. Trifaro.
1988.
A discontinuous and highly porous sodium dodecyl sulfate-polyacrylamide slab gel system of high resolution.
Anal. Biochem.
168:265-271[Medline].
|
| 11.
|
Elbers, K.,
N. Tautz,
P. Becher,
D. Stoll,
T. Rümenapf, and H.-J. Thiel.
1996.
Processing in the pestivirus E2-NS2 region: identification of proteins p7 and E2p7.
J. Virol.
70:4131-4135[Abstract].
|
| 12.
|
Fuerst, T. R.,
E. G. Niles,
F. W. Studier, and B. Moss.
1986.
Eukaryotic transient expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase.
Proc. Natl. Acad. Sci. USA
83:8122-8126[Abstract/Free Full Text].
|
| 13.
|
Gorbalenya, A. E.,
A. P. Donchenko,
E. V. Koonin, and V. M. Blinov.
1989.
N-terminal domains of putative helicases of flavi- and pestiviruses may be serine proteases.
Nucleic Acids Res.
17:3889-3897[Abstract/Free Full Text].
|
| 14.
|
Gunn, M., and E. Weavers.
1992.
Mucosal disease in cattle housed in isolation.
Vet. Rec.
131:376[Medline].
|
| 15.
|
Harlow, E., and D. Lane.
1988.
In
Antibodies: a laboratory manual, p. 460.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 16.
|
Henikoff, S.
1987.
Unidirectional digestion with exonuclease III in DNA sequence analysis.
Methods Enzymol.
155:156-165[Medline].
|
| 17.
|
Jarvis, T. C., and K. Kirkegaard.
1991.
The polymerase in its labyrinth: mechanisms and implications of RNA recombination.
Trends Genet.
7:186-191[Medline].
|
| 18.
|
Kessler, S. W.
1981.
Use of protein A-bearing staphylococci for the immunoprecipitation and isolation of antigens from cells.
Methods Enzymol.
73:442-459[Medline].
|
| 19.
|
King, A. M. Q.,
S. A. Ortlepp,
J. W. I. Newman, and D. McCahon.
1987.
Genetic recombination in RNA viruses, p. 129-152.
In
D. J. Rowlands, M. A. Mayo, and B. W. J. Mahy (ed.), molecular biology of positive strand RNA viruses. Academic Press, London, United Kingdom.
|
| 20.
|
Kümmerer, B. M.,
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[Abstract/Free Full Text].
|
| 21.
|
Kunkel, T. A.,
J. D. Roberts, and R. A. Zakour.
1987.
Rapid and efficient site-specific mutagenesis without phenotypic selection.
Methods Enzymol.
154:367-392[Medline].
|
| 22.
|
Lai, M. M. C.
1992.
RNA recombination in animal and plant viruses.
Microbiol. Rev.
56:61-79[Abstract/Free Full Text].
|
| 23.
|
Mann, S. S., and J. A. Hammerback.
1994.
Molecular characterization of light chain 3.
J. Biol. Chem.
269:11492-11497[Abstract/Free Full Text].
|
| 24.
|
McClurkin, A. W.,
S. R. Bolin, and M. F. Coria.
1985.
Isolation of cytopathic and noncytopathic bovine viral diarrhea virus from the spleen of cattle acutely and chronically affected with bovine viral diarrhea.
J. Am. Vet. Med. Assoc.
186:568-569[Medline].
|
| 25.
|
McKercher, D. G.,
J. K. Saito,
G. L. Crenshaw, and R. B. Bushnell.
1968.
Complications in cattle following vaccination with a combined bovine viral diarrhea-infectious bovine rhinotracheitis vaccine.
J. Am. Vet. Med. Assoc.
152:1621-1624[Medline].
|
| 26.
|
Meyers, G.,
T. Rümenapf, and H.-J. Thiel.
1989.
Ubiquitin in a togavirus.
Nature (London)
341:491[Medline].
|
| 27.
|
Meyers, G.,
T. Rümenapf, and H.-J. Thiel.
1990.
Insertion of ubiquitin-coding sequence identified in the RNA genome of a togavirus, p. 25-30.
In
M. A. Brinton, and F. X. Heinz (ed.), New aspects of positive-strand RNA viruses. American Society for Microbiology, Washington, D.C.
|
| 28.
|
Meyers, G.,
N. Tautz,
P. Becher,
H.-J. Thiel, and B. M. Kümmerer.
1996.
Recovery of cytopathogenic and noncytopathogenic bovine viral diarrhea viruses from cDNA constructs.
J. Virol.
70:8606-8613[Abstract].
|
| 29.
|
Meyers, G.,
N. Tautz,
E. J. Dubovi, and H.-J. Thiel.
1991.
Viral cytopathogenicity correlated with integration of ubiquitin-coding sequences.
Virology
180:602-616[Medline].
|
| 30.
|
Meyers, G.,
N. Tautz,
R. Stark,
J. Brownlie,
E. J. Dubovi,
M. S. Collett, and H.-J. Thiel.
1992.
Rearrangement of viral sequences in cytopathogenic pestiviruses.
Virology
191:368-386[Medline].
|
| 31.
|
Meyers, G., and H.-J. Thiel.
1996.
Molecular characterization of pestiviruses.
Adv. Virus Res.
47:53-117[Medline].
|
| 32.
|
Meyers, G.,
H.-J. Thiel, and T. Rümenapf.
1996.
Classical swine fever virus: recovery of infectious viruses from cDNA constructs and generation of recombinant cytopathogenic defective interfering particles.
J. Virol.
70:1588-1595[Abstract].
|
| 33.
|
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[Medline].
|
| 34.
|
Qi, F.,
J. F. Ridpath,
T. Lewis,
S. R. Bolin, and E. S. Berry.
1992.
Analysis of the bovine viral diarrhea virus genome for possible insertions.
Virology
189:285-292[Medline].
|
| 35.
|
Rice, C. M.
1996.
Flaviviridae: the viruses and their replication, p. 931-959.
In
B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology. Lippincott-Raven, Philadelphia, Pa.
|
| 36.
|
Rümenapf, T.,
G. Unger,
J. H. Strauss, and H.-J. Thiel.
1993.
Processing of the envelope glycoproteins of pestiviruses.
J. Virol.
67:3288-3294[Abstract/Free Full Text].
|
| 37.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
In
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 38.
|
Sanger, F.,
S. Nicklen, and A. R. Coulson.
1977.
DNA sequencing with chain terminating inhibitors.
Proc. Natl. Acad. Sci. USA
74:5463-5467[Abstract/Free Full Text].
|
| 39.
|
Schägger, H., and G. von Jagow.
1987.
Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1-100 kDa.
Anal. Biochem.
166:368-379[Medline].
|
| 40.
|
Stark, R.,
G. Meyers,
T. Rümenapf, and H.-J. Thiel.
1993.
Processing of pestivirus polyprotein: cleavage site between autoprotease and nucleocapsid protein of classical swine fever virus.
J. Virol.
67:7088-7095[Abstract/Free Full Text].
|
| 41.
|
Tautz, N.,
K. Elbers,
D. Stoll,
G. Meyers, and H.-J. Thiel.
1997.
Serine protease of pestiviruses: determination of cleavage sites.
J. Virol.
71:5415-5422[Abstract].
|
| 42.
|
Tautz, N.,
G. Meyers,
R. Stark,
E. J. Dubovi, and H.-J. Thiel.
1996.
Cytopathogenicity of a pestivirus correlates with a 27-nucleotide insertion.
J. Virol.
70:7851-7858[Abstract].
|
| 43.
|
Tautz, N.,
G. Meyers, and H.-J. Thiel.
1993.
Processing of poly-ubiquitin in the polyprotein of an RNA virus.
Virology
197:74-85[Medline].
|
| 44.
|
Tautz, N.,
H.-J. Thiel,
E. Dubovi, and G. Meyers.
1994.
Pathogenesis of mucosal disease: a cytopathogenic pestivirus generated by an internal deletion.
J. Virol.
68:3289-3297[Abstract/Free Full Text].
|
| 45.
|
Thiel, H.-J.,
G. W. Plagemann, and V. Moennig.
1996.
The pestiviruses, p. 1059-1073.
In
B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology. Lippincott-Raven, Philadelphia, Pa.
|
| 46.
|
Thiel, H.-J.,
R. Stark,
E. Weiland,
T. Rümenapf, and G. Meyers.
1991.
Hog cholera virus: molecular composition of virions from a pestivirus.
J. Virol.
65:4705-4712[Abstract/Free Full Text].
|
| 47.
|
Van der Werf, S.,
J. Bradley,
E. Wimmer,
F. W. Studier, and J. J. Dunn.
1986.
Synthesis of infectious poliovirus RNA by purified T7 RNA polymerase.
Proc. Natl. Acad. Sci. USA
83:2330-2334[Abstract/Free Full Text].
|
| 48.
|
Warrener, P., and M. S. Collett.
1995.
Pestivirus NS3 (p80) protein possesses RNA helicase activity.
J. Virol.
69:1720-1726[Abstract].
|
| 49.
|
Wiskerchen, M., and M. S. Collett.
1991.
Pestivirus gene expression: protein p80 of bovine viral diarrhea virus is a proteinase involved in polyprotein processing.
Virology
184:341-350[Medline].
|
| 50.
|
Xu, J.,
E. Mendez,
P. R. Caron,
C. Lin,
M. A. Murcko,
M. S. Collett, and C. M. Rice.
1997.
Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication.
J. Virol.
71:5312-5322[Abstract].
|
J Virol, May 1998, p. 4139-4148, Vol. 72, No. 5
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Pankraz, A., Preis, S., Thiel, H.-J., Gallei, A., Becher, P.
(2009). A Single Point Mutation in Nonstructural Protein NS2 of Bovine Viral Diarrhea Virus Results in Temperature-Sensitive Attenuation of Viral Cytopathogenicity. J. Virol.
83: 12415-12423
[Abstract]
[Full Text]
-
Gallei, A., Blome, S., Gilgenbach, S., Tautz, N., Moennig, V., Becher, P.
(2008). Cytopathogenicity of Classical Swine Fever Virus Correlates with Attenuation in the Natural Host. J. Virol.
82: 9717-9729
[Abstract]
[Full Text]
-
Gallei, A., Orlich, M., Thiel, H.-J., Becher, P.
(2005). Noncytopathogenic Pestivirus Strains Generated by Nonhomologous RNA Recombination: Alterations in the NS4A/NS4B Coding Region. J. Virol.
79: 14261-14270
[Abstract]
[Full Text]
-
Gallei, A., Rumenapf, T., Thiel, H.-J., Becher, P.
(2005). Characterization of Helper Virus-Independent Cytopathogenic Classical Swine Fever Virus Generated by an In Vivo RNA Recombination System. J. Virol.
79: 2440-2448
[Abstract]
[Full Text]
-
Lackner, T., Muller, A., Pankraz, A., Becher, P., Thiel, H.-J., Gorbalenya, A. E., Tautz, N.
(2004). Temporal Modulation of an Autoprotease Is Crucial for Replication and Pathogenicity of an RNA Virus. J. Virol.
78: 10765-10775
[Abstract]
[Full Text]
-
Fricke, J., Voss, C., Thumm, M., Meyers, G.
(2004). Processing of a Pestivirus Protein by a Cellular Protease Specific for Light Chain 3 of Microtubule-Associated Proteins. J. Virol.
78: 5900-5912
[Abstract]
[Full Text]
-
Agapov, E. V., Murray, C. L., Frolov, I., Qu, L., Myers, T. M., Rice, C. M.
(2004). Uncleaved NS2-3 Is Required for Production of Infectious Bovine Viral Diarrhea Virus. J. Virol.
78: 2414-2425
[Abstract]
[Full Text]
-
Hemelaar, J., Lelyveld, V. S., Kessler, B. M., Ploegh, H. L.
(2003). A Single Protease, Apg4B, Is Specific for the Autophagy-related Ubiquitin-like Proteins GATE-16, MAP1-LC3, GABARAP, and Apg8L. J. Biol. Chem.
278: 51841-51850
[Abstract]
[Full Text]
-
Muller, A., Rinck, G., Thiel, H.-J., Tautz, N.
(2003). Cell-Derived Sequences in the N-Terminal Region of the Polyprotein of a Cytopathogenic Pestivirus. J. Virol.
77: 10663-10669
[Abstract]
[Full Text]
-
Nagai, M., Sakoda, Y., Mori, M., Hayashi, M., Kida, H., Akashi, H.
(2003). Insertion of cellular sequence and RNA recombination in the structural protein coding region of cytopathogenic bovine viral diarrhoea virus. J. Gen. Virol.
84: 447-452
[Abstract]
[Full Text]
-
Becher, P., Thiel, H.-J., Collins, M., Brownlie, J., Orlich, M.
(2002). Cellular Sequences in Pestivirus Genomes Encoding Gamma-Aminobutyric Acid (A) Receptor-Associated Protein and Golgi-Associated ATPase Enhancer of 16 Kilodaltons. J. Virol.
76: 13069-13076
[Abstract]
[Full Text]
-
Qu, L., McMullan, L. K., Rice, C. M.
(2001). Isolation and Characterization of Noncytopathic Pestivirus Mutants Reveals a Role for Nonstructural Protein NS4B in Viral Cytopathogenicity. J. Virol.
75: 10651-10662
[Abstract]
[Full Text]
-
Becher, P., Orlich, M., Thiel, H.-J.
(2001). RNA Recombination between Persisting Pestivirus and a Vaccine Strain: Generation of Cytopathogenic Virus and Induction of Lethal Disease. J. Virol.
75: 6256-6264
[Abstract]
[Full Text]
-
Kirisako, T., Ichimura, Y., Okada, H., Kabeya, Y., Mizushima, N., Yoshimori, T., Ohsumi, M., Takao, T., Noda, T., Ohsumi, Y.
(2000). The Reversible Modification Regulates the Membrane-Binding State of Apg8/Aut7 Essential for Autophagy and the Cytoplasm to Vacuole Targeting Pathway. JCB
151: 263-276
[Abstract]
[Full Text]
-
Ridpath, J. F., Neill, J. D.
(2000). Detection and Characterization of Genetic Recombination in Cytopathic Type 2 Bovine Viral Diarrhea Viruses. J. Virol.
74: 8771-8774
[Abstract]
[Full Text]
Top
Abstract
Introduction
