The genetic basis for cytopathogenicity of pestiviruses

The genetic basis for cytopathogenicityof pestiviruses

Beate M. KuÈmmerera, Norbert Tautzb, Paul Becherb,Heinz-JuÈrgen Thielb, Gregor Meyersa,*

aFederal Research Center for Virus Diseases of Animals, Paul-Ehrlich-Str. 28, D-72076 TuÈbingen, GermanybInstitut fuÈr Virologie (FB VeterinaÈrmedizin), Justus-Liebig-UniversitaÈt Giessen, Frankfurter Str. 107,

D-35392 Giessen, Germany

Abstract

Two biotypes of pestiviruses, cytopathogenic (cp) and noncp viruses, can be distinguished by

their effects on tissue culture cells. Identi®cation of cp bovine viral diarrhea virus (BVDV) has been

frequently reported since antigenically closely related noncp and cp BVDV can be isolated from

cattle with fatal mucosal disease (MD) and are called a virus pair. In contrast to the BVDV system,

only few cp border disease virus (BDV) and cp classical swine fever virus (CSFV) strains have been

described. Serological analyses and sequence comparison studies showed that cp pestiviruses arise

from noncp viruses by mutation. Elaborate studies during the last 10 years revealed that in most

cases RNA recombination is responsible for the generation of the cp viruses. Recent results showed

a second way for the development of a cp pestivirus which is based on the introduction of a set of

point mutations within the NS2 gene. # 2000 Elsevier Science B.V. All rights reserved.

Keywords: Cytopathogenic virus; Bovine viral diarrhea virus; Mucosal disease; Border disease virus; Classical

swine fever virus

1. Introduction

Pestiviruses are currently classi®ed as one genus within the family Flaviviridae, which

also includes the genera Flavivirus and hepatitis C virus group (HCV) (Wengler et al.,

1995). The genomes of pestiviruses consist of positive-stranded RNAs, which usually

have a length of 12.3 kb and code for polyproteins of about 4000 amino acids. Entire or

partial genomic sequences of numerous pestivirus isolates have been determined (Meyers

and Thiel, 1996). Phylogenetic analyses have made it possible to determine the genetic

Veterinary Microbiology 77 (2000) 117±128

* Corresponding author. Tel.: �49-7071-967-207; fax: �49-7071-967-305

E-mail address: [email protected] (G. Meyers).

0378-1135/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.

PII: S 0 3 7 8 - 1 1 3 5 ( 0 0 ) 0 0 2 6 8 - 6

relatedness of pestiviruses resulting in the proposition of a classi®cation scheme

consisting of four pestivirus species. These include the three formerly known species

bovine viral diarrhea virus (BVDV), classical swine fever virus (CSFV) and border

disease virus (BDV) of sheep, and a fourth species (BVDV 2) which comprises ovine and

bovine isolates (Becher et al., 1997).

Pestivirus virions consist, together with the RNA, of four structural proteins, the

nucleocapsid C protein and the envelope glycoproteins Erns, E1 and E2. Eleven or twelve

pestiviral proteins have been identi®ed as products of polyprotein processing which

occurs co- and post-translationally by viral and host cell proteases (Fig. 1). In the

hypothetical polyprotein, the proteins are arranged in the order Npro/C/Erns/E1/E2/p7/

NS2-3/NS4A/NS4B/NS5A/NS5B; NS2-3 can be processed to yield NS2 and NS3

(Meyers and Thiel, 1996; Rice, 1996).

One remarkable property of pestiviruses is the existence of two biotypes that were

recognized according to morphological changes they induce after infection of tissue

culture cells. Noncytopathogenic (noncp) pestiviruses replicate without a cytopathic

effect whereas cytopathogenic (cp) viruses lead to the death of appropriate infected cells

(Fig. 2) (Thiel et al., 1996). In the BVDV system, a well-known molecular difference

between cp and noncp viruses concerns the proteins present within the infected cells. For

cp BVDV strains, the nonstructural protein NS3 is found together with NS2-3 whereas

after infection with noncp BVDV only NS2-3 can be detected (Fig. 2) (Meyers and Thiel,

1996).

Mucosal disease (MD) is a sporadic disease in cattle which often occurs between the

ages of 6 months and 2 years. The disease is characterized by high case fatality with death

occurring usually within 2 weeks after the onset of clinical signs (Baker, 1987). MD

occurs only in cattle that have been infected in utero with a noncp BVDV during the ®rst

trimester of gestation (Liess et al., 1984). Such animals acquire immunological tolerance

with regard to the infecting BVDV strains and develop a persistent infection. The

immunotolerance is restricted to the particular noncp virus strain and there is no

indication of either formation of antibodies or cytolytic T-cells against the persisting virus

(Thiel et al., 1996). Experimental infection with cp BVDV has shown that the respective

biotype is not capable of inducing a persistent infection (Brownlie, 1991).

Fig. 1. Genome organization of pestiviruses.

118 B.M. KuÈmmerer et al. / Veterinary Microbiology 77 (2000) 117±128

Both viral biotypes, noncp BVDV and cp BVDV, are consistently found in animals that

come down with MD (Bolin et al., 1985; Brownlie et al., 1984). For a single animal with

MD, the noncp virus together with the cp virus is called a `̀ virus pair''; the partners of

such a `̀ pair'' are in general antigenetically very closely related (Corapi et al., 1988;

Pocock et al., 1987). The cp virus can either be introduced into the persistently infected

animals by superinfection or it can arise from the noncp virus by different mutations.

Also for BDV and CSFV cp isolates have been identi®ed. The present manuscript

summarizes the data with regard to the nature of these mutations obtained in different

laboratories.

2. Results and discussion

Differentiation of noncp and cp pestiviruses was ®rst based on the effect on tissue

culture cells. As a second criterium, the expression of NS3 was recognized as a speci®c

marker for cp viruses in the BVDV system (Fig. 2). Data concerning differences of the

viral genomes were not available for a long time. Our group demonstrated that two

widely used cp BVDV strains, namely Osloss and NADL, contain insertions of cellular

sequences in the NS2-3 gene and proposed the integration of the cellular sequences as the

molecular basis for cytopathogenicity (Meyers et al., 1989; Meyers et al., 1990). For cp

BVDV strain Osloss, the insertion was identi®ed as a ubiquitin±coding sequence. The

Fig. 2. Differentiation of cp and noncp BVDV.

B.M. KuÈmmerer et al. / Veterinary Microbiology 77 (2000) 117±128 119

cellular homologue of the cp BVDV strain NADL, termed cIns, has also been identi®ed

but the cellular gene product(s) has not been further characterized. In the following,

several BVDV `̀ pairs'' were analyzed and it could be shown that the genomes of the

respective cp strains exhibited a surprising variety of alterations which were absent from

the genomes of noncp strains (Meyers and Thiel, 1996).

Thus, the following changes have been identi®ed in the genomes of cp viruses when

compared to the genomic RNAs of corresponding noncp viruses (Fig. 3):

1. Insertions of cellular sequences coding for ubiquitin, parts of the cIns protein, part of

light chain 3 of microtubule-associated proteins (LC3) or SMT3B, sometimes ¯anked

by duplications of viral sequences including NS3 (>2 kb) (Meyers and Thiel, 1996;

Becher et al., 1998; Meyers et al., 1998; Qi et al., 1998).

Fig. 3. Genome structures of some cp BVDV strains.


2. Duplications and rearrangement of viral sequences including the NS3 gene (>2 kb) in

conjunction with sequence rearrangements (Fig. 3); in these cases, the insertion of

several hundred nucleotides is not a host-cell-derived element but represents

sequences which code for the pestiviral autoprotease Npro (Meyers and Thiel, 1996).

3. Deletions that led to generation of cp defective interfering particles (DIs) (Meyers and

Thiel, 1996).

4. A small duplication of a viral sequence (27 nucleotides); the insertion is identical with

a sequence located about 300 nucleotides upstream. The 27 duplicated nucleotides are

inserted in a different reading frame and therefore encode nine amino acids which are

not found elsewhere in the polyprotein (Tautz et al., 1996).

All the different possibilities were identi®ed for cp BVDV. Cytopathogenic isolates of

CSFV were found to contain cp DIs (Meyers and Thiel, 1995; Mittleholzer et al., 1997).

In the case of BDV, cytopathogenicity is correlated with the insertion of cellular

sequences homologous to the cIns sequence ®rst found in the genome of BVDV NADL

(Becher et al., 1996).

It is assumed that noncp BVDV strains undergo nonhomologous RNA recombination

by template switching during RNA replication which results in the observed genome

alterations that lead to the cp phenotype of the viruses. However, the generation of a cp

pestiviruses is not always due to RNA recombination. Rather soon after publication of the

`̀ recombination hypothesis'', there were reports on cp BVDV isolates for which PCR-

based investigations did not result in identi®cation of recombination induced genome

alterations (de Moerlooze et al., 1990; Greiser-Wilke et al., 1993; Qi et al., 1992). A

putative pitfall of these studies was that only small parts of the genomes were analyzed

and the choice of primers for ampli®cation restricted the detectable genomic changes. We

therefore cloned and sequenced the complete genome of one of these cp BVDV isolates,

namely strain Oregon. Indeed, the sequence analysis revealed that no genome alteration

due to recombination was present (KuÈmmerer et al., 1998). Nevertheless, the virus was

clearly cytopathogenic and NS3, the marker protein of cp BVDV, was detectable in

infected cells. To de®ne the genetic basis for this phenotype, we ®rst tried to identify the

region of the genome responsible for the observed cleavage of NS2-3. Transient

expression of cDNA constructs allowed to determine the NS2 gene as causative for the

generation of NS3. To further narrow down the necessary part of the sequence, chimeric

constructs were established that contained sequences from BVDV Oregon together with

cDNA of a noncp BVDV (CP7/Ins-). After expression of these plasmids and

determination of the NS2-3 cleavage ef®ciency, it was shown that indeed the NS2 gene

determined whether NS2-3 cleavage occurred or not. When exchanging only parts of the

NS2 gene, cleavage was observed if the construct contained the 30 terminal third of the

Oregon NS2 gene (Fig. 4). However, in any construct encompassing only parts of the

Oregon NS2 sequence, cleavage ef®ciency was lower than in the wild type with a level of

about 80%.

In order to more precisely de®ne the amino acid exchanges in the Oregon NS2

responsible for the observed cleavage, single site exchanges were introduced into the

Oregon sequence as well as into the CP7/Ins- sequence. One residue that corresponds to

position 1555 of the polyprotein was found to have major impact on cleavage ef®ciency.


Fig. 4. NS2-3 cleavage ef®ciency of different chimeric cDNA constructs containing sequences from cp BVDV Oregon and noncp BVDV CP7/Ins-. The processing was

analyzed after transient expression, radioimmunoprecipitation and phosphoimager analysis.

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In the Oregon protein this position is occupied by a serine, in contrast to the

phenylalanine present in all other known pestivirus sequences. Exchange of the S to F

resulted in reduction of cleavage ef®ciency to 50% of the wild type level. Introduction of

additional exchanges led to a further decrease of processing ef®ciency (Fig. 5). On the

other hand, a change of the F to S in the sequence of CPV7/Ins- resulted in a low level of

cleavage (about 10% of the value observed for BVDV Oregon). Thus, position 1555 of

the polyprotein must play an important role for processing of NS2-3.

Taken together the data obtained during the expression experiments with chimeric

constructs as well as point mutants, it can be concluded that in the case of BVDV Oregon

processing of NS2-3 results from a set of point mutations within the NS2 gene.

Experiments with infectious cDNA clones showed that these point mutations are not only

responsible for expression of NS3 but also for the cp phenotype (KuÈmmerer and Meyers,

2000). Thus, BVDV Oregon represents the ®rst pestivirus isolate, for which the existence

of a second way to a cp phenotype not relying on RNA recombination was de®nitely

proven.

The cp phenotype of BVDV strains is strictly correlated with the appearance of the

nonstructural protein NS3. In contrast, CSFV expresses NS3 regardless whether it is cp or

noncp. Also for BDV, the presence of NS3 within cells infected with noncp isolates has

been reported. Nevertheless, cp-speci®c genome alterations apparently always in¯uence

the expression of NS3 (Fig. 6), since for CSFV and BDV it is obvious that cells infected

with cp isolates contain much more NS3 than those inoculated with noncp variants of

these viruses that are able to express NS3. The mechanism how the identi®ed genome

alterations induce NS3 expression is well understood for some types of alterations. For

example, the integration of ubiquitin-coding sequences into BVDV genomes leads to an

additional protease cleavage site in the viral polyprotein. The respective sequence is cut

by a cellular protease, most likely by ubiquitin carboxy terminal hydrolases (UCH) (Tautz

et al., 1993). The cleavage results in release of the N-terminus of the cp marker protein

NS3 (Fig. 7). Interestingly, integration of cellular ubiquitin-coding sequences at a certain

position of the NS2-3 gene of BVDV represents a frequently used way to generate a cp

BVD virus (Fig. 8) (Meyers and Thiel, 1996). For those viruses expressing fusions of Npro

and NS3 either due to duplication of the respective genes or to a deletion as observed for

the CP9 DI, the aminoterminus of NS3 is generated by the autoproteolytic activity of Npro

that cleaves at its own carboxyterminus and thereby releases NS3 (Fig. 7) (Meyers and

Thiel, 1996). In several other cases, the mechanism leading to the expression of NS3 is

still not known and further efforts are aiming at identi®cation of the responsible

protease(s).

Importantly, for some of the cp viruses NS2-3 is not processed. Instead NS3 is

translated either from the duplicated genomic region downstream of the insertion or from

the DI genomes. It can be concluded that the cytopathic effect is not due to generation of

NS2. One interesting aspect concerning the N-terminus of NS3 is that it is conserved in

almost all cases investigated so far. There is no support for the hypothesis that this

conservation is due to a hot spot of recombination located at this site, and it rather seems

likely that the correct aminoterminus of NS3 is functionally important either for viability

or cytopathogenicity of the viruses. This conclusion is supported by the fact that NS3 of

BVDV Oregon starts with the same sequence as that hypothesized for isolates expressing


Fig. 5. NS2-3 cleavage ef®ciency of NS2-3 proteins containing different point mutations within NS2.

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Fig. 6. All the different genome alterations speci®c for cp pestiviruses have an in¯uence on expression of NS3.

Fig. 7. Mechanisms responsible for generation of NS3.


fusion proteins composed of ubiquitin and NS3 or Npro and NS3 (KuÈmmerer et al., 1998).

This ®nding cannot be explained by a recombination hot spot since the cp phenotype of

BVDV Oregon is not due to RNA recombination.

Our results demonstrate that two different mechanisms can be responsible for the

generation of cp pestiviruses, namely RNA recombination or introduction of point

mutations. Experiments with infectious clones have proven the linkage between the

Fig. 8. Genome organization of different viruses containing ubiquitin-coding insertions (data from Meyers et al.,

1991; Qi et al., 1992; Tautz et al., 1993; Becher et al., 1998).


cp-speci®c genome alterations, the cp phenotype and the generation of NS3. The latter

protein represents the prime candidate for induction of cytopathogenicity. Further studies

employing infectious cDNA constructs, subgenomic autonomous replicons and cell

biological approaches will hopefully help to elucidate the role of NS3 with regard to

cytopathogenicity and pathogenesis of pestivirus-induced diseases.

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The genetic basis for cytopathogenicity of pestiviruses