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Proteins encoded in the 5′ region of the pestivirus genome—considerations concerning taxonomy
Veterinary Microbiology, 33 ( 1992 ) 213-219 Elsevier Science Publishers B.V., Amsterdam
213
Proteins encoded in the 5' region of the pestivirus genome considerations concerning taxonomy Heinz-Jiirgen Thiel, Robert Stark, Gregor Meyers, Emilie Weiland and Tillmann Riimenapf Federal Research Centre for Virus Diseases of Animals, Tiibingen, FRG (Accepted 26 June 1992 )
ABSTRACT Thiel, H.-J., Stark, R., Meyers, G., Weiland, E. and Riimenapf, T., 1992. Proteins encoded in the 5' region of the pestivirus genome---considerations concerning taxonomy. Vet. Microbiol., 33: 213219. The first protein encoded within the pestivirus open reading frame is a nonstructural protein which removes itself from the polyprotein by autoproteolytic cleavage. The following nucleocapsid protein ends just before a putative signal sequence preceding three glycosylated proteins. All three glycoproteins are part of the viral envelope and exist in the form of disulfide-linked dimers. Pestiviruses have recently been reclassified as members of the family Flaviviridae which now comprises three genera, namely flavivirus, hepatitis C virus group and pestivirus. All members of the family have certain characteristics in common like the overall genome organization and the strategy ofgene expression. Major differences exist, however, between the genera; the most obvious ones concern proteins encoded in the 5' region of the respective genomes.
INTRODUCTION
Hog cholera virus (HCV), bovine viral diarrhea virus (BVDV) and border disease virus of sheep are grouped together in the genus pestivirus (Westaway et al., 1985 ). Pestiviruses have a single-stranded RNA genome which is about 12.5 kb in length (Collett et al., 1988a; Riimenapf et al., 1989). The RNA is of positive polarity and comprises a single large open reading frame (ORF) (Collett et al., 1988a,b; Meyers et al., 1989; Moormann et al., 1990). Viral gene expression is believed to occur via synthesis of a polyprotein which is processed co- and posttranslationally. The different proteins encoded by pestiviruses were mostly identified by using antibodies against bacterial fusion Correspondence to: H.-J. Thiel, Federal Research Centre for Virus Diseases of Animals, P.O. Box 11 49, D-7400 Tiibingen, FRG.
0378-1135/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
214
H.-J. THIEL ET AL.
proteins encompassing defined regions of the viral genome (Collett et al., 1988b; Stark et al., 1990). From these analyses it was concluded that the structural proteins are encoded in the 5' terminal part of the pestiviral ORF while RNA-dependent RNA polymerase is encoded in the 3' region. Until recently pestiviruses held generic status in the family Togaviridae (Westaway et al., 1985). Since genome organization and strategy of gene expression are more similar to flaviviruses than to togaviruses it has been decided to include pestiviruses as an additional genus in the family Flaviviridae. The molecular composition of virions from the genus pestivirus has long been a matter of controversy. According to the data obtained after immunoprecipitation from virus-infected cells by using antisera against bacterial fusion proteins, pestiviruses probably possess four structural proteins, three of which are glycosylated (Collett et al., 1988b; Stark et al., 1990). The 5' terminal part of the pestiviral ORF is assumed to code for the nucleocapsid protein (Collett et al., 1988b; RiJmenapfet al., 1991 a). Accordingly, all previous reports about structural proteins have been either wrong or at least incomplete (Pritchett and Zee, 1975; Matthaeus, 1979; Enzman, 1982, 1988; Purchio et al., 1984; Wensvoort et al., 1990). One of the glycoproteins, BVDV gp53/HCV gp 55, has been indirectly shown to be located at the surface of virions since it mediates neutralization by monoclonal antibodies (Donis et al., 1988; Weiland et al., 1990; Wensvoort et al., 1990).
Structural proteins of pestiviruses Using an efficient cell culture system for HCV propagation (Riimenapf et al., 1991b) as well as specific antibodies against different components assumed to be part of the virion, the molecular composition of virions from the genus pestivirus has recently been elucidated (Thiel et al., 1991 ). The different serological reagents employed to identify (glyco)proteins encoded in the 5' terminal third of the HCV genome are outlined in Fig. 1; among these were
, pz3
:p 4 H gp44/,m.__, gF,=
[,.......
"
t
t
~bodi~
mabs
mal~
antisera against bacterial fusion proteins
: putative signal sequence
Fig. 1. Antibodies against putative structural proteins.
STRUCTURE
AND TAXONOMIC
STATUS OF PESTIVIRUSES
t iiJis IIll .
215
216
H.-J. THIEL ET AL.
monodonal antibodies (MAbs) against gp55 and gp44/48, antisera against two bacterial fusion proteins encompassing the first part of the ORF (amino acids 1-55 and 102-292, respectively) as well as antibodies against peptides corresponding to the region just before the putative signal sequence of the glycoprotein region (amino acids 224-240 and 237-248, respectively). Extracts from radioactively labeled virions were used for immunoprecipitation experiments employing these highly specific reagents. As a result the molecular composition of HCV virions and the organization of the 5' region of the pestivirus genome was elucidated: the first protein of the ORF, HCV p23, is followed by the nucleocapsid protein p14 and a putative signal sequence which probably initiates translocation of HCV gp44/48 and two additional glycoproteins, HCV gp33 and HCV gp55 (Fig. 2 ). All three glycoproreins form parts of disulfide-linked dimers which are present in infected cells as well as virions (Thiel et al., 1991 ). First protein of HCV ORF is a nonstructural protein
The first protein of the pestivirus ORF is represented by HCV p23/BVDV p20 which does not form part of the virion (Thiel et al., 1991 ). According to pulse chase experiments, the cleavage between HCV p23 and p14 is not only complete but also extremely rapid. This result suggests an autoproteolytic activity. Additional experiments which included in vitro transcription/translation of defined cDNA constructs indicated that the proteolytic activity resides in HCV p23 (unpubl. results, Fig. 3 ). For BVDV p20 it has been recently demonstrated that the respective protein represents an autoprotease (Wiskerchen et al., 1991 ). Further efforts will be directed toward characterization 1
164/165 (?)
,. . . .
249/250 (?)
i
1
1
._p23 . . . .
o 23 - Evidence
for
n_
p14
&otoorotemse
- in vitro transcriplio~/translation of eDNA constructs •
?
-similarity to conserved motif of serine i r ~ J i r ~ alpt~virus capsid
pro~ea~ ( GDSG, GSDG ) - ck~raOe see : a l p ~ r u s
po~mus
1
WS
w v (?)
Fig. 3. Autoprotease and nucleocapsid protein of hog cholera virus.
217
STRUCTURE AND TAXONOMIC STATUS OF PESTIVIRUSES
of both, the cleavage site and the protease itself. With regard to the former it will be important to rely not only on site-directed mutagenesis but also to purify and sequence the N-terminus of p14 and possibly also the C-terminus of HCV p23.
Taxonomy With regard to classification of pestiviruses, some characteristics of the structural proteins are noteworthy. The nucleocapsid protein represents an internal fragment of the polyprotein. It is postulated that generation of the Nterminus of the nucleocapsid protein is due to a proteolytic activity of the nonstructural protein HCV p23. In addition, three glycoproteins, all of which form covalently linked dimers, are apparently part of the virus envelope (Fig. 2 ). With respect to the strategy of gene expression and genome organization, pestiviruses resemble flaviviruses (Collett et al., 1988c, 1989), and it has therefore been decided that the genus Pestivirus be included in the family Flaviviridae. Hepatitis C virus, a nonA nonB hepatitis virus, shows similar characteristics and was also included in the same family. However, some objections to this decision have been put forward (Meyers et al., 1989; Moormann et al., 1990; Weiland et al., 1990) and the study about HCV structural proteins (Thiel et al., 1991 ) indicates additional fundamental differences between the three groups. The differences concern virion composition and probably also virion structure, as well as a nonstructural autoprotease which represents the first protein from only the pestivirus ORF. Fig. 4 shows schematically the genomic localization of the structural proteins from all three groups (for hepatitis C: Hijikata et al., 1991 ). The differences are obvious and it appears justified to consider separate virus families. On the other hand
.
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.
FLAVMRUS ~ a t
[ - - . ._C . . . .
M
][_ . . . . . . . . . . . . . . . .
E.....
nsl
.....
]
PIESTM~
m~,~/~, _i.-]l---~-~,,
IF_
~,s6
HEPATITIS C V I R U S
P-±i~--Jl---~.-i-i
lIT
__
~ _ _
-. . . .
!
Fig. 4. Comparisonof proteins encodedin 5'-terminal part of ORF.
l
,~
218
H.-J. THIEL ET AL.
o n e c o u l d also use the following criteria to include d i f f e r e n t viruses in the family Flaviviridae: ( 1 ) e n v e l o p e d plus s t r a n d e d R N A viruses; ( 2 ) strategy o f g e n e expression; ( 3 ) structural p r o t e i n s e n c o d e d in the 5' t e r m i n a l region; ( 4 ) p o l y m e r a s e e n c o d e d in 3' region; ( 5 ) structural a n d p r o b a b l y also functional similarity between s o m e n o n s t r u c t u r a l proteins ( C h a m b e r s et al., 1990; W i s k e r c h e n a n d Collett, 1991 ). Interestingly, the n u c l e o t i d e s e q u e n c e in the 5' u n t r a n s l a t e d region o f hepatitis C virus c o n t a i n s blocks o f s e q u e n c e h o m o l o g y with pestiviruses but not with o t h e r viruses ( H a n et al., 1991 ). T h i s finding indicates t h a t pestiviruses are m o r e closely related to hepatitis C virus t h a n to flaviviruses. W i t h o u t any d o u b t we h a v e m u c h m o r e to learn a b o u t the t h r e e groups, especially pestiviruses and hepatitis C virus. A m o n g o t h e r benefits the studies will c e r t a i n l y help to b r o a d e n o u r view c o n c e r n i n g classification o f viruses in general. ACKNOWLEDGEMENTS T h i s s t u d y was j o i n t l y s u p p o r t e d by the B u n d e s m i n i s t e r f'tir F o r s c h u n g u n d T e c h n o l o g i e a n d I n t e r v e t I n t e r n a t i o n a l BV ( p r o j e c t No. 0 3 1 9 0 2 8 A ) .
REFERENCES Chambers, T.J., Weir, R.C., Grakoui, A., McCourt, D.W., Bazan, J.F., Fletterick, R.J. and Rice, C.M., 1990. Evidence that the N-terminal domain ofnonstructural protein NS3 from yellow fever virus is a serine protease responsible for site-specific cleavages in the viral polyprotein. Proc. Natl. Acad. Sci. USA, 87: 8898-8902. Collett, M.S., Larson, R., Gold, C., Strick, D., Anderson, D.K. and Purchio, A.F., 1988a. Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus. Virology, 165: 191-199. Collett, M.S., Larson, R., Belzer, S.K. and Retzel, E., 1988b. Proteins encoded by bovine viral diarrhea virus: the genomic organization ofa pestivirus. Virology, 165: 200-208. Collett, M.S., Anderson, D.K. and Retzel, R., 1988c. Comparisons ofthe pestivirus bovine viral diarrhea virus with members of the flaviviridae. J. Gen. Virol., 69: 2637-2643. Collett, M.S., Moennig, V. and Horzinek, M.C., 1989. Review article: Recent advances in pestivirus research. J. Gen. Virol., 70: 253-266. Donis, R.O., Corapi, W. and Dubovi, E.J., 1988. Neutralizing monoclonal antibodies to bovine diarrhoea virus bind to the 56K to 58K glycoprotein. J. Gen. Virol., 69: 77-86. Enzmann, P.-J., 1982. In vivo labelling of viral proteins with 75 Selenomethionine. J. Virol. Methods, 5: 243-246. Enzmann, P.-J., 1988. Molecular biology of the virus. In: B. Liess (Editor), Classical Swine Fever and Related Viral Infections. Martinus Nijhoff, Boston, MA, pp. 81-98. Han, J.H., Shymala, V., Richman, K.H., Brauer, M.H., Irvine, B., Urdea, M.S., Tekamp-Oison, P., Kuo, G., Choo, Q.-L. and Houghton, M., 1991. Characterization of the terminal regions of hepatitis C viral RNA: identification of conserved sequences in the 5' untranslated region and poly(A ) tails at the 3' end. Proc. Natl. Acad. Sci. USA, 88:1711-1715. Hijikata, M.N., Kato, N., Ootsuyama, Y., Nakagawa, M. and Shimotohno, K., 1991. Gene map-
STRUCTURE AND TAXONOMIC STATUS OF PESTIVIRUSES
2 !9
ping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis. Proc. Natl. Acad. Sci. USA, 88: 5547-5551. Matthaeus, W., 1979. Detection of three polypeptides in preparations of bovine viral diarrhea virus. Arch. Virol., 59: 299-305. Meyers, G., Riimenapf, T. and Thiel, H.-J., 1989. Molecular cloning and nucleotide scquence of the genome of hog cholera virus. Virology, 17 l: 555-567. Moormann, R.J.M., Warmerdam, P.A., Van der Meer, B., Schaaper, W.M.M., Wensvoort, G. and Hulst, M.M., 1990. Molecular cloning and nucleotide sequence of hog cholera virus strain Brescia and mapping of the genomic region encoding envelope protein El. Virology, 177: 184-198. Pritchett, R.F. and Zee, Y.C., 1975. Structural proteins of bovine viral diarrhea virus. Am. J. Vct. Res., 36: 1731-1734. Purchio, A.F., Larson, R. and Collett, M.S., 1984. Characterization of bovine viral diarrhea virus proteins. J. Virol., 50: 666-669. Riimenapf, T., Meyers, G., Stark, R. and Thiel, H.-J., 1989. Hog cholera virus---characterization of specific antiserum and identification ofcDNA clones. Virology, 17 l: 18-27. Riimenapf, T., Stark, R., Meyers, G. and Thiel, H.-J., 1991 a. Structural proteins of hog cholera virus expressed by vaccinia virus: further characterization and induction of protective immunity. J. Virol., 65: 589-597. RiJmenapf, T., Meyers, G., Stark, R. and Thiel, H.-J., 1991b. Molecular characterization of hog cholera virus. Arch. Virol., Suppl. 3: 7-18. Stark, R., Riimenapf, T., Meyers, G. and Thiel, H.-J., 1990. Genomic localization of hog cholera virus glycoproteins. Virology, 174: 286-289. Thiel, H.-J., Stark, R., Weiland, E., Riimenapf, T. and Meyers, G., 1991. Hog cholera virus: molecular composition of virions from a pestivirus. J. Virol., 65:4705-4712. Weiland, E., Stark, R., Haas, B., RiJmenapf, T., Meyers, G. and Thiel, H.-J., 1990. Pestivirus glycoprotein which induces neutralizing antibodies forms part of a disulfide linked heterodimer. J. Virol., 64: 3563-3569. Wensvoort, G., Boonstra, J. and Bodzinga, B.G., 1990. Immunoaffinity purification and characterization of the envelope protein E 1 of hog cholera virus. J. Gen. Virol., 71: 531-540. Westaway, E.G., Brinton, M.A., Gaidamovich, S.Y.A., Horzinek, M.C., lgarashi, A., K/i/iriiSinen, L., Lvov, D.K., Porterfieid, J.S., Russel, P.K. and Trent, D.W., 1985. Togaviridae. Intervirology, 24: 125-139. Wiskerchen, M. and Collett, M.S., 1991. Pestivirus gene expression: protein p80 of bovine viral diarrhea virus is a proteinase involved in polyprotein processing. Virology, 184:341-350. Wiskerchen, M., Belzer, S.L. and Collett, M.S., 1991. Pestivirus gene expression: the first protein product of the bovine viral diarrhea virus large open reading frame, p20, possesses proteolytic activity, J. Virol., 65:4508-4514.
213
Proteins encoded in the 5' region of the pestivirus genome considerations concerning taxonomy Heinz-Jiirgen Thiel, Robert Stark, Gregor Meyers, Emilie Weiland and Tillmann Riimenapf Federal Research Centre for Virus Diseases of Animals, Tiibingen, FRG (Accepted 26 June 1992 )
ABSTRACT Thiel, H.-J., Stark, R., Meyers, G., Weiland, E. and Riimenapf, T., 1992. Proteins encoded in the 5' region of the pestivirus genome---considerations concerning taxonomy. Vet. Microbiol., 33: 213219. The first protein encoded within the pestivirus open reading frame is a nonstructural protein which removes itself from the polyprotein by autoproteolytic cleavage. The following nucleocapsid protein ends just before a putative signal sequence preceding three glycosylated proteins. All three glycoproteins are part of the viral envelope and exist in the form of disulfide-linked dimers. Pestiviruses have recently been reclassified as members of the family Flaviviridae which now comprises three genera, namely flavivirus, hepatitis C virus group and pestivirus. All members of the family have certain characteristics in common like the overall genome organization and the strategy ofgene expression. Major differences exist, however, between the genera; the most obvious ones concern proteins encoded in the 5' region of the respective genomes.
INTRODUCTION
Hog cholera virus (HCV), bovine viral diarrhea virus (BVDV) and border disease virus of sheep are grouped together in the genus pestivirus (Westaway et al., 1985 ). Pestiviruses have a single-stranded RNA genome which is about 12.5 kb in length (Collett et al., 1988a; Riimenapf et al., 1989). The RNA is of positive polarity and comprises a single large open reading frame (ORF) (Collett et al., 1988a,b; Meyers et al., 1989; Moormann et al., 1990). Viral gene expression is believed to occur via synthesis of a polyprotein which is processed co- and posttranslationally. The different proteins encoded by pestiviruses were mostly identified by using antibodies against bacterial fusion Correspondence to: H.-J. Thiel, Federal Research Centre for Virus Diseases of Animals, P.O. Box 11 49, D-7400 Tiibingen, FRG.
0378-1135/92/$05.00 © 1992 Elsevier Science Publishers B.V. All rights reserved.
214
H.-J. THIEL ET AL.
proteins encompassing defined regions of the viral genome (Collett et al., 1988b; Stark et al., 1990). From these analyses it was concluded that the structural proteins are encoded in the 5' terminal part of the pestiviral ORF while RNA-dependent RNA polymerase is encoded in the 3' region. Until recently pestiviruses held generic status in the family Togaviridae (Westaway et al., 1985). Since genome organization and strategy of gene expression are more similar to flaviviruses than to togaviruses it has been decided to include pestiviruses as an additional genus in the family Flaviviridae. The molecular composition of virions from the genus pestivirus has long been a matter of controversy. According to the data obtained after immunoprecipitation from virus-infected cells by using antisera against bacterial fusion proteins, pestiviruses probably possess four structural proteins, three of which are glycosylated (Collett et al., 1988b; Stark et al., 1990). The 5' terminal part of the pestiviral ORF is assumed to code for the nucleocapsid protein (Collett et al., 1988b; RiJmenapfet al., 1991 a). Accordingly, all previous reports about structural proteins have been either wrong or at least incomplete (Pritchett and Zee, 1975; Matthaeus, 1979; Enzman, 1982, 1988; Purchio et al., 1984; Wensvoort et al., 1990). One of the glycoproteins, BVDV gp53/HCV gp 55, has been indirectly shown to be located at the surface of virions since it mediates neutralization by monoclonal antibodies (Donis et al., 1988; Weiland et al., 1990; Wensvoort et al., 1990).
Structural proteins of pestiviruses Using an efficient cell culture system for HCV propagation (Riimenapf et al., 1991b) as well as specific antibodies against different components assumed to be part of the virion, the molecular composition of virions from the genus pestivirus has recently been elucidated (Thiel et al., 1991 ). The different serological reagents employed to identify (glyco)proteins encoded in the 5' terminal third of the HCV genome are outlined in Fig. 1; among these were
, pz3
:p 4 H gp44/,m.__, gF,=
[,.......
"
t
t
~bodi~
mabs
mal~
antisera against bacterial fusion proteins
: putative signal sequence
Fig. 1. Antibodies against putative structural proteins.
STRUCTURE
AND TAXONOMIC
STATUS OF PESTIVIRUSES
t iiJis IIll .
215
216
H.-J. THIEL ET AL.
monodonal antibodies (MAbs) against gp55 and gp44/48, antisera against two bacterial fusion proteins encompassing the first part of the ORF (amino acids 1-55 and 102-292, respectively) as well as antibodies against peptides corresponding to the region just before the putative signal sequence of the glycoprotein region (amino acids 224-240 and 237-248, respectively). Extracts from radioactively labeled virions were used for immunoprecipitation experiments employing these highly specific reagents. As a result the molecular composition of HCV virions and the organization of the 5' region of the pestivirus genome was elucidated: the first protein of the ORF, HCV p23, is followed by the nucleocapsid protein p14 and a putative signal sequence which probably initiates translocation of HCV gp44/48 and two additional glycoproteins, HCV gp33 and HCV gp55 (Fig. 2 ). All three glycoproreins form parts of disulfide-linked dimers which are present in infected cells as well as virions (Thiel et al., 1991 ). First protein of HCV ORF is a nonstructural protein
The first protein of the pestivirus ORF is represented by HCV p23/BVDV p20 which does not form part of the virion (Thiel et al., 1991 ). According to pulse chase experiments, the cleavage between HCV p23 and p14 is not only complete but also extremely rapid. This result suggests an autoproteolytic activity. Additional experiments which included in vitro transcription/translation of defined cDNA constructs indicated that the proteolytic activity resides in HCV p23 (unpubl. results, Fig. 3 ). For BVDV p20 it has been recently demonstrated that the respective protein represents an autoprotease (Wiskerchen et al., 1991 ). Further efforts will be directed toward characterization 1
164/165 (?)
,. . . .
249/250 (?)
i
1
1
._p23 . . . .
o 23 - Evidence
for
n_
p14
&otoorotemse
- in vitro transcriplio~/translation of eDNA constructs •
?
-similarity to conserved motif of serine i r ~ J i r ~ alpt~virus capsid
pro~ea~ ( GDSG, GSDG ) - ck~raOe see : a l p ~ r u s
po~mus
1
WS
w v (?)
Fig. 3. Autoprotease and nucleocapsid protein of hog cholera virus.
217
STRUCTURE AND TAXONOMIC STATUS OF PESTIVIRUSES
of both, the cleavage site and the protease itself. With regard to the former it will be important to rely not only on site-directed mutagenesis but also to purify and sequence the N-terminus of p14 and possibly also the C-terminus of HCV p23.
Taxonomy With regard to classification of pestiviruses, some characteristics of the structural proteins are noteworthy. The nucleocapsid protein represents an internal fragment of the polyprotein. It is postulated that generation of the Nterminus of the nucleocapsid protein is due to a proteolytic activity of the nonstructural protein HCV p23. In addition, three glycoproteins, all of which form covalently linked dimers, are apparently part of the virus envelope (Fig. 2 ). With respect to the strategy of gene expression and genome organization, pestiviruses resemble flaviviruses (Collett et al., 1988c, 1989), and it has therefore been decided that the genus Pestivirus be included in the family Flaviviridae. Hepatitis C virus, a nonA nonB hepatitis virus, shows similar characteristics and was also included in the same family. However, some objections to this decision have been put forward (Meyers et al., 1989; Moormann et al., 1990; Weiland et al., 1990) and the study about HCV structural proteins (Thiel et al., 1991 ) indicates additional fundamental differences between the three groups. The differences concern virion composition and probably also virion structure, as well as a nonstructural autoprotease which represents the first protein from only the pestivirus ORF. Fig. 4 shows schematically the genomic localization of the structural proteins from all three groups (for hepatitis C: Hijikata et al., 1991 ). The differences are obvious and it appears justified to consider separate virus families. On the other hand
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.
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.
.
FLAVMRUS ~ a t
[ - - . ._C . . . .
M
][_ . . . . . . . . . . . . . . . .
E.....
nsl
.....
]
PIESTM~
m~,~/~, _i.-]l---~-~,,
IF_
~,s6
HEPATITIS C V I R U S
P-±i~--Jl---~.-i-i
lIT
__
~ _ _
-. . . .
!
Fig. 4. Comparisonof proteins encodedin 5'-terminal part of ORF.
l
,~
218
H.-J. THIEL ET AL.
o n e c o u l d also use the following criteria to include d i f f e r e n t viruses in the family Flaviviridae: ( 1 ) e n v e l o p e d plus s t r a n d e d R N A viruses; ( 2 ) strategy o f g e n e expression; ( 3 ) structural p r o t e i n s e n c o d e d in the 5' t e r m i n a l region; ( 4 ) p o l y m e r a s e e n c o d e d in 3' region; ( 5 ) structural a n d p r o b a b l y also functional similarity between s o m e n o n s t r u c t u r a l proteins ( C h a m b e r s et al., 1990; W i s k e r c h e n a n d Collett, 1991 ). Interestingly, the n u c l e o t i d e s e q u e n c e in the 5' u n t r a n s l a t e d region o f hepatitis C virus c o n t a i n s blocks o f s e q u e n c e h o m o l o g y with pestiviruses but not with o t h e r viruses ( H a n et al., 1991 ). T h i s finding indicates t h a t pestiviruses are m o r e closely related to hepatitis C virus t h a n to flaviviruses. W i t h o u t any d o u b t we h a v e m u c h m o r e to learn a b o u t the t h r e e groups, especially pestiviruses and hepatitis C virus. A m o n g o t h e r benefits the studies will c e r t a i n l y help to b r o a d e n o u r view c o n c e r n i n g classification o f viruses in general. ACKNOWLEDGEMENTS T h i s s t u d y was j o i n t l y s u p p o r t e d by the B u n d e s m i n i s t e r f'tir F o r s c h u n g u n d T e c h n o l o g i e a n d I n t e r v e t I n t e r n a t i o n a l BV ( p r o j e c t No. 0 3 1 9 0 2 8 A ) .
REFERENCES Chambers, T.J., Weir, R.C., Grakoui, A., McCourt, D.W., Bazan, J.F., Fletterick, R.J. and Rice, C.M., 1990. Evidence that the N-terminal domain ofnonstructural protein NS3 from yellow fever virus is a serine protease responsible for site-specific cleavages in the viral polyprotein. Proc. Natl. Acad. Sci. USA, 87: 8898-8902. Collett, M.S., Larson, R., Gold, C., Strick, D., Anderson, D.K. and Purchio, A.F., 1988a. Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus. Virology, 165: 191-199. Collett, M.S., Larson, R., Belzer, S.K. and Retzel, E., 1988b. Proteins encoded by bovine viral diarrhea virus: the genomic organization ofa pestivirus. Virology, 165: 200-208. Collett, M.S., Anderson, D.K. and Retzel, R., 1988c. Comparisons ofthe pestivirus bovine viral diarrhea virus with members of the flaviviridae. J. Gen. Virol., 69: 2637-2643. Collett, M.S., Moennig, V. and Horzinek, M.C., 1989. Review article: Recent advances in pestivirus research. J. Gen. Virol., 70: 253-266. Donis, R.O., Corapi, W. and Dubovi, E.J., 1988. Neutralizing monoclonal antibodies to bovine diarrhoea virus bind to the 56K to 58K glycoprotein. J. Gen. Virol., 69: 77-86. Enzmann, P.-J., 1982. In vivo labelling of viral proteins with 75 Selenomethionine. J. Virol. Methods, 5: 243-246. Enzmann, P.-J., 1988. Molecular biology of the virus. In: B. Liess (Editor), Classical Swine Fever and Related Viral Infections. Martinus Nijhoff, Boston, MA, pp. 81-98. Han, J.H., Shymala, V., Richman, K.H., Brauer, M.H., Irvine, B., Urdea, M.S., Tekamp-Oison, P., Kuo, G., Choo, Q.-L. and Houghton, M., 1991. Characterization of the terminal regions of hepatitis C viral RNA: identification of conserved sequences in the 5' untranslated region and poly(A ) tails at the 3' end. Proc. Natl. Acad. Sci. USA, 88:1711-1715. Hijikata, M.N., Kato, N., Ootsuyama, Y., Nakagawa, M. and Shimotohno, K., 1991. Gene map-
STRUCTURE AND TAXONOMIC STATUS OF PESTIVIRUSES
2 !9
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