Tài liệu Báo cáo khoa học: Specific interaction between the classical swine fever virus NS5B protein

Specific interaction between the classical swine fever virus NS5B

protein and the viral genome

Ming Xiao1,2, Jufang Gao2

, Wei Wang2

, Yujing Wang2

, Jun Chen2

, Jiakuan Chen1 and Bo Li1

1

Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, The Institute of Biodiversity Science,

Fudan University, Shanghai, China; 2

College of Life and Environment Sciences, Shanghai Normal University, China

The NS5B protein of the classical swine fever virus (CSFV) is

the RNA-dependent RNA polymerase of the virus and is

able to catalyze the viral genome replication. The 3¢ un￾translated region is most likely involved in regulation of the

Pestivirus genome replication. However, little is known

about the interaction between the CSFV NS5B protein and

the viral genome. We used different RNA templates derived

from the plus-strand viral genome, or the minus-strand viral

genome and the CSFV NS5B protein obtained from the

Escherichia coli expression system to address this problem.

We first showed that the viral NS5B protein formed a

complex with the plus-strand genome through the genomic

3¢ UTR and that the NS5B protein was also able to bind the

minus-strand 3¢ UTR. Moreover, it was found that viral

NS5B protein bound the minus-strand 3¢ UTR more effi￾ciently than the plus-strand 3¢ UTR. Further, we observed

that the plus-strand 3¢ UTR with deletion of CCCGG or 21

continuous nucleotides at its 3¢ terminal had no binding

activity and also lost the activity for initiation of minus￾strand RNA synthesis, which similarly occurred in the

minus-strand 3¢ UTR with CATATGCTC or the 21 nuc￾leotide fragment deleted from the 3¢ terminal. Therefore, it is

indicated that the 3¢ CCCGG sequence of the plus-strand

3¢ UTR, and the 3¢ CATATGCTC fragment of the minus￾strand are essential to in vitro synthesis of the minus-strand

RNA and the plus-strand RNA, respectively. The same

conclusion is also appropriate for the 3¢ 21 nucleotide

terminal site of both the 3¢ UTRs.

Keywords: CSFV; RdRp; replication; RNA synthesis;

3¢ UTR.

Classical swine fever virus (CSFV) is the causative agent of

swine fever, which is a highly contagious and fatal viral

disease of pigs. CSFV, bovine viral diarrhea virus (BVDV),

and Border Disease virus (BDV) are members of the

Pestivirus genus within the Flaviviridae family. BVDV and

BDV can infect both ruminants and pigs. The hepatitis C

virus (HCV), an etiological agent of non-A, non-B hepatitis,

also belongs to the Flaviviridae family.

Pestiviruses are small, enveloped, plus-strand RNA

viruses, similar to HCV. The RNA genome is
12.5 kb in

length, consisting of a large and continual open reading

frame (ORF), a 5¢ untranslated region (5¢ UTR) and a 3¢

untranslated region (3¢ UTR). The ORF is translated into a

polyprotein, which is further processed into 12 mature

proteins by viral and host cell proteases. The 12 proteins

comprise four structure proteins (C, Erns, E1, and E2) and

eight nonstructure proteins (Npro, P7, NS2, NS3, NS4A,

NS4B, NS5A, and NS5B). In the CSFV genome, the genes

encoding Npro, C, Erns, E1, E2, p7, and NS2 have proved to

be dispensable for RNA replication [1]. The 3¢ UTR and the

5¢ UTR are believed to regulate Pestivirus genome replica￾tion [2,3]. The Pestivirus genomic replication consists of

several consecutive processes. Repliase first recognizes and

binds the 3¢ UTR and starts RNA synthesis, in which a

minus-strand RNA is produced with the plus-strand

genomic RNA as a template. Then, a progeny plus-RNA

is produced with the novel minus-RNA as a template [4]. The

5¢ UTR is also the site for initiating translation of the viral

genomes, at which an internal ribosomal entry site (IRES) is

observed [5]. Short 3¢ terminal extensions do not interfere

with infectivity of in vitro transcript whereas 5¢ extensions

sometimes do and sometimes do not [6]. The CSFV NS5B

gene is located at the 3¢ end of the genome adjacent to the

3¢ UTR. The CSFV NS5B protein has an RNA-dependent

RNA polymerase (RdRp) activity, and thus plays a central

role in viral RNA replication [7–10]. Even NS5B as a fusion

protein with the green fluorescent protein still displays an

RdRp activity [11]. The NS5B proteins of BVDV and HCV

have been expressed in different systems and their biochemi￾cal properties have been studied [12–17]. NS5B protein is

able to catalyze RNA elongation by a primer-dependent or

copy-back mechanism, and can initiate RNA synthesis from

the 3¢ end of different RNA templates in vitro [7,9,15]. It is

reported that the mechanism for de novo initiation of RNA

synthesis is also associated with the NS5B proteins [17–21].

Moreover, the crystal structure of HCV NS5B protein has

been characterized [22].

Correspondence to B. Li, Ministry of Education Key Laboratory for

Biodiversity Science and Ecological Engineering, The Institute of

Biodiversity Science, Fudan University, Shanghai, 200433, China.

Fax: +86 21 6564246, Tel.: +86 21 65642178,

E-mail: [email protected]

Abbreviations: BDV, Border disease virus; BVDV, bovine viral

diarrhea virus; CSFV, classical swine fever virus; EMSA, electro￾phoretic mobility shift assay; HCV, hepatitis C virus; IRES, internal

ribosome entry site; RdRp, RNA-dependent RNA polymerase;

TNTase, terminal nucleotidyl transferase.

(Received 25 June 2004, revised 28 July 2004, accepted 6 August 2004)

Eur. J. Biochem. 271, 3888–3896 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2004.04325.x