The NS5A protein of hepatitis C virus is a zinc metalloprotein

被引:272
作者
Tellinghuisen, TL
Marcotrigiano, J
Gorbalenya, AE
Rice, CM
机构
[1] Rockefeller Univ, Ctr Study Hepatitis C, Lab Virol & Infect Dis, New York, NY 10021 USA
[2] Leiden Univ, Med Ctr, Ctr Infect Dis, Dept Med Microbiol, NL-2300 RC Leiden, Netherlands
关键词
D O I
10.1074/jbc.M407787200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The NS5A protein of hepatitis C virus is believed to be an integral part of the viral replicase. Despite extensive investigation, the role of this protein remains elusive. Only limited biochemical characterization of NS5A has been performed, with most research to date involving the myriad of host proteins and signaling cascades that interact with NS5A. The need for better characterization of NS5A is paramount for elucidating the role of this protein in the virus life cycle. Examination of NS5A using bioinformatics tools suggested the protein consisted of three domains and contained an unconventional zinc binding motif within the N-terminal domain. We have developed a method to produce NS5A and performed limited proteolysis to confirm the domain organization model. The zinc content of purified NS5A and the N-terminal domain of NS5A was determined, and each of these proteins was found to coordinate one zinc atom per protein. The predicted zinc binding motif consists of four cysteine residues, conserved among the Hepacivirus and Pestivirus genera, fitting the formula of CX17CXCX20C. Mutation of any of the four cysteine components of this motif reduced NS5A zinc coordination and led to a lethal phenotype for HCV RNA replication, whereas mutation of other potential metal coordination residues in the N-terminal domain of NS5A, but outside the zinc binding motif, had little effect on zinc binding and, aside from one exception, were tolerated for replication. Collectively, these results indicate that NS5A is a zinc metalloprotein and that zinc coordination is likely required for NS5A function in the hepatitis C replicase.
引用
收藏
页码:48576 / 48587
页数:12
相关论文
共 64 条
[1]   Analysis of zinc binding sites in protein crystal structures [J].
Alberts, IL ;
Nadassy, K ;
Wodak, SJ .
PROTEIN SCIENCE, 1998, 7 (08) :1700-1716
[2]   Modulation of cell growth by the hepatitis C virus nonstructural protein NS5A [J].
Arima, N ;
Kao, CY ;
Licht, T ;
Padmanabhan, R ;
Sasaguri, Y ;
Padmanabhan, R .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2001, 276 (16) :12675-12684
[3]  
BERG JM, 1990, J BIOL CHEM, V265, P6513
[4]   The galvanization of biology: A growing appreciation for the roles of zinc [J].
Berg, JM ;
Shi, YG .
SCIENCE, 1996, 271 (5252) :1081-1085
[5]   Highly permissive cell lines for subgenomic and genomic hepatitis C virus RNA replication [J].
Blight, KJ ;
McKeating, JA ;
Rice, CM .
JOURNAL OF VIROLOGY, 2002, 76 (24) :13001-13014
[6]   Efficient initiation of HCV RNA replication in cell culture [J].
Blight, KJ ;
Kolykhalov, AA ;
Rice, CM .
SCIENCE, 2000, 290 (5498) :1972-1974
[7]   An amino-terminal amphipathic α-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A [J].
Brass, V ;
Bieck, E ;
Montserret, R ;
Wölk, B ;
Hellings, JA ;
Blum, HE ;
Penin, F ;
Moradpour, D .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2002, 277 (10) :8130-8139
[8]   ISOLATION OF A CDNA CLONE DERIVED FROM A BLOOD-BORNE NON-A, NON-B VIRAL-HEPATITIS GENOME [J].
CHOO, QL ;
KUO, G ;
WEINER, AJ ;
OVERBY, LR ;
BRADLEY, DW ;
HOUGHTON, M .
SCIENCE, 1989, 244 (4902) :359-362
[9]   Nonstructural protein 5A of hepatitis C virus inhibits the function of karyopherin β3 [J].
Chung, KM ;
Lee, J ;
Kim, JE ;
Song, OK ;
Cho, S ;
Lim, J ;
Seedorf, M ;
Hahm, B ;
Jang, SK .
JOURNAL OF VIROLOGY, 2000, 74 (11) :5233-5241
[10]   Hepatitis C virus NS5A as a potential viral Bcl-2 homologue interacts with Bax and inhibits apoptosis in hepatocellular carcinoma [J].
Chung, YL ;
Sheu, ML ;
Yen, SH .
INTERNATIONAL JOURNAL OF CANCER, 2003, 107 (01) :65-73