The Amino Terminal Deletion Mutants of Hepatitis C Virus Nonstructural Protein NS5A Function as Transcriptional Activators in Yeast

doi:10.1006/bbrc.1997.6967

Biochemical and Biophysical Research Communications

Volume 236, Issue 2, 18 July 1997, Pages 360-364

https://doi.org/10.1006/bbrc.1997.6967 Get rights and content

Abstract

To investigate the biological function of hepatitis C virus (HCV)-NS5A, the NS5A was fused at its N-terminus with the DNA binding domain (DBD) of yeast transcriptional activator GAL4 (GAL4-DBD). The GAL4-DBD alone had no transcriptional activation function. However, a mutant of the GAL4-DBD/NS5A fusion protein, in which 129 amino acid residues were deleted from the N-terminus of NS5A, exhibited strong transcriptional activation in yeast cells, bearing the Escherichia colilacZreporter gene encoding the β-galactosidase under the transcriptional control of GAL4 promoter and TATA box. Further mutational analysis of NS5A revealed that the region between the amino acid residues 130 to 352 were critical for optimal level of transactivation. This region includes two acidic domains and one proline-rich region which have been shown to be involved in the function of several transcriptional activators.

References (36)

T. Kaneko et al.
Biochem. Biophys. Res. Commun.
(1994)
J. Ma et al.
Cell
(1987)
N. Mermod et al.
Cell
(1989)
S. Hahn
Cell
(1993)
K.K. Leuther et al.
Cell
(1993)
K. Struhl
Cell
(1987)
Y.S. Lin et al.
Cell
(1991)
Y. Ide et al.
Gene
(1996)
J. Ma et al.
Cell
(1987)
R.H. Purcell
Proc. Natl. Acad. Sci. U.S.A.
(1994)

K. Shimotohno

Intervirology

(1995)

Q.L. Choo et al.

Science

(1989)

A. Grakoui et al.

J. Virol.

(1993)

M. Hijikata et al.

Proc. Natl. Acad. Sci. U.S.A.

(1993)

L. Tomei et al.

J. Virol.

(1993)

K. Shimotohno et al.

J. Hepatol.

(1995)

S.E. Behrens et al.

Embo. J.

(1996)

Cited by (70)

Sequence diversity of hepatitis C virus 6a within the extended interferon sensitivity-determining region correlates with interferon-alpha/ribavirin treatment outcomes
2010, Virus Research
Studies on the association between sequence variability of the interferon sensitivity-determining region (ISDR) of hepatitis C virus and the outcome of treatment have reached conflicting results. In this study, 25 patients infected with HCV 6a who had received interferon-alpha/ribavirin combination treatment were analyzed for the sequence variations. 14 of them had the full genome sequences obtained from a previous study, whereas the other 11 samples were sequenced for the extended ISDR (eISDR). This eISDR fragment covers 192 bp (64 amino acids) upstream and 201 bp (67 amino acids) downstream from the ISDR previously defined for HCV 1b. The comparison between interferon-alpha resistance and response groups for the amino acid mutations located in the full genome (6 and 8 patients respectively) as well as the mutations located in the eISDR (10 and 15 patients respectively) showed that the mutations I2160V, I2256V, V2292I (P < 0.05) within eISDR were significantly associated with resistance to treatment. However, the extent of amino acid variations within previously defined ISDR was not associated with resistance to treatment as previously reported. Four amino acid variations I248V (P = 0.03–0.06) within E1, R445K (P = 0.02–0.05) and S747T (P = 0.03) within E2, I861V (P = 0.01) within NS2 which located outside the eISDR may also associate with treatment outcome as identified by a prescreening of variations within 14 HCV 6a full genomes.
Phosphorylation of hepatitis C virus NS5A nonstructural protein: A new paradigm for phosphorylation-dependent viral RNA replication?
2007, Virology
Citation Excerpt :
Interestingly, nuclear localization of NS5A has also been reported (Ide et al., 1996), and caspase-mediated cleavage products of NS5A, which lack the membrane-anchor region, are also detected in the nucleus under certain apoptotic conditions (Goh et al., 2001; Kalamvoki and Mavromara, 2004; Satoh et al., 2000). Coincidentally, the carboxyl-terminal domain of NS5A, including the ISDR, has been shown to exert potent trans-activating activity, suggesting that NS5A might function as a viral transcriptional activator (Chung et al., 1997; Kato et al., 1997; Tanimoto et al., 1997). Moreover, the transcriptional activity of NS5A appeared to be affected by mutations in the ISDR.
The hepatitis C virus (HCV) nonstructural 5A (NS5A) phosphoprotein has been intensely studied due to its ability to subvert the host interferon-induced antiviral response. However, more recent studies suggest that it may also play an important regulatory role in HCV RNA replication as well as modulate host intracellular signaling pathways. Phosphorylation of NS5A appears to be a highly regulated process and several cellular protein kinases responsible for NS5A phosphorylation have been identified in vitro. Studies utilizing the HCV replicon cell culture system have suggested a provocative role for the differential phosphorylation of NS5A in the regulation of viral RNA replication through its association with the viral replication complex, including several host cell factors. Importantly, recent in vivo data linking loss of NS5A hyperphosphorylation to non-productive HCV replication in the chimpanzee model have provided high validation for targeting the cellular kinases involved, particularly the kinases responsible for NS5A phosphorylation, for antiviral therapeutic intervention. Understanding the process of NS5A phosphorylation and the definite identification of the culprit cellular protein kinase(s) will shed light on the mechanisms of HCV RNA replication and/or pathogenesis.
The NS5A protein of the hepatitis C virus genotype 1a is cleaved by caspases to produce C-terminal-truncated forms of the protein that reside mainly in the cytosol
2006, Journal of Biological Chemistry
The nonstructural 5A (NS5A) protein of the hepatitis C virus (HCV) is a multifunctional protein that is implicated in viral replication and pathogenesis. We report here that NS5A of HCV-1a is cleaved at multiple sites by caspase proteases in transfected cells. Two cleavage sites at positions Asp¹⁵⁴ and ²⁴⁸DXXD²⁵¹ were mapped. Cleavage at Asp¹⁵⁴ has been previously recognized as one of the caspase cleavage sites for the NS5A protein of HCV genotype 1b (1, 2) and results in the production of a 17-kDa fragment. The sequence ²⁴⁸DXXD²⁵¹ is a novel caspase recognition motif for NS5A and is responsible for the production of a 31-kDa fragment. Furthermore, we show that Arg²¹⁷ is implicated in the production of the previously described 24-kDa product, whose accumulation is affected by both calpain and caspase inhibitors. We also showed that caspase-mediated cleavage occurs in the absence of exogenous proapoptotic stimuli and is not related to the accumulation of the protein in the endoplasmic reticulum. Interestingly, our data indicate that NS5A is targeted by at least two different caspases and suggest that caspase 6 is implicated in the production of the 17-kDa fragment. Most importantly, we report that, all the detectable NS5A fragments following caspase-mediated cleavage are C-terminal-truncated forms of NS5A and are mainly localized in the cytosol. Thus, in sharp contrast to the current view we found no evidence supporting a role for caspase-mediated cleavage in the transport of the NS5A protein to the nucleus, which could lead to transcriptional activation.
Characterization of a HCV NS5A protein derived from a patient with hepatoma
2005, Biochemical and Biophysical Research Communications
A cDNA encoding hepatitis C virus NS5A protein was isolated from the serum of a patient with hepatocellular carcinoma. The NS5A^HCC was localized in both the cytoplasmic and nuclear fractions of Huh-7 cells. Immunoprecipitation and electrophoresis experiments showed four major phosphorylated species of NS5A^HCC, p58, p56, p53, and p50. Two mutants (NS5A^HCC-NLSmt and NS5A^HCC-TSmt) carrying mutations on the putative nuclear localization signal were engineered. NS5A^HCC-NLSmt was localized exclusively in the cytoplasm, whereas some forms of NS5A^HCC-TSmt can be transported into the nucleus. These NS5A^HCC mutant proteins were capable of transactivating c-fos and SV40 promoters. However, the transactivation efficiency was not dependent on its capability of nuclear localization. Subsequently, interaction between NS5A^HCC mutants and Grb2 was studied. While capable of transactivating oncogenic promoters, NS5A^HCC-TSmt could not interact with Grb2. Our results suggested that other cytosolic pathways independent of Grb2-mediated mechanisms were involved in the transactivation activity of HCV NS5A.
Purification and characterization of hepatitis C virus non-structural protein 5A expressed in Escherichia coli
2004, Protein Expression and Purification
We have employed a pET-ubiquitin expression system to produce two his-tagged forms of hepatitis C virus (HCV) non-structural protein 5A (NS5A) in Escherichia coli. One derivative contains the full-length protein extended to include a carboxy-terminal hexahistidine tag; the other derivative contains an amino-terminal hexahistidine tag in place of the 32 amino acid amphipathic helix that mediates membrane association. At least 1 mg of each derivative at a purity of 90% could be produced from a 1-L culture. The purified derivatives produced high titer antibody that recognized both p56 and p58 forms of NS5A in Huh-7.5 cells expressing an HCV subgenomic replicon. The NS5A derivatives were efficiently phosphorylated by casein kinase II, leading to at least 5 mol of phosphate incorporated per mole of protein. Interestingly, this level of phosphorylation did not alter the migration of the protein in an SDS–polyacrylamide gel, suggesting that hyperphosphorylation alone is not sufficient to generate the p58 form of NS5A observed in Huh-7 cells. Neither NS5A derivative was capable of inhibiting the eIF2α-phosphorylation activity of the activated form of the double-stranded RNA-activated protein kinase, PKR, suggesting that NS5A phosphorylation may be required for this function of NS5A. However, both unphosphorylated derivatives were shown to interact with NS5B, the HCV RNA-dependent RNA polymerase, in solution by using a novel kinase-protection assay. The availability of purified HCV NS5A will permit rigorous biochemical and biophysical characterization of this protein, ultimately providing insight into the function of this protein during HCV genome replication.
Structural biology of hepatitis C virus
2003, Clinics in Liver Disease

View all citing articles on Scopus

¹: Corresponding Author: Dr. R. Padmanabhan, Department of Biochemistry and Molecular Biology, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7421.

View full text

Regular ArticleThe Amino Terminal Deletion Mutants of Hepatitis C Virus Nonstructural Protein NS5A Function as Transcriptional Activators in Yeast

Abstract

Biochem. Biophys. Res. Commun.

Cell

Cell

Cell

Cell

Cell

Cell

Gene

Cell

Proc. Natl. Acad. Sci. U.S.A.

Intervirology

Science

J. Virol.

Proc. Natl. Acad. Sci. U.S.A.

J. Virol.

J. Hepatol.

Embo. J.

Regular Article
The Amino Terminal Deletion Mutants of Hepatitis C Virus Nonstructural Protein NS5A Function as Transcriptional Activators in Yeast