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. 1999 Apr;73(4):2841-53.
doi: 10.1128/JVI.73.4.2841-2853.1999.

Hepatitis C virus core protein interacts with cellular putative RNA helicase

L R You  1 , C M Chen, T S Yeh, T Y Tsai, R T Mai, C H Lin, Y H Lee
Affiliations

Hepatitis C virus core protein interacts with cellular putative RNA helicase

L R You et al. J Virol. 1999 Apr.

Abstract

The nucleocapsid core protein of hepatitis C virus (HCV) has been shown to trans-act on several viral or cellular promoters. To get insight into the trans-action mechanism of HCV core protein, a yeast two-hybrid cloning system was used for identification of core protein-interacting cellular protein. One such cDNA clone encoding the DEAD box family of putative RNA helicase was obtained. This cellular putative RNA helicase, designated CAP-Rf, exhibits more than 95% amino acid sequence identity to other known RNA helicases including human DBX and DBY, mouse mDEAD3, and PL10, a family of proteins generally involved in translation, splicing, development, or cell growth. In vitro binding or in vivo coimmunoprecipitation studies demonstrated the direct interaction of the full-length/matured form and C-terminally truncated variants of HCV core protein with this targeted protein. Additionally, the protein's interaction domains were delineated at the N-terminal 40-amino-acid segment of the HCV core protein and the C-terminal tail of CAP-Rf, which encompassed its RNA-binding and ATP hydrolysis domains. Immunoblotting or indirect immunofluorescence analysis revealed that the endogenous CAP-Rf was mainly localized in the nucleus and to a lesser extent in the cytoplasm, and when fused with FLAG tag, it colocalized with the HCV core protein either in the cytoplasm or in the nucleus. Similar to other RNA helicases, this cellular RNA helicase has nucleoside triphosphatase-deoxynucleoside triphosphatase activity, but this activity is inhibited by various forms of homopolynucleotides and enhanced by the HCV core protein. Moreover, transient expression of HCV core protein in human hepatoma HuH-7 cells significantly potentiated the trans-activation effect of FLAG-tagged CAP-Rf or untagged CAP-Rf on the luciferase reporter plasmid activity. All together, our results indicate that CAP-Rf is involved in regulation of gene expression and that HCV core protein promotes the trans-activation ability of CAP-Rf, likely via the complex formation and the modulation of the ATPase-dATPase activity of CAP-Rf. These findings provide evidence that HCV may have evolved a distinct mechanism in alteration of host cellular gene expression regulation via the interaction of its nucleocapsid core protein and cellular putative RNA helicase known to participate in all aspects of cellular processes involving RNA metabolism. This feature of core protein may impart pleiotropic effects on host cells, which may partially account for its role in HCV pathogenesis.

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Figures

FIG. 1
FIG. 1
Alignment of amino acid sequence or 5′ and 3′ noncoding region sequences of CAP-Rf with those of the DEAD family of RNA helicases. (A) Alignment of amino acid sequence of CAP-Rf with those of other known RNA helicases. The amino acid sequence of CAP-Rf is aligned with those of human DBX and DBY (48) (GenBank accession no. AF000982 and AF000985, respectively), mouse PL10 (54) (GenBank accession no. J04847), and mDEAD3 (24) (GenBank accession no. L25126). A consensus sequence is shown at the bottom of the alignment. Positions of identical amino acids in the six proteins are boxed. The roman numerals indicate the positions of conserved motifs found in RNA helicases. (B and C) Alignment of 5′ (B) and 3′ (C) noncoding regions of CAP-Rf cDNA with those of other known RNA helicases. The 5′ or 3′ noncoding sequence of CAP-Rf is compared with the corresponding region of human RNA helicases from DBX and DBY (48) (GenBank accession no. AF000982 and AF000985, respectively) or DDX14 (14) (accession no. U50553). The initiation or stop codon for these four RNA helicases is boldface and underlined. Only the different positions are indicated. The dotted lines represent identical nucleotides, while the dashed lines indicate deletions.
FIG. 1
FIG. 1
Alignment of amino acid sequence or 5′ and 3′ noncoding region sequences of CAP-Rf with those of the DEAD family of RNA helicases. (A) Alignment of amino acid sequence of CAP-Rf with those of other known RNA helicases. The amino acid sequence of CAP-Rf is aligned with those of human DBX and DBY (48) (GenBank accession no. AF000982 and AF000985, respectively), mouse PL10 (54) (GenBank accession no. J04847), and mDEAD3 (24) (GenBank accession no. L25126). A consensus sequence is shown at the bottom of the alignment. Positions of identical amino acids in the six proteins are boxed. The roman numerals indicate the positions of conserved motifs found in RNA helicases. (B and C) Alignment of 5′ (B) and 3′ (C) noncoding regions of CAP-Rf cDNA with those of other known RNA helicases. The 5′ or 3′ noncoding sequence of CAP-Rf is compared with the corresponding region of human RNA helicases from DBX and DBY (48) (GenBank accession no. AF000982 and AF000985, respectively) or DDX14 (14) (accession no. U50553). The initiation or stop codon for these four RNA helicases is boldface and underlined. Only the different positions are indicated. The dotted lines represent identical nucleotides, while the dashed lines indicate deletions.
FIG. 2
FIG. 2
Northern blot and immunoblot analysis of CAP-Rf expression in various cell lines. (A) The poly(A)+ mRNAs (5 μg) extracted from cell lines HuH-7 (lane 1), HepG2 (lane 2), and HeLa (lane 3) were used for Northern blot analysis with the 32P-labeled CAP-Rd DNA fragment (2.1-kb EcoRI fragment of pGAD/CAP-Rd) as a probe. The same blot was rehybridized with glyceraldehyde-3-phosphate dehydrogenase DNA probe and served as the control. (B) Immunoblot analysis of CAP-Rf expression. Total cell pellets from various cell lines were solubilized by the sample buffer (47) and analyzed by immunoblotting with rabbit anti-His · CAP-Rd antiserum for detection. The protein amount loaded in the gel is 20 μg in each lane. (C) Analysis of the subcellular localization of CAP-Rf by immunoblotting. The total cell extracts, cytoplasmic fractions, or nuclear extracts (50 μg each) prepared from various cell lines (see Materials and Methods) were analyzed by immunoblotting with mouse monoclonal anti-chicken α-tubulin (Amersham; 1:6,700 dilution), goat anti-human B23 (Santa Cruz; 1:2,000 dilution), or rabbit anti-His · CAP-Rd (1:2,000 dilution) antibodies for detection.
FIG. 3
FIG. 3
Expression and purification of His · CAP-Rd and His · CAP-Rf proteins. (A, B, D, and E) Analysis of the His · CAP-Rd or His · CAP-Rf expression in E. coli. E. coli BL21(DE3) harboring the vector pET15b or CAP-Rf expression vector pET/His · CAP-Rd or pET/His · CAP-Rf was induced with (lanes 2 and 4) or without (lanes 1 and 3) 1 mM IPTG for 3 h. Cells were harvested, solubilized in the sample buffer (47), subsequently analyzed by SDS-PAGE, and stained with Coomassie brilliant blue (A and D) or processed for immunoblotting with anti-His · CAP-Rd antisera (lanes 1 to 4) with the ECL detection system. The CAP-Rd (C) and CAP-Rf (F) proteins affinity purified by Ni2+ prebound His-Bind resin (see Materials and Methods) were analyzed by SDS-PAGE and detected by Coomassie brilliant blue staining. The position for His · CAP-Rd or His · CAP-Rf is indicated.
FIG. 4
FIG. 4
Analysis of the interaction between CAP-Rf and HCV core variants. (A) Analysis of in vitro translation products of HCV core protein. The 35S-labeled HCV core proteins of various lengths (C195, C122, and C101) prepared by in vitro transcription and translation (lanes 2 to 4) (86) (also see Materials and Methods) (5 to 10 μl) were individually precipitated by HCV-positive patients’ sera and analyzed by SDS-PAGE (13.5% polyacrylamide gel) and autoradiography. (B) Analysis of the interaction between His · CAP-Rf and in vitro-translated HCV core protein. The in vitro-translated HCV core proteins (10 to 20 μl) were loaded onto His-Bind resin prebound with His · CAP-Rd (4 μg in 20-μl resins) (see Materials and Methods). The bound proteins were boiled in the sample buffer, analyzed by SDS-PAGE (13.5% polyacrylamide gel), and detected by autoradiography. Lane 1, in vitro-translated products without HCV core mRNA; lane 2, C195; lane 3, C122; lane 4, C101. (C) In vitro binding analysis of endogenous CAP-Rf and the various truncated forms of HCV core protein. HepG2 cell lysates were incubated with glutathione-Sepharose 4B beads which were prebound with GST, GST/HCVc122, or GST/HCVc122Δ(41-107) (see Materials and Methods). The bound proteins retained on the resins were immunoblotted with antiserum against His · CAP-Rf (see Materials and Methods).
FIG. 5
FIG. 5
Both FLAG · CAP-Rf and HCV core protein colocalize inside a cell. HuH-7 cells were transfected with FLAG-tagged CAP-Rf construct pFLAG/Rf (A) or together with various forms of HCV core construct (pSRα/HCVc195, pSRα/HCVc122, and pSRα/HCVc101) (B to D, respectively). The distributions of CAP-Rf and HCV core protein were assessed by indirect immunofluorescence staining (see Materials and Methods). For double immunofluorescence staining, cells were stained with rabbit anti-HCV core protein antiserum (1:1,000 dilution) and mouse anti-FLAG-tagged M2 monoclonal antibody (1:250 dilution), followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG or rhodamine-conjugated goat anti-mouse IgG. Cell nuclei were also visualized by Hoechst 33258 staining (blue). These immunofluorescence patterns were recorded by confocal laser scanning microscopy. As noted, the top and bottom sections of panels B to D show the colocalization of core protein with FLAG · CAP-Rf in the cytoplasmic or nuclear compartment, respectively.
FIG. 6
FIG. 6
In vivo coimmunoprecipitation of FLAG · CAP-Rf and HCV core protein. (A) Immunoblot analysis of the HCV core protein expression in HCV core-producing HuH-7 cells (HuH-7/C190). The total cell extracts (20 μg) prepared from HuH-7 and HuH-7/C190 cells (9) were examined for the expression of HCV core protein by immunoblotting with rabbit antisera against HCV core protein. (B and C) HuH-7 cells were transfected with FLAG · CAP-Rf construct pFLAG/Rf (lanes 1 to 4) together with various forms of HCV core construct (pSRα/HCVc195 [C195], pSRα/HCVc122 [C122], and pSRα/HCVc101 [C101]) (lanes 2 to 4, respectively) or its vector pSRα (lanes 1) (see Materials and Methods). Two days after transfection, cells were lysed and subjected to immunoprecipitation (IP) with anti-FLAG monoclonal antibody (M2) (see Materials and Methods). Immunoprecipitates were analyzed by SDS-PAGE followed by Western blotting (WB) assay with rabbit antibody against HCV core protein. Panels B and C show identical sets of experiments but with different exposure times. Numbers at left of each panel indicate molecular mass in kilodaltons.
FIG. 7
FIG. 7
NTP and dNTP hydrolysis activities of His · CAP-Rf. For detection of the nucleotide hydrolysis activity of the purified His · CAP-Rf, four α-32P-labeled ribonucleotides (3 μM) (A) and four α-32P-labeled deoxyribonucleotides (3 μM) (B) were used as substrates. All the enzymatic activities were assayed at indicated concentrations of purified His · CAP-Rf as described in Materials and Methods. The top of each panel indicates the representative TLC plate used for analyzing the ADP or dATP conversion from [α-32P]ATP or [α-32P]dATP, respectively. CIP, the reaction mixtures of [α-32P]ATP or [α-32P]dATP treated with calf intestinal phosphatase as a control. Graphs of the data from panels A and B are shown in panels C and D, respectively.
FIG. 8
FIG. 8
HCV core protein enhances the NTPase-dNTPase activities of His · CAP-Rf. The purified His · CAP-Rf (0.5 μg) was preincubated in NTPase buffer (50 mM MOPS-KCl [pH 6.5], 2 mM EDTA, 10 mM NaCl) alone or with 0.25 μg of GST, GST/HCVc122, or GST/HCVc101, respectively, for 30 min at 37°C and subsequently assayed for nucleotide hydrolyzing activity with [α-32P]ATP (A) or [α-32P]dATP (B) (3 μM) as the substrate.
FIG. 9
FIG. 9
HCV core protein affects the luciferase reporter activity trans activated by CAP-Rf. (A) HuH-7 cells were transfected with a reporter plasmid, pCMV-Luc (0.15 μg), and various amounts of pFLAG/Rf or pECE/Rf or their control vector. After day 2 posttransfection, cells were assayed for luciferase activity as described in Materials and Methods. The luciferase activity is represented as fold induction relative to that for cells transfected with reporter plasmid and control vector. (B) All experimental conditions were similar to those described for panel A except that HuH-7 cells were transfected with pCMV-Luc (0.15 μg) and 0.3 μg of pFLAG/Rf (or pFLAG-CMV-2) together with 0.3 or 0.6 μg of HCV core construct (pSRα/HCVc122 or pSRα/HCVc101) or their control vector pSRα. (C) All experimental conditions were similar to those described for panel A except that HuH-7 cells were transfected with pCMV-Luc (0.15 μg) and pECE/Rf (or pECE) together with the HCV core construct (pSRα/HCVc122 or pSRα/HCVc101) or their control vector pSRα (0.3 μg each). Values shown in all panels are averages (means ± standard deviations) of one representative experiment in which each transfection was performed in triplicate.
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