Interaction with a ubiquitin-like protein enhances the ubiquitination and degradation of hepatitis C virus RNA-dependent RNA polymerase - PubMed (original) (raw)
Interaction with a ubiquitin-like protein enhances the ubiquitination and degradation of hepatitis C virus RNA-dependent RNA polymerase
Lu Gao et al. J Virol. 2003 Apr.
Abstract
To identify potential cellular regulators of hepatitis C virus (HCV) RNA-dependent RNA polymerase (NS5B), we searched for cellular proteins interacting with NS5B protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a ubiquitin-like protein, hPLIC1 (for human homolog 1 of protein linking intergrin-associated protein and cytoskeleton), which is expressed in the liver (M. F. Kleijnen, A. H. Shih, P. Zhou, S. Kumar, R. E. Soccio, N. L. Kedersha, G. Gill, and P. M. Howley, Mol. Cell 6: 409-419, 2000). In vitro binding assays and in vivo coimmunoprecipitation studies confirmed the interaction between hPLIC1 and NS5B, which occurred through the ubiquitin-associated domain at the C terminus of the hPLIC1 protein. As hPLICs have been shown to physically associate with two E3 ubiquitin protein ligases as well as proteasomes (Kleijnen et al., Mol. Cell 6: 409-419, 2000), we investigated whether the stability and posttranslational modification of NS5B were affected by hPLIC1. A pulse-chase labeling experiment revealed that overexpression of hPLIC1, but not the mutant lacking the NS5B-binding domain, significantly shortened the half-life of NS5B and enhanced the polyubiquitination of NS5B. Furthermore, in Huh7 cells that express an HCV subgenomic replicon, the amounts of both NS5B and the replicon RNA were reduced by overexpression of hPLIC1. Thus, hPLIC1 may be a regulator of HCV RNA replication through interaction with NS5B.
Figures
FIG. 1.
Interaction of hPLIC1 with NS5B. (A) Interaction of NS5B with the N-terminally deleted hPLIC1 in yeast. The original cDNA clone from the two-hybrid screening was used. A colony lift filter assay of β-Gal is shown. (B) In vitro interaction of the full-length hPLIC1 with NS5B in GST-fusion protein binding assay. GST-hPLIC1 was incubated with in vitro-translated 35S-labeled NS5B and precipitated with glutathione beads (lane 2). GST protein alone was used as a negative control (lane 3). Lane 1 represents 10% of the input NS5B protein used in lanes 2 and 3. (C) In vivo coimmunoprecipitation of hPLIC1 with NS5B. The Flag-tagged full-length hPLIC1 was cotransfected with NS5B into Huh7 cells. Cell lysates were immunoprecipitated (IP) with anti-Flag-cross-linked Sepharose 4B beads. An HCV patient serum was used to detect NS5B (lanes 1 to 4) in Western blotting. Polyclonal anti-Flag antibody was used to detect Flag-hPLIC1 (lanes 5 to 8). Huh7 cells transfected with NS5B alone (lanes 3, 4, 7, and 8) were used as the negative control. Lanes 1, 3, 5, and 7 represent 10% of lysates prior to immunoprecipitation. A duplicate gel loaded with similar samples was stained with Coomassie blue (lanes 9 to 13). Lanes 9 and 10 represent 5% of lysates prior to immunoprecipitation.
FIG. 2.
Mapping the NS5B-binding domain on hPLIC1. (A) A schematic drawing indicates the structure of the Flag-tagged, truncated hPLIC1 constructs used and the results of binding. (B) In vivo coimmunoprecipitation of the truncated hPLIC1 with NS5B. Various Flag-tagged truncated hPLIC1 proteins were cotransfected with NS5B into Huh7 cells. Cell lysates were immunoprecipitated (IP) with anti-Flag-cross-linked Sepharose 4B beads. In the upper panels, an HCV patient serum was used to detect NS5B by immunoblotting. In the lower panels, polyclonal anti-Flag antibody was used to detect truncated hPLIC1 proteins. Lanes 1, 3, 5, 7, and 9 represent 10% of lysates prior to immunoprecipitation.
FIG. 3.
Detection of in vivo degradation of NS5B by pulse-chase labeling. (A) The transfected Huh7 cells were pulse-labeled with 35S protein labeling mix for 15 min, followed by chase in medium containing excessive cold amino acids for 0 to 5 h. Flag-tagged proteins were immunoprecipitated on an anti-Flag M2-agarose gel prior to SDS-PAGE. The positions of Flag-NS5B, Flag-hPLIC-1, and Flag-hPLIC-1ΔC (aa 1 to 542) are indicated by a circle, arrow, and arrowhead, respectively. This is a representative gel from three independent experiments. Lane mock, untransfected cells precipitated with anti-Flag antibody. (B) Quantification of the intensity of each band from the gel shown in panel A by Alpha Innotech software.
FIG. 4.
In vivo ubiquitination of NS5B in the presence of hPLIC1. (A) Huh7 cells were cotransfected with Flag-NS5B and hPLIC1 or vector. Flag-NS5B was immunoprecipitated with anti-Flag M2-agarose affinity gel, separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with antiubiquitin monoclonal antibody (Ab). Cells used for lanes 3, 4, 8, 9, and 10 were treated with 5 μM lactacystin for 1 h before harvesting. Vector-transfected cells (lanes 7 and 10) served as negative control. (B) Experiments similar to those in panel A were performed except that Huh7 cells transfected with Flag-NS5A were used.
FIG. 5.
Steady-state level of NS5B protein in the presence of hPLIC1. Huh7-replicon cells transfected with hPLIC1 or vector were harvested at 48 h posttransfection. (A) Immunofluorescence staining of NS5B in Huh7-replicon cells after transfection with hPLIC1. Cells seeded on chamber slides were fixed in 4% formaldehyde-PBS, permeabilized by 0.1% Triton X-100-PBS, and immunostained with monoclonal antibodies against NS5A or NS5B, followed by secondary antibodies conjugated with TRITC. Images were taken under fluorescence microscopy with the Zeiss program for the same length of exposure time. (B) Western blot analysis of protein expression in transfected Huh7-replicon cells. Thirty micrograms of postnuclear cell lysate from each transfection was separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with a monoclonal antibody against either NS5B or NS5A. The same membrane was reprobed with antitubulin antibody as loading control. (C) Immunofluorescence staining of NS5B in Huh7-replicon cells transfected with pEGFPC1-hPLIC1. Huh7-replicon cells on chamber slides were transfected with pEGFPC1-hPLIC1 or pEGFPC1 vector. Cells were immunostained with monoclonal antibodies against NS5A or NS5B, followed by secondary antibodies conjugated with TRITC at 48 h posttransfection. The green cells indicate GFP-hPLIC1 or GFP expression.
FIG. 6.
Steady-state level of HCV subgenomic replicon RNA in the presence of hPLIC1. (A) Northern blot analysis of total RNA extracted from Huh7-replicon cells transfected with hPLIC1 or vector at various times posttransfection (p. t.). This is a representative gel from three experiments. Ten micrograms of total RNA from each plate was separated by electrophoresis on a 1% agarose gel and transferred to a nylon membrane. A specific RNA probe complementary to the HCV 5′-untranslated region RNA was used in hybridization at 70°C. The 28S rRNA was visualized by staining the membrane with 0.03% methylene blue in 0.3 M sodium acetate, pH 5.2. (B) Quantification of the gel shown in panel A by phosphorimaging screen. (C) hPLIC1 mRNA expression determined by Northern blot analysis. Lane 1 contained 5 μg of total RNA from Huh7 replicon cells transfected with hPLIC1. The strong band in lane 1 probably represents plasmid cDNA. Twenty micrograms of total RNA was loaded in both lanes 2 and 3.
References
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