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Interaction with a Ubiquitin-Like Protein Enhances the Ubiquitination and Degradation of Hepatitis C Virus RNA-Dependent RNA Polymerase

Lu Gao,¹ Hong Tu,^1, Stephanie T. Shi,^1, Ki-Jeong Lee,¹ Miyuki Asanaka,² Soon B. Hwang,³ and Michael M. C. Lai^1,4*

Department of Molecular Microbiology and Immunology,¹ Howard Hughes Medical Institute, Keck School of Medicine, University of Southern California, Los Angeles, California 90033,⁴ Tampa Bay Research Institute, St. Petersburg, Florida 33716,² Institute of Life Science, Hallym Academy of Sciences, Hallym University, Chuncheon 200-702, Korea³

Received 16 August 2002/ Accepted 12 December 2002

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.

Present address: Shanghai Cancer Institute, Shanghai, China.

Present address: Pfizer Global Research & Development, La Jolla Laboratories, San Diego, CA 92121.

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