Ubiquitin-independent degradation of hepatitis C virus F protein.
Hepatitis C virus (HCV) F protein is encoded by the +1 reading frame of the viral genome. It overlaps with the core protein coding sequence, and multiple mechanisms for its expression have been proposed. The full-length F protein that is synthesized by translational ribosomal frameshift at codons 9 to 11 of the core protein sequence is a labile protein. By using a combination of genetic, biochemical, and cell biological approaches, we demonstrate that this HCV F protein can bind to the proteasome subunit protein alpha3, which reduces the F-protein level in cells in a dose-dependent manner. Deletion-mapping analysis identified amino acids 40 to 60 of the F protein as the alpha3-binding domain. This alpha3-binding domain of the F protein together with its upstream sequence could significantly destabilize the green fluorescent protein, an otherwise stable protein. Further analyses using an F-protein mutant lacking lysine and a cell line that contained a temperature-sensitive E1 ubiquitin-activating enzyme indicated that the degradation of the F protein was ubiquitin independent. Based on these observations as well as the observation that the F protein could be degraded directly by the 20S proteasome in vitro, we propose that the full-length HCV F protein as well as the F protein initiating from codon 26 is degraded by an ubiquitin-independent pathway that is mediated by the proteasome subunit alpha3. The ability of the F protein to bind to alpha3 raises the possibility that the HCV F protein may regulate protein degradation in cells.
Yuksek
Kamile
K
Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA.
Chen
Wen-Ling
WL
Chien
David
D
Ou
Jing-Hsiung James
JH
D000818:Animals; D019677:Artificial Gene Fusion; D002460:Cell Line; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins; D017384:Sequence Deletion; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000502:physiology; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D014758:Viral Core Proteins; C433031:hepatitis C protein F, Hepatitis C virus; D049452:Green Fluorescent Proteins; C488165:PSMA3 protein, human; D046988:Proteasome Endopeptidase Complex