Ubiquitin-Independent Degradation of Hepatitis C Virus F Protein

Abstract

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 ␣3, 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 ␣3-binding domain. This ␣3-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 temperaturesensitive 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 ␣3. The ability of the F protein to bind to ␣3 raises the possibility that the HCV F protein may regulate protein degradation in cells.

Changes in proteasome pool in human papillary thyroid carcinoma development

“…The 20S proteasomes, on the contrary, degrade proteins and short peptides independently of ATP and ubiquitin. The list of proteins identified as substrates of 20S proteasomes extends every year; it includes, for example, proteins with a damaged tertiary structure [13] and some virus proteins [14,15].…”

The Intrinsically Disordered N-terminal Domain of Thymidylate Synthase Targets the Enzyme to the Ubiquitin-independent Proteasomal Degradation Pathway

“…3). Such substrates include ornithine decarboxylase (ODC) 3 (4 -6), c-Fos (7,8), p21 Cip1 (9, 10), hepatitis virus F protein (11), and c-Jun (12), among others. Although the number of substrates identified as degraded by a ubiquitin-independent mechanism remains small, recent biochemical analyses indicate that the process may be more widespread than previously thought and contributes significantly to the regulation of protein turnover (13).…”

Cooperation between an Intrinsically Disordered Region and a Helical Segment Is Required for Ubiquitin-independent Degradation by the Proteasome

“…For ubiquitin-dependent degrons, the recognition/binding phase is accomplished by covalently attached polyubiquitin moieties that recognize one or more subunits of the proteasomal complex (46,47). For ubiquitin-independent degradation, mechanisms that directly involve the target substrate's polypeptide chain are utilized (12,18,44). The initiation phase involves unfolding of the proteasome-bound substrate, "threading" of the substrate through the pore that leads to the proteolytic chamber, and entry into the chamber itself, where proteolysis ensues (11,38,46).…”

“…Waf1/Cip1 (16,17), the F protein of hepatitis C virus (18), and the NFB inhibitor IB␣ (19,20). Recent proteomic studies have indicated that as many as 20% of cellular proteins may be degraded in the absence of a requirement for ubiquitin modification (21), suggesting that many more ubiquitin-independent substrates remain to be discovered.…”