Abstract
Human T-cell leukemia virus type 1 (HTLV-1) encodes an antisense viral gene product termed HTLV-1 basic leucine-zipper factor (HBZ). HBZ forms heterodimers with c-Jun, a member of the AP-1 family, and promotes its proteasomal degradation. Although most proteasomal substrates are targeted for degradation via conjugation of polyubiquitin chains, we show that ubiquitination is not required for HBZ-mediated proteasomal degradation of c-Jun. We demonstrate that HBZ directly interacts with both the 26 S proteasome and c-Jun and facilitates the delivery of c-Jun to the proteasome without ubiquitination. HBZ acts as a tethering factor between the 26 S proteasome and its substrate, thereby bypassing the targeting function of ubiquitination. These findings disclose a novel viral strategy to utilize the cellular proteolytic system for viral propagation.
Highlights
The proteasome is a major nonlysosomal proteolytic apparatus
Human papillomavirus type-16 E6 protein and adenovirus E1B55k-E4orf6 proteins act as part of E3 ubiquitin ligase complexes to promote ubiquitination and subsequent degradation of the tumor suppressor, p53 [14, 38]
The human immunodeficiency virus type-1 Vif protein acts as an E3 ubiquitin ligase that targets APOBEC3G, a potent cellular anti
Summary
Cell Culture, Transfection, and Reagents—HEK-293T cells were cultured in Dulbecco’s modified Eagle’s medium (Nissui) supplemented with 10% fetal bovine serum (Invitrogen), 4 mM L-glutamine, and 0.1 mg/ml kanamycin sulfate at 37 °C in a 5% CO2 atmosphere. Deletions and point mutants of HBZ and c-Jun were generated by PCR amplification of pcDNA3-FLAG-HBZ and pcDNA3HA-c-Jun, respectively These PCR fragments were subcloned into expression vectors such as pcDNA3-FLAG, pcDNA3-HA, pcDNA3-His (Invitrogen), pGEX-6P-1 (Amersham Biosciences), or pGBT9 (Clontech). The cDNAs were amplified by reverse transcription-PCR using total RNA from MT-2 cells (for c-Jun, JunB, JunD, c-Fos, ATF1, ATF2, ATF4, CREB, p53, and GADD34), 293T cells (for ubiquitin, Rpn, MCM5, MCM7, and DET1), and Saos-2 cells (for COP1) They were subcloned into pcDNA3-FLAG, pcDNA3-Myc, pcDNA3-HA, pcDNA3-His, or pCAG vectors. Immunoprecipitations—Cells were lysed in 1 ml of lysis buffer, which contained 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 50 mM sodium fluoride, 2 mM sodium orthovanadate, and a protease inhibitor mixture (Complete EDTA-free, Roche Applied Science) at 4 °C for 30 min. MM Hepes (pH 7.5)) and incubated with 20 l of Ni-NTA beads (Qiagen) for 2 h at room temperature
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
