Elana S. Ehrlich

DDB1 and Cul4A Are Required for Human Immunodeficiency Virus Type 1 Vpr-Induced G2 Arrest

ABSTRACT Vpr-mediated induction of G2 cell cycle arrest has been postulated to be important for human immunodeficiency virus type 1 (HIV-1) replication, but the precise role of Vpr in this cell cycle arrest is unclear. In the present study, we have shown that HIV-1 Vpr interacts with damaged DNA binding protein 1 (DDB1) but not its partner DDB2. The interaction of Vpr with DDB1 was inhibited when DCAF1 (VprBP) expression was reduced by short interfering RNA (siRNA) treatment. The Vpr mutant (Q65R) that was defective for DCAF1 interaction also had a defect in DDB1 binding. However, Vpr binding to DDB1 was not sufficient to induce G2 arrest. A reduction in DDB1 or DDB2 expression in the absence of Vpr also did not induce G2 arrest. On the other hand, Vpr-induced G2 arrest was impaired when the intracellular level of DDB1 or Cullin 4A was reduced by siRNA treatment. Furthermore, Vpr-induced G2 arrest was largely abolished by a proteasome inhibitor. These data suggest that Vpr assembles with DDB1 through interaction with DCAF1 to form an E3 ubiquitin ligase that targets cellular substrates for proteasome-mediated degradation and G2 arrest.

Structural Insight into the Human Immunodeficiency Virus Vif SOCS Box and Its Role in Human E3 Ubiquitin Ligase Assembly

ABSTRACT Human immunodeficiency virus (HIV) virion infectivity factor (Vif) causes the proteasome-mediated destruction of human antiviral protein APOBEC3G by tethering it to a cellular E3 ubiquitin ligase composed of ElonginB, ElonginC, Cullin5, and Rbx2. It has been proposed that HIV Vif hijacks the E3 ligase through two regions within its C-terminal domain: a BC box region that interacts with ElonginC and a novel zinc finger motif that interacts with Cullin5. We have determined the crystal structure of the HIV Vif BC box in complex with human ElonginB and ElonginC. This complex presents direct structural evidence of the recruitment of a human ubiquitin ligase by a viral BC box protein that mimics the conserved interactions of cellular ubiquitin ligases. We further mutated conserved hydrophobic residues in a region downstream of the Vif BC box. These mutations demonstrate that this region, the Vif Cullin box, composes a third E3-ligase recruiting site critical for interaction between Vif and Cullin5. Furthermore, our homology modeling reveals that the Vif Cullin box and zinc finger motif may be positioned adjacent to the N terminus of Cullin5 for interaction with loop regions in the first cullin repeat of Cullin5.

Regulation of Hsp90 client proteins by a Cullin5-RING E3 ubiquitin ligase

We report a link between Cullin5 (Cul5) E3 ubiquitin ligase and the heat shock protein 90 (Hsp90) chaperone complex. Hsp90 participates in the folding of its client proteins into their functional conformation. Many Hsp90 clients have been reported to be aberrantly expressed in a number of cancers. We demonstrate Cul5 interaction with members of the Hsp90 chaperone complex as well as the Hsp90 client, ErbB2. We observed recruitment of Cul5 to the site of ErbB2 at the plasma membrane and subsequent induction of polyubiquitination and proteasomal degradation. We also demonstrate Cul5 involvement in regulation of another Hsp90 client, Hif-1α. We observed Cul5 degradation of ErbB2 to occur independently of ElonginB-ElonginC function. The involvement of Cul5 in Hsp90 client regulation has implications in the effectiveness of Hsp90 targeted chemotherapy, which is currently undergoing clinical trials. The link between Cul5 and Hsp90 client regulation may represent an avenue for cancer drug development.

Adenovirus E4orf6 assembles with Cullin5-ElonginB-ElonginC E3 ubiquitin ligase through an HIV/SIV Vif-like BC-box to regulate p53

The adenovirus protein E4orf6 targets p53 for polyubiquitination and proteasomal degradation and is known to form a complex with the Cul5‐ElonginB‐ElonginC E3 ubiquitin ligase. However, whether Cul5 is directly responsible for the E4orf6‐mediated degradation of p53 remains unclear. By using a dominant‐negative mutant of Cul5 and silencing Cul5 expression through RNA interference, we have now demonstrated that E4orf6‐mediated p53 degradation requires Cul5. Furthermore, we have identified a lenti‐viral Vif‐like BC‐box motif in E4orf6 that is highly conserved among adenoviruses from multiple species. More importantly, we have shown that this Vif‐like BC‐box is essential for the recruitment of Cul5‐Elong‐inB‐ElonginC E3 ubiquitin ligase by E4orf6 and is also required for E4orf6‐mediated p53 degradation. E4orf6 selectively recruited Cul5 despite the lack of either a Cul5‐box, which is used by cellular substrate receptors to recruit Cul5, or a newly identified HCCH zinc‐binding motif, which is used by primate lentiviral Vif to recruit Cul5. Therefore, adenovirus E4orf6 molecules represent a novel family of viral BC‐box proteins the cellular ancestor of which is as yet unknown.—Luo K., Ehrlich, E., Xiao, Z., Zhang, W., Ketner, G., Yu X.‐F. Adenovirus E4orf6 assembles with Cullin5‐ElonginB‐ElonginC E3 ubiquitin ligase through an HIV/SIV Vif‐like BC‐box to regulate p53. FASEB J. 21, 1742–1750 (2007)

E1B 55k-independent dissociation of the DNA ligase IV/XRCC4 complex by E4 34k during adenovirus infection

The ligase IV/XRCC4 complex plays a central role in DNA double-strand break repair by non-homologous end joining (NHEJ). During adenovirus infection, NHEJ is inhibited by viral proteins E4 34k and E1B 55k, which redirect the Cul5/Rbx1/Elongin BC ubiquitin E3 ligase to polyubiquitinate and promote degradation of ligase IV. In cells infected with E1B 55k-deficient adenovirus, ligase IV could not be found in XRCC4-containing complexes and was observed in a novel ligase IV/E4 34k/Cul5/Elongin BC complex. These observations suggest that dissociation of the ligase IV/XRCC4 complex occurs at an early stage in E4 34k-mediated degradation of ligase IV and indicate a role for E4 34k in dissociation of the ligase IV/XRCCC4 complex. Expression of E4 34k alone was not sufficient to dissociate the ligase IV/XRCC4 complex, which indicates a requirement for an additional, as yet unidentified, factor in E1B 55k-independent dissociation of the ligase IV/XRCC4 complex.

Lentiviral Vif: Viral Hijacker of the Ubiquitin-Proteasome System

Since the beginning of time, microorganisms have been devising ways to bypass detection and destruction by our immune system. Therefore, it is no surprise that along with the identification of the cellular antiviral protein APOBEC3G (A3G) has come the recognition of the viral solution to this assault. Here, we review the research that led up to the identification of A3G and the mechanism that the human immunodeficiency virus protein Vif developed to evade A3G’s antiviral activities.