E3 Ubiquitin Ligases in Gammaherpesviruses and HIV: A Review of Virus Adaptation and Exploitation.

For productive infection and replication to occur, viruses must control cellular machinery and counteract restriction factors and antiviral proteins. Viruses can accomplish this, in part, via the regulation of cellular gene expression and post-transcriptional and post-translational control. Many viruses co-opt and counteract cellular processes via modulation of the host post-translational modification machinery and encoding or hijacking kinases, SUMO ligases, deubiquitinases, and ubiquitin ligases, in addition to other modifiers. In this review, we focus on three oncoviruses, Epstein-Barr virus (EBV), Kaposi's sarcoma herpesvirus (KSHV), and human immunodeficiency virus (HIV) and their interactions with the ubiquitin-proteasome system via viral-encoded or cellular E3 ubiquitin ligase activity.

Gold nanoparticles loaded with cullin-5 DNA increase sensitivity to 17-AAG in cullin-5 deficient breast cancer cells.

HSP90 inhibitors have the potential to treat many types of cancer due to the dependence of tumor cells on HSP90 for cell growth and proliferation. The Cullin-5 (Cul5) E3 ubiquitin ligase is required for HSP90 inhibitors to induce client protein degradation and subsequent cell death. Cul5 is expressed at low levels in breast cancer cells compared to patient matched controls. This observed low Cul5 expression may play a role in the reported decreased efficacy of 17-AAG and related HSP90 inhibitors as a monotherapy. We have developed a method for delivery of 17-AAG plus Cul5 DNA to cells via gold nanoparticles (AuNPs). Delivery of AuNPs containing Cul5 DNA increases the sensitivity of Cul5 deficient AU565 cells to 17-AAG. Characterization of AuNPs by UV-vis spectrum, TEM, gel electrophoresis assay and 1H NMR indicate attachment of both 17-AAG and DNA payload as well as AuNP stability. Studies in Cul5 deficient AU565 cells reveal that delivery of Cul5 and 17-AAG together increase cytotoxicity. Our results provide evidence that delivery of DNA with drug may serve as a method to sensitize drug resistant tumor cells.

The Itch ubiquitin ligase is required for KSHV RTA induced vFLIP degradation.

Expression of Kaposi's sarcoma herpesvirus vFLIP, a potent activator of NFkB signaling, promotes latency. Inhibition of NFkB signaling promotes lytic reactivation. We previously reported that lytic inducer, RTA, inhibits vFLIP induced NFkB signaling by inducing the degradation of vFLIP via the proteasome. Here we report that the cellular ubiquitin ligase, Itch, is required for RTA induced degradation of vFLIP. Expression of either Itch targeting shRNA or a dominant negative mutant of the ubiquitin ligase both increased the stability of vFLIP in the presence of RTA. Itch potently ubiquitinated vFLIP in vivo and in vitro. We provide evidence for interaction between RTA, vFLIP and Itch and we identified an RTA resistant mutant of vFLIP that is unable to interact with Itch. These observations contribute to our understanding of how RTA counteracts the activities of vFLIP.

Coxsackievirus A16 infection induces neural cell and non-neural cell apoptosis in vitro.

Coxsackievirus A16 (CA16) is one of the main causative pathogens of hand, foot and mouth disease (HFMD). Viral replication typically results in host cell apoptosis. Although CA16 infection has been reported to induce apoptosis in the human rhabdomyosarcoma (RD) cell line, it remains unclear whether CA16 induces apoptosis in diverse cell types, especially neural cells which have important clinical significance. In the current study, CA16 infection was found to induce similar apoptotic responses in both neural cells and non-neural cells in vitro, including nuclear fragmentation, DNA fragmentation and phosphatidylserine translocation. CA16 generally is not known to lead to serious neurological symptoms in vivo. In order to further clarify the correlation between clinical symptoms and cell apoptosis, two CA16 strains from patients with different clinical features were investigated. The results showed that both CA16 strains with or without neurological symptoms in infected patients led to neural and muscle cell apoptosis. Furthermore, mechanistic studies showed that CA16 infection induced apoptosis through the same mechanism in both neural and non-neural cells, namely via activation of both the mitochondrial (intrinsic) pathway-related caspase 9 protein and the Fas death receptor (extrinsic) pathway-related caspase 8 protein. Understanding the mechanisms by which CA16 infection induces apoptosis in both neural and non-neural cells will facilitate a better understanding of CA16 pathogenesis.

KSHV RTA abolishes NFκB responsive gene expression during lytic reactivation by targeting vFLIP for degradation via the proteasome.

Kaposi's sarcoma herpesvirus (KSHV) is a gamma-2 herpesvirus present in all cases of Kaposi's sarcoma, primary effusion lymphoma (PEL), and some cases of multicentric Castleman's disease. Viral FLICE inhibitory protein (vFLIP) is a latently expressed gene that has been shown to be essential for survival of latently infected PEL cells by activating the NFκB pathway. Inhibitors of either vFLIP expression or the NFĸB pathway result in enhanced lytic reactivation and apoptosis. We have observed a decrease in vFLIP protein levels and of NFκB activation in the presence of the KSHV lytic switch protein RTA. Whereas vFLIP alone induced expression of the NFĸB responsive genes ICAM1 and TNFα, inclusion of RTA decreased vFLIP induced ICAM1 and TNFα expression in both co-transfected 293T cells and in doxycycline induced TREx BCBL1 cells. RTA expression resulted in proteasome dependent destabilization of vFLIP. Neither RTA ubiquitin E3 ligase domain mutants nor a dominant-negative RAUL mutant abrogated this effect, while RTA truncation mutants did, suggesting that RTA recruits a novel cellular ubiquitin E3 ligase to target vFLIP for proteasomal degradation, allowing for inhibition of NFĸB responsive gene expression early during lytic reactivation.

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-1alpha. 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.

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.

Structural insight into the human immunodeficiency virus Vif SOCS box and its role in human E3 ubiquitin ligase assembly.

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.

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.

Selective assembly of HIV-1 Vif-Cul5-ElonginB-ElonginC E3 ubiquitin ligase complex through a novel SOCS box and upstream cysteines.

APOBEC3G, which induces hypermutations in newly synthesized viral DNA, is suppressed by HIV-1 Vif, acting through Cul5-ElonginB-ElonginC E3 ubiquitin ligase. We have now characterized a novel SOCS box in HIV-1 Vif that mediates its interaction with ElonginC. In this SOCS box, alanine replaces the consensus cysteine in the previously identified SOCS box. This new motif was necessary but insufficient for interaction with Cul5-ElonginB-ElonginC, as two highly conserved Cys residues outside the SOCS box were required to interact with Cul5 but not ElonginC. Therefore, selective assembly with Cul5 versus Cul2 E3 may require protein interfaces besides the SOCS-box-ElonginC interaction.