Norwalk virus open reading frame 3 encodes a minor structural protein

Norwalk virus (NV) is a causative agent of acute epidemic nonbacterial gastroenteritis in humans. The inability to cultivate NV has required the use of molecular techniques to examine the genome organization and functions of the viral proteins. The function of the NV protein encoded by open reading frame 3 (ORF 3) has been unknown. In this paper, we report the characterization of the NV ORF 3 protein expressed in a cell-free translation system and in insect cells and show its association with recombinant virus-like particles (VLPs) and NV virions. Expression of the ORF 3 coding region in rabbit reticulocyte lysates resulted in the production of a single protein with an apparent molecular weight of 23,000 (23K protein), which is not modified by N-linked glycosylation. The ORF 3 protein was expressed in insect cells by using two different baculovirus recombinants; one recombinant contained the entire 3' end of the genome beginning with the ORF 2 coding sequences (ORFs 2+3), and the second recombinant contained ORF 3 alone. Expression from the construct containing both ORF 2 and ORF 3 resulted in the expression of a single protein (23K protein) detected by Western blot analysis with ORF 3-specific peptide antisera. However, expression from a construct containing only the ORF 3 coding sequences resulted in the production of multiple forms of the ORF 3 protein ranging in size from 23,000 to 35,000. Indirect-immunofluorescence studies using an ORF 3 peptide antiserum showed that the ORF 3 protein is localized to the cytoplasm of infected insect cells. The 23K ORF 3 protein was consistently associated with recombinant VLPs purified from the media of insect cells infected with a baculovirus recombinant containing the entire 3' end of the NV genome. Western blot analysis of NV purified from the stools of NV-infected volunteers revealed the presence of a 35K protein as well as multiple higher-molecular-weight bands specifically recognized by an ORF 3 peptide antiserum. These results indicate that the ORF 3 protein is a minor structural protein of the virion.     

E3 ubiquitin ligase SIAH1 mediates ubiquitination and degradation of TRB3

Tribbles 3 homolog (TRB3) is recently identified as a scaffold-like regulator of various signal transducers and has been implicated in several processes including insulin signaling, NF-kappaB signaling, lipid metabolism and BMP signaling. To further understand cellular mechanisms of TRB3 regulation, we performed a yeast two-hybrid screen to identify novel TRB3 interacting proteins and totally obtained ten in-frame fused preys. Candidate interactions were validated by co-immunoprecipitation assays in mammalian cells. We further characterized the identified proteins sorted by Gene Ontology Annotation. Its interaction with the E3 ubiquitin ligase SIAH1 was further investigated. SIAH1 could interact with TRB3 both in vitro and in vivo. Importantly, SIAH1 targeted TRB3 for proteasome-dependent degradation. Cotransfection of SIAH1 could withdraw up-regulation of TGF-beta signaling by TRB3, suggesting SIAH1-induced degradation of TRB3 represents a potential regulatory mechanism for TGF-beta signaling.     

SCFβ-TRCP E3 ubiquitin ligase targets the tumor suppressor ZNRF3 for ubiquitination and degradation

Wnt signaling has emerged as a major regulator of tissue development by governing the self-renewal and maintenance of stem cells in most tissue types. As a key upstream regulator of the Wnt pathway, the transmembrane E3 ligase ZNRF3 has recently been established to play a role in negative regulation of Wnt signaling by targeting Frizzled (FZD) receptor for ubiquitination and degradation. However, the upstream regulation of ZNRF3, in particular the turnover of ZNRF3, is still unclear. Here we report that ZNRF3 is accumulated in the presence of proteasome inhibitor treatment independent of its E3-ubiquitin ligase activity. Furthermore, the Cullin 1-specific SCF complex containing β-TRCP has been identified to directly interact with and ubiquitinate ZNRF3 thereby regulating its protein stability. Similar with the degradation of β-catenin by β-TRCP, ZNRF3 is ubiquitinated by β-TRCP in both CKI-phosphorylation-and degron-dependent manners. Thus, our findings not only identify a novel substrate for β-TRCP oncogenic regulation, but also highlight the dual regulation of Wnt signaling by β-TRCP in a contextdependent manner where β-TRCP negatively regulates Wnt signaling by targeting β-catenin, and positively regulates Wnt signaling by targeting ZNRF3.     

The open reading frame 3 gene of hepatitis E virus contains a cis-reactive element and encodes a protein required for infection of macaques

An infectious cDNA clone of hepatitis E virus was mutated in order to prevent synthesis of either open reading frame 2 (ORF2) protein or ORF3 protein. HuH-7 cells transfected with an ORF2-null mutant produced ORF3, and those transfected with an ORF3-null mutant produced ORF2. Silent mutations introduced into a highly conserved nucleotide sequence in the ORF3 coding region eliminated the synthesis of both ORF2 and ORF3 proteins, suggesting that it comprised a cis-reactive element. A mutant that was not able to produce ORF3 protein did not produce a detectable infection in rhesus macaques. However, a mutant that encoded an ORF3 protein lacking a phosphorylation site reported to be critical for function was able to replicate its genome in cell culture and to induce viremia and seroconversion in rhesus monkeys, suggesting that phosphorylation of ORF3 protein was not necessary for genome replication or for production of infectious virions.     

The equine herpesvirus 2 E1 open reading frame encodes a functional chemokine receptor

Several herpesviruses contain open reading frames (ORFs) that encode potential homologs of eucaryotic genes. Equine herpesvirus 2 (EHV-2) is a gammaherpesvirus related to other lymphotropic herpesviruses such as herpesvirus saimiri and Epstein-Barr virus. The E1 ORF of EHV-2, a G protein-coupled receptor homolog, shows 31 to 47% amino acid identity with known CC chemokine receptors. To investigate whether E1 may encode a functional receptor, we cloned the E1 ORF and expressed it in stably transfected cell lines. We report here the identification of the CC chemokine eotaxin as a functional ligand for the EHV-2 E1 receptor. Chemokines are likely to play a role in the regulation of immune functions in equine hosts during EHV-2 infection and, via interaction with E1, may affect viral replication and/or escape from immune responses.     

The E3 ubiquitin ligase HECTD3 regulates ubiquitination and degradation of Tara

Tara was identified as an interacting partner of guanine nucleotide exchange factor Trio and TRF1. Tara is proposed to be involved in many important fundamental cellular processes, ranging from actin remodeling, directed cell movement, to cell cycle regulation. Yet, its exact roles required further elucidation. Here, we identify a novel Tara-binding protein HECTD3, a putative member of HECT E3 ubiquitin ligases. HECTD3 directly binds Tara in vitro and forms a complex with Tara in vivo. Overexpression of HECTD3 enhances the ubiquitination of Tara in vivo and promotes the turnover of Tara, whereas depletion of HECTD3 by small interfering RNA decreases Tara degradation. Furthermore, depletion of HECTD3 leads to multipolar spindle formation. All these findings suggest that HECTD3 may facilitate cell cycle progression via regulating ubiquitination and degradation of Tara.     

HECT ubiquitin ligase Smurf1 targets the tumor suppressor ING2 for ubiquitination and degradation

Inhibitor of growth 2 (ING2) gene encodes a candidate tumor suppressor and is frequently reduced in many tumors. However, the mechanisms underlying the regulation of ING2, in particular its protein stability, are still unclear. Here we show that the homologous to E6AP carboxyl terminus (HECT)-type ubiquitin ligase Smad ubiquitination regulatory factor 1 (Smurf1) interacts with and targets ING2 for poly-ubiquitination and proteasomal degradation. Intriguingly, the ING2 binding domain in Smurf1 was mapped to the catalytic HECT domain. Furthermore, the C-terminal PHD domain of ING2 was required for Smurf1-mediated degradation. This study provided the first evidence that the stability of ING2 could be regulated by ubiquitin-mediated degradation.

Structured summary

MINT-7894271: ING2 (uniprotkb:Q9H160) binds (MI:0407) to Smurf1 (uniprotkb:Q9HCE7) by pull-down (MI:0096)MINT-7894319, MINT-7894339: ING2 (uniprotkb:Q9H160) physically interacts (MI:0915) with Smurf1 (uniprotkb:Q9HCE7) by anti tag co-immunoprecipitation (MI:0007)MINT-7894301: Smurf1 (uniprotkb:Q9HCE7) physically interacts (MI:0915) with ING2 (uniprotkb:Q9H160) by anti bait co-immunoprecipitation (MI:0006)MINT-7894358: ING1b (uniprotkb:Q9UK53-2) physically interacts (MI:0915) with Smurf1 (uniprotkb:Q9HCE7) by anti tag co-immunoprecipitation (MI:0007)MINT-7894249: ING2 (uniprotkb:Q9H160) physically interacts (MI:0915) with ubiquitin (uniprotkb:P62988) by anti tag co-immunoprecipitation (MI:0007)     

E5 open reading frame of bovine papillomavirus type 1 encodes a transforming gene.

We have previously shown that the early region of the bovine papillomavirus type 1 genome contains two nonoverlapping segments that can independently induce the morphological transformation of cultured cells. The transforming gene from the 5' end of the early region is encoded by the E6 open reading frame. The second transforming segment was previously localized to a 2.3-kilobase fragment (2.3T) from the 3' end of the early region. To determine which of the four open reading frames (E2, E3, E4, and E5) located within 2.3T encodes a transforming gene, we have now introduced a series of insertion and deletion mutations into a clone (pHLB1) in which 2.3T is activated by the Harvey viral long terminal repeat, and we tested the mutants for their ability to induce focal transformation. Our results indicate that the E5 open reading frame, which could encode a low-molecular-weight hydrophobic peptide, is required for pHLB1-induced transformation of NIH 3T3 cells, but that the E2, E3, and E4 open reading frames are not.     

Calcium-sensing receptor ubiquitination and degradation mediated by the E3 ubiquitin ligase dorfin

Calcium-sensing receptors (CaR) contribute to regulation of systemic calcium homeostasis by activation of G(q)- and G(i)-linked signaling pathways in the parathyroids, kidney, and intestine. Little is known about the mechanisms regulating CaR synthesis and degradation. Screening of a human kidney yeast two-hybrid library identified the E3 ubiquitin ligase dorfin as a binding partner for the intracellular carboxyl terminus of CaR. Interaction between CaR and dorfin was confirmed by coimmunoprecipitation from HEK293 cells. Ubiquitination of CaR was observed in the presence of the proteasomal inhibitor MG132; mutation of all putative intracellular loop and carboxyl-terminal lysine residues abolished ubiquitination of CaR. Coexpression with dorfin decreased the amount of total CaR protein and increased CaR ubiquitination, whereas a dominant negative fragment of dorfin had opposite effects. The AAA-ATPase p97/valosin-containing protein associates with both CaR and dorfin in HEK293 cells. Treatment with tunicamycin, an inhibitor of N-linked glycosylation, induced the appearance of the unglycosylated 115-kDa CaR form, which was further increased by exposure to MG132, or upon transfection with a dorfin dominant negative construct, suggesting that dorfin-mediated proteasomal degradation of immature CaR occurs from the endoplasmic reticulum. Because endogenous CaR in Madin-Darby canine kidney cells is also subject to degradation from the endoplasmic reticulum, dorfin-mediated ubiquitination may contribute to a general mechanism for CaR quality control during biosynthesis.     

Human papillomavirus type 16 open reading frame E7 encodes a transforming gene for rat 3Y1 cells.

Human papillomavirus type 16 (HPV 16) DNA is capable of morphologically transforming rat 3Y1 cells. The expression plasmids, constructed from the simian virus 40-based expression vector pSV2-0 and specific DNA fragments from the putative early region of the HPV 16 genome, were tested for their transforming capacity. Among the various pSV2 plasmids, only those containing the intact E7 coding region were found to produce foci of the transformed rat cells which could grow in a soft-agar medium. The data indicate that expression of the HPV 16 E7 open reading frame is sufficient to induce focal transformation of rat cells.     

The PHD domain of MEKK1 acts as an E3 ubiquitin ligase and mediates ubiquitination and degradation of ERK1/2

ERK1/2 MAP kinases are important regulators in cellular signaling, whose activity is normally reversibly regulated by threonine-tyrosine phosphorylation. In contrast, we have found that stress-induced ERK1/2 activity is downregulated by ubiquitin/proteasome-mediated degradation of ERK1/2. The PHD domain of MEKK1, a RING finger-like structure, exhibited E3 ubiquitin ligase activity toward ERK2 in vitro and in vivo. Moreover, both MEKK1 kinase activity and the docking motif on ERK1/2 were involved in ERK1/2 ubiquitination. Significantly, cells expressing ERK2 with the docking motif mutation were resistant to sorbitol-induced apoptosis. Therefore, MEKK1 functions not only as an upstream activator of the ERK and JNK through its kinase domain, but also as an E3 ligase through its PHD domain, providing a negative regulatory mechanism for decreasing ERK1/2 activity.     

E6AP ubiquitin ligase mediates ubiquitylation and degradation of hepatitis C virus core protein

Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.     

The internal open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication.

The coronavirus mouse hepatitis virus (MHV) contains a large open reading frame embedded entirely within the 5' half of its nucleocapsid (N) gene. This internal gene (designated I) is in the +1 reading frame with respect to the N gene, and it encodes a mostly hydrophobic 23-kDa polypeptide. We have found that this protein is expressed in MHV-infected cells and that it is a previously unrecognized structural protein of the virion. To analyze the potential biological importance of the I gene, we disrupted its expression by site-directed mutagenesis using targeted RNA recombination. The start codon for I was replaced by a threonine codon, and a stop codon was introduced at a short interval downstream. Both alterations created silent changes in the N reading frame. In vitro translation studies showed that these mutations completely abolished synthesis of I protein, and immunological analysis of infected cell lysates confirmed this conclusion. The MHV I mutant was viable and grew to high titer. However, the I mutant had a reduced plaque size in comparison with its isogenic wild-type counterpart, suggesting that expression of I confers some minor growth advantage to the virus. The engineered mutations were stable during the course of experimental infection in mice, and the I mutant showed no significant differences from wild type in its ability to replicate in the brains or livers of infected animals. These results demonstrate that I protein is not essential for the replication of MHV either in tissue culture or in its natural host.     

Initiation at the third in-frame AUG codon of open reading frame 3 of the hepatitis E virus is essential for viral infectivity in vivo

To determine the initiation strategy of the hepatitis E virus (HEV) open reading frame 3 (ORF3), we constructed five HEV mutants with desired mutations in the ORF1 and ORF2 junction region and tested their levels of in vivo infectivity in pigs. A mutant with a C-terminally truncated ORF3 is noninfectious in pigs, indicating that an intact ORF3 is required for in vivo infectivity. Mutations with substitutions in the first in-frame AUG in the junction region or with the same T insertion at the corresponding position of HEV genotype 4 did not affect the virus infectivity or rescue, although mutations with combinations of the two affected virus recovery efficiency, and a single mutation at the third in-frame AUG completely abolished virus infectivity in vivo, indicating that the third in-frame AUG in the junction region is required for virus infection and is likely the authentic initiation site for ORF3. A conserved double stem-loop RNA structure, which may be important for HEV replication, was identified in the junction region. This represents the first report of using a unique homologous pig model system to study the molecular mechanism of HEV replication and to systematically and definitively identify the authentic ORF3 initiation site.     

Vaccinia virus A17L open reading frame encodes an essential component of nascent viral membranes that is required to initiate morphogenesis.

We generated an antiserum to the predicted C-terminal peptide of the A17L open reading frame (ORF), which encodes a 23-kDa polypeptide with hydrophobic regions characteristic of membrane proteins. Immuno-electron microscopy of infected cells indicated that the A17L protein is intimately associated with the earliest characteristic viral membranes, even those formed in the presence of the drug rifampin. To study the role of the A17L protein in morphogenesis, we constructed recombinant vaccinia viruses in which the endogenous A17L ORF was deleted and a copy of the ORF under the control of the bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor was inserted into an alternative site in the vaccinia virus genome. Growth of these recombinant viruses was entirely dependent on the induction of A17L expression by isopropyl-beta-D-thiogalactopyranoside. Electron microscopic examination of cells infected in the absence of inducer revealed the accumulation of large, well-demarcated electron-dense aggregates but no characteristic membrane-associated viral structures. Viral late protein synthesis occurred under these conditions, although the maturational proteolytic processing of structural proteins was inhibited. We conclude that the product of the A17L gene is an essential component of the immature viral membrane and has an early function in viral morphogenesis.     

Chaperone-dependent E3 ligase CHIP ubiquitinates and mediates proteasomal degradation of soluble guanylyl cyclase

The nitric oxide receptor soluble guanylyl cyclase (sGC) exists in multimeric protein complexes, including heat shock protein (HSP) 90 and endothelial nitric oxide synthase. Inhibition of HSP90 by geldanamycin causes proteasomal degradation of sGC protein. In this study, we have investigated whether COOH terminus of heat shock protein 70-interacting protein (CHIP), a co-chaperone molecule that is involved in protein folding but is also a chaperone-dependent ubiquitin E3 ligase, could play a role in the process of degradation of sGC. Transient overexpression of CHIP in COS-7 cells degraded heterologous sGC in a concentration-related manner; this downregulation of sGC was abrogated by the proteasome inhibitor MG-132. Transfection of tetratricopeptide repeats and U-box domain CHIP mutants attenuated sGC degradation, suggesting that both domains are indispensable for CHIP function. Results from immunoprecipitation and indirect immunofluorescent microscopy experiments demonstrated that CHIP is associated with sGC, HSP90, and HSP70 in COS-7 cells. Furthermore, CHIP increased the association of HSP70 with sGC. In in vitro ubiquitination assays using purified proteins and ubiquitin enzymes, E3 ligase CHIP directly ubiquitinated sGC; this ubiquitination was potentiated by geldanamycin in COS-7 cells, followed by proteasomal degradation. In rat aortic smooth muscle cells, endogenous sGC was also degraded by adenovirus-infected wild-type CHIP but not by the chaperone interaction-deficient K30A CHIP, whereas CHIP, but not K30A, attenuated sGC expression in, and nitric oxide donor-induced relaxation of, rat aortic rings, suggesting that CHIP plays a regulatory role under physiological conditions. This study reveals a new mechanism for the regulation of sGC, an important mediator of cellular and vascular function.