An Excellent Monitoring System for Surface Ubiquitination-Induced Internalization in Mammals

Background. At present, it is difficult to visualize the internalization of surface receptors induced by ubiquitination that is taken place at the plasma membrane in mammals. This problem makes it difficult to reveal molecular basis for ubiquitinationmediated internalization in mammals. Methodology/Principle Findings. In order to overcome it, we have generated T-REx-c-MIR, a novel mammalian Tet-on B cell line using a constitutively active E3 ubiquitin ligase, c-MIR, and its artificial target molecule. By applying the surface biotinylation method to T-REx-c-MIR, we succeeded to monitor the fate of surface target molecules after initiation of ubiquitination process by doxycycline (Dox)-induced c-MIR expression. Target molecules that preexisted at the plasma membrane before induction of c-MIR expression were oligo-ubiquitinated and degraded by Dox-induced c-MIR expression. Dox-induced c-MIR expression initiated rapid internalization of surface target molecules, and blockage of the internalization induced the accumulation of the surface target molecules that were newly ubiquitinated by c-MIR. Inhibition of the surface ubiquitination by down-regulating ubiquitin conjugating enzyme E2 impaired the internalization of target molecules. Finally, a complex of c-MIR and target molecule was detected at the plasma membrane. Conclusions/ Significances. These results demonstrate that in T-REx-c-MIR, surface target molecule is ubiquitinated at the plasma membrane and followed by being internalized from the plasma membrane. Thus, T-REx-c-MIR is a useful experimental tool t

The Epstein-Barr Virus G-Protein-Coupled Receptor Contributes to Immune Evasion by Targeting MHC Class I Molecules for Degradation

Epstein-Barr virus (EBV) is a human herpesvirus that persists as a largely subclinical infection in the vast majority of adults worldwide. Recent evidence indicates that an important component of the persistence strategy involves active interference with the MHC class I antigen processing pathway during the lytic replication cycle. We have now identified a novel role for the lytic cycle gene, BILF1, which encodes a glycoprotein with the properties of a constitutive signaling G-protein-coupled receptor (GPCR). BILF1 reduced the levels of MHC class I at the cell surface and inhibited CD8+ T cell recognition of endogenous target antigens. The underlying mechanism involves physical association of BILF1 with MHC class I molecules, an increased turnover from the cell surface, and enhanced degradation via lysosomal proteases. The BILF1 protein of the closely related CeHV15 c1-herpesvirus of the Rhesus Old World primate (80% amino acid sequence identity) downregulated surface MHC class I similarly to EBV BILF1. Amongst the human herpesviruses, the GPCR encoded by the ORF74 of the KSHV c2-herpesvirus is most closely related to EBV BILF1 (15% amino acid sequence identity) but did not affect levels of surface MHC class I. An engineered mutant of BILF1 that was unable to activate G protein signaling pathways retained the ability to downregulate MHC class I, indicating that the immune-modulating and GPCR-signaling properties are two distinct functions of BILF1. These findings extend our understanding of the normal biology of an important human pathogen. The discovery of a third EBV lytic cycle gene that cooperates to interfere with MHC class I antigen processing underscores the importance of the need for EBV to be able to evade CD8+ T cell responses during the lytic replication cycle, at a time when such a large number of potential viral targets are expressed

The RING-CH ligase K5 antagonizes restriction of KSHV and HIV-1 particle release by mediating ubiquitin-dependent endosomal degradation of tetherin

Tetherin (CD317/BST2) is an interferon-induced membrane protein that inhibits the release of diverse enveloped viral particles. Several mammalian viruses have evolved countermeasures that inactivate tetherin, with the prototype being the HIV-1 Vpu protein. Here we show that the human herpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) is sensitive to tetherin restriction and its activity is counteracted by the KSHV encoded RING-CH E3 ubiquitin ligase K5. Tetherin expression in KSHV-infected cells inhibits viral particle release, as does depletion of K5 protein using RNA interference. K5 induces a species-specific downregulation of human tetherin from the cell surface followed by its endosomal degradation. We show that K5 targets a single lysine (K18) in the cytoplasmic tail of tetherin for ubiquitination, leading to relocalization of tetherin to CD63-positive endosomal compartments. Tetherin degradation is dependent on ESCRT-mediated endosomal sorting, but does not require a tyrosine-based sorting signal in the tetherin cytoplasmic tail. Importantly, we also show that the ability of K5 to substitute for Vpu in HIV-1 release is entirely dependent on K18 and the RING-CH domain of K5. By contrast, while Vpu induces ubiquitination of tetherin cytoplasmic tail lysine residues, mutation of these positions has no effect on its antagonism of tetherin function, and residual tetherin is associated with the trans-Golgi network (TGN) in Vpu-expressing cells. Taken together our results demonstrate that K5 is a mechanistically distinct viral countermeasure to tetherin-mediated restriction, and that herpesvirus particle release is sensitive to this mode of antiviral inhibition

Inhibition of Atrogin-1/MAFbx Mediated MyoD Proteolysis Prevents Skeletal Muscle Atrophy In Vivo

Ubiquitin ligase Atrogin1/Muscle Atrophy F-box (MAFbx) up-regulation is required for skeletal muscle atrophy but substrates and function during the atrophic process are poorly known. The transcription factor MyoD controls myogenic stem cell function and differentiation, and seems necessary to maintain the differentiated phenotype of adult fast skeletal muscle fibres. We previously showed that MAFbx mediates MyoD proteolysis in vitro. Here we present evidence that MAFbx targets MyoD for degradation in several models of skeletal muscle atrophy. In cultured myotubes undergoing atrophy, MAFbx expression increases, leading to a cytoplasmic-nuclear shuttling of MAFbx and a selective suppression of MyoD. Conversely, transfection of myotubes with sh-RNA-mediated MAFbx gene silencing (shRNAi) inhibited MyoD proteolysis linked to atrophy. Furthermore, overexpression of a mutant MyoDK133R lacking MAFbx-mediated ubiquitination prevents atrophy of mouse primary myotubes and skeletal muscle fibres in vivo. Regarding the complex role of MyoD in adult skeletal muscle plasticity and homeostasis, its rapid suppression by MAFbx seems to be a major event leading to skeletal muscle wasting. Our results point out MyoD as the second MAFbx skeletal muscle target by which powerful therapies could be developed

Murine Gammaherpesvirus-68 Inhibits Antigen Presentation by Dendritic Cells

Dendritic cells (DCs) play a central role in initiating adaptive immunity. Murine gammaherpesvirus-68 (MHV-68), like many persistent viruses, infects DCs during normal host colonization. It therefore provides a means to understanding what host and viral genes contribute to this aspect of pathogenesis. The infected DC phenotype is likely to depend on whether viral gene expression is lytic or latent and whether antigen presentation is maintained. For MHV-68, neither parameter has been well defined. Here we show that MHV-68 infects immature but not mature bone marrow-derived DCs. Infection was predominantly latent and these DCs showed no obvious defect in antigen presentation. Lytically infected DCs were very different. These down-regulated CD86 and MHC class I expression and presented a viral epitope poorly to CD8+ T cells. Antigen presentation improved markedly when the MHV-68 K3 gene was disrupted, indicating that K3 fulfils an important function in infected DCs. MHV-68 infects only a small fraction of the DCs present in lymphoid tissue, so K3 expression is unlikely to compromise significantly global CD8+ T cell priming. Instead it probably helps to maintain lytic gene expression in DCs once CD8+ T cell priming has occurred

Membrane-Associated RING-CH Proteins Associate with Bap31 and Target CD81 and CD44 to Lysosomes

Membrane-associated RING-CH (MARCH) proteins represent a family of transmembrane ubiquitin ligases modulating intracellular trafficking and turnover of transmembrane protein targets. While homologous proteins encoded by gamma-2 herpesviruses and leporipoxviruses have been studied extensively, limited information is available regarding the physiological targets of cellular MARCH proteins. To identify host cell proteins targeted by the human MARCH-VIII ubiquitin ligase we used stable isotope labeling of amino-acids in cell culture (SILAC) to monitor MARCH-dependent changes in the membrane proteomes of human fibroblasts. Unexpectedly, we observed that MARCH-VIII reduced the surface expression of Bap31, a chaperone that predominantly resides in the endoplasmic reticulum (ER). We demonstrate that Bap31 associates with the transmembrane domains of several MARCH proteins and controls intracellular transport of MARCH proteins. In addition, we observed that MARCH-VIII reduced the surface expression of the hyaluronic acid-receptor CD44 and both MARCH-VIII and MARCH-IV sequestered the tetraspanin CD81 in endo-lysosomal vesicles. Moreover, gene knockdown of MARCH-IV increased surface levels of endogenous CD81 suggesting a constitutive involvement of this family of ubiquitin ligases in the turnover of tetraspanins. Our data thus suggest a role of MARCH-VIII and MARCH-IV in the regulated turnover of CD81 and CD44, two ubiquitously expressed, multifunctional proteins

Francisella tularensis Elicits IL-10 via a PGE2-Inducible Factor, to Drive Macrophage MARCH1 Expression and Class II Down-Regulation

Francisella tularensis is a bacterial pathogen that uses host-derived PGE2 to subvert the host's adaptive immune responses in multiple ways. Francisella-induced PGE2 acts directly on CD4 T cells to blunt production of IFN-γ. Francisella-induced PGE2 can also elicit production of a >10 kDa soluble host factor termed FTMØSN (F. tularensis macrophage supernatant), which acts on IFN-γ pre-activated MØ to down-regulate MHC class II expression via a ubiquitin-dependent mechanism, blocking antigen presentation to CD4 T cells. Here, we report that FTMØSN-induced down-regulation of MØ class II is the result of the induction of MARCH1, and that MØ expressing MARCH1 “resistant” class II molecules are resistant to FTMØSN-induced class II down-regulation. Since PGE2 can induce IL-10 production and IL-10 is the only reported cytokine able to induce MARCH1 expression in monocytes and dendritic cells, these findings suggested that IL-10 is the active factor in FTMØSN. However, use of IL-10 knockout MØ established that IL-10 is not the active factor in FTMØSN, but rather that Francisella-elicited PGE2 drives production of a >10 kDa host factor distinct from IL-10. This factor then drives MØ IL-10 production to induce MARCH1 expression and the resultant class II down-regulation. Since many human pathogens such as Salmonella typhi, Mycobacterium tuberculosis and Legionella pneumophila also induce production of host PGE2, these results suggest that a yet-to-be-identified PGE2-inducible host factor capable of inducing IL-10 is central to the immune evasion mechanisms of multiple important human pathogens

Constructing and Multilateralizing the Regional Comprehensive Economic Partnership: An Asian Perspective

In May 2013 the ASEAN+6 countries began to negotiate the Regional Comprehensive Economic Partnership (RCEP). The objective of this paper is to analyze the feasibility of constructing such a region-wide agreement and to examine ways to multilateralize it. The paper first reviews free trade agreement (FTA) developments, and discusses the characteristics and motives of FTAs in East Asia. It then analyzes the contents of major plurilateral FTAs in East Asia, that is ASEAN's five FTAs each with the People's Republic of China, Japan, the Republic of Korea, India, and Australia/New Zealand, which are considered as a base for a region-wide FTA. Finally, the paper examines the feasibility of the RCEP by consolidating the ASEAN+1 FTAs and discusses the possible ways to multilateralize the RCEP

Detection of Mycobacterium leprae DNA from Archaeological Skeletal Remains in Japan Using Whole Genome Amplification and Polymerase Chain Reaction

BACKGROUND: Identification of pathogen DNA from archaeological human remains is a powerful tool in demonstrating that the infectious disease existed in the past. However, it is very difficult to detect trace amounts of DNA remnants attached to the human skeleton, especially from those buried in a humid atmosphere with a relatively high environmental temperature such as in Asia. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate Mycobacterium leprae DNA from archaeological skeletal remains in Japan by polymerase chain reaction, DNA sequencing and single nucleotide polymorphism (SNP) analysis. In addition, we have established a highly sensitive method of detecting DNA using a combination of whole genome amplification and polymerase chain reaction, or WGA-PCR, which provides superior sensitivity and specificity in detecting DNA from trace amounts of skeletal materials. CONCLUSION/SIGNIFICANCE: We have detected M. leprae DNA in archaeological skeletal remains for the first time in the Far East. Its SNP genotype corresponded to type 1; the first detected case worldwide of ancient M. leprae DNA. We also developed a highly sensitive method to detect ancient DNA by utilizing whole genome amplification