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

Classical swine fever virus : analysis of the capsid protein core intracellular distribution and identification of its cellular partners

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Classical Swine Fever Virus: Analysis of the Capsid Protein Core Intracellular Distribution and Identification of its Cellular Partners.

Classical swine fever virus (CSFV) causes a highly contagious haemorrhagic disease of pigs leading to large-scale economic losses in Europe. Acute, subacute, and chronic infections are produced depending on the virulence of the strain and host genetic factors. CSFV is a small enveloped-positive-strand RNA virus classified in the Pestivirus genus of the Flaviviridae. The generation of a persistent infection suggests CSFV is able to modulate innate immune responses and/or apotosis. CSFV shares many similarities with the human pathogen hepatitis C virus (HCV) which also causes persistent infections. The core (capsid) protein of HCV is a multifunctional protein able to modulate cellular transcription, signal transduction pathways, and contribute to viral immune escape. This thesis has carried out a yeast two-hybrid (Y2H) analysis of cellular proteins that bind CSFV core, and generated antibodies to follow its processing and its intracellular distribution. CSFV core bound proteins topors and Sp100 in the Y2H screen. These are nuclear proteins that reside in promyelocytic leukaemia (PML) oncogenic domains (PODs) and are involved in p53 ubiquitination, transcription, and DNA replication. Interestingly, Sp110b, a homologue of Sp100 and co-repressor of retinoic acid nuclear receptor retinoic α (RARα), has been shown to bind HCV core. This interaction was shown responsible for tissue transglutaminase gene activation and all-frans-retinoic acid (ATRA)-induced cell death sensitisation through cytoplasmic retention of Sp110b. CSFV core expressed alone located to nuclear dots that resemble PODs and partially co-localised with topors and Sp100 in cotransfection experiments. For HCV core, nuclear localisation requires processing and removal of the hydrophobic amino acids that form the signal peptide of E1; otherwise it binds the endoplasmic reticulum (ER). For CSFV, both the core protein fused to the signal peptide, and, a truncated form lacking this signal sequence located to the nucleus suggesting differences in protein trafficking between the two viruses