What Has the Study of the K3 and K5 Viral Ubiquitin E3 Ligases Taught Us about Ubiquitin-Mediated Receptor Regulation?

Cells communicate with each other and the outside world through surface receptors, which need to be tightly regulated to prevent both overstimulation and receptor desensitization. Understanding the processes involved in the homeostatic control of cell surface receptors is essential, but we are not alone in trying to regulate these receptors. Viruses, as the ultimate host pathogens, have co-evolved over millions of years and have both pirated and adapted host genes to enable viral pathogenesis. K3 and K5 (also known as MIR1 and MIR2) are viral ubiquitin E3 ligases from Kaposi’s Sarcoma Associated Herpesvirus (KSHV) which decrease expression of a number of cell surface receptors and have been used to interrogate cellular processes and improve our understanding of ubiquitin-mediated receptor endocytosis and degradation. In this review, we summarize what has been learned from the study of these viral genes and emphasize their role in elucidating the complexity of ubiquitin in receptor regulation

A non-canonical ESCRT pathway, including His domain phosphotyrosine phosphatase (HD-PTP), is used for down-regulation of virally ubiquitinated MHC Class I

The Kaposi’s sarcoma-associated herpes virus (KSHV) K3 viral gene product effectively down-regulates cell surface MHC Class I. K3 is an E3 ubiquitin ligase that promotes K63-linked polyubiquitination of MHC Class I, providing the signal for clathrin mediated endocytosis. Endocytosis is followed by sorting into the intralumenal vesicles (ILVs) of multivesicular bodies (MVBs) and eventual delivery to lysosomes. The sorting of MHC Class I into MVBs requires many individual proteins of the four endosomal sorting complexes required for transport (ESCRTs). In HeLa cells expressing the KSHV K3 ubiquitin ligase, the effect of RNA interference-mediated depletion of individual proteins of the ESCRT-0 and ESCRT-I complexes and three ESCRT-III proteins showed that these are required to down-regulate MHC Class I. However, depletion of proteins of the ESCRT-II complex or of the ESCRT-III protein, VPS20/CHMP6, failed to prevent the loss of MHC Class I from the cell surface. Depletion of His domain phosphotyrosine phosphatase (HD-PTP) resulted in an increase in the cell surface concentration of MHC Class I in HeLa cells expressing the KSHV K3 ubiquitin ligase. Rescue experiments with wild type and mutant HD-PTP supported the conclusion that HD-PTP acts as an alternative to ESCRT-II and VPS20/CHMP6 as a link between the ESCRT-I and those ESCRT-III protein(s) necessary for ILV formation. Thus, the down-regulation of cell surface MHC Class I, polyubiquitinated by the KSHV K3 ubiquitin ligase, does not employ the canonical ESCRT pathway, but instead utilizes an alternative pathway in which HD-PTP replaces ESCRT-II and VPS20/CHMP6

A non-canonical ESCRT pathway, including histidine domain phosphotyrosine phosphatase (HD-PTP), is used for down-regulation of virally ubiquitinated MHC class I.

The Kaposi's sarcoma-associated herpes virus (KSHV) K3 viral gene product effectively down-regulates cell surface MHC class I. K3 is an E3 ubiquitin ligase that promotes Lys(63)-linked polyubiquitination of MHC class I, providing the signal for clathrin-mediated endocytosis. Endocytosis is followed by sorting into the intralumenal vesicles (ILVs) of multivesicular bodies (MVBs) and eventual delivery to lysosomes. The sorting of MHC class I into MVBs requires many individual proteins of the four endosomal sorting complexes required for transport (ESCRTs). In HeLa cells expressing the KSHV K3 ubiquitin ligase, the effect of RNAi-mediated depletion of individual proteins of the ESCRT-0 and ESCRT-I complexes and three ESCRT-III proteins showed that these are required to down-regulate MHC class I. However, depletion of proteins of the ESCRT-II complex or of the ESCRT-III protein, VPS20 (vacuolar protein sorting 20)/CHMP6 (charged MVB protein 6), failed to prevent the loss of MHC class I from the cell surface. Depletion of histidine domain phosphotyrosine phosphatase (HD-PTP) resulted in an increase in the cell surface concentration of MHC class I in HeLa cells expressing the KSHV K3 ubiquitin ligase. Rescue experiments with wild-type (WT) and mutant HD-PTP supported the conclusion that HD-PTP acts as an alternative to ESCRT-II and VPS20/CHMP6 as a link between the ESCRT-I and those ESCRT-III protein(s) necessary for ILV formation. Thus, the down-regulation of cell surface MHC class I, polyubiquitinated by the KSHV K3 ubiquitin ligase, does not employ the canonical ESCRT pathway, but instead utilizes an alternative pathway in which HD-PTP replaces ESCRT-II and VPS20/CHMP6.This work was supported by an MRC research grant to J.P.L. (G0900113). M.D.J.P. and J.L.E. were MRC research students and S.P. a Wellcome Trust research student. K.B. was a British Heart Foundation Intermediate Fellow and P.J.L. is a Wellcome Trust Principal Fellow. The CIMR is supported by a Wellcome Trust Strategic Award 100140 and an electron microscope was purchased with Wellcome Trust grant 093026.This is the final version of the article. It first appeared from Portland Press via http://dx.doi.org/10.1042/BJ2015033

Cleavage by signal peptide peptidase is required for the degradation of selected tail-anchored proteins.

The regulated turnover of endoplasmic reticulum (ER)-resident membrane proteins requires their extraction from the membrane lipid bilayer and subsequent proteasome-mediated degradation. Cleavage within the transmembrane domain provides an attractive mechanism to facilitate protein dislocation but has never been shown for endogenous substrates. To determine whether intramembrane proteolysis, specifically cleavage by the intramembrane-cleaving aspartyl protease signal peptide peptidase (SPP), is involved in this pathway, we generated an SPP-specific somatic cell knockout. In a stable isotope labeling by amino acids in cell culture-based proteomics screen, we identified HO-1 (heme oxygenase-1), the rate-limiting enzyme in the degradation of heme to biliverdin, as a novel SPP substrate. Intramembrane cleavage by catalytically active SPP provided the primary proteolytic step required for the extraction and subsequent proteasome-dependent degradation of HO-1, an ER-resident tail-anchored protein. SPP-mediated proteolysis was not limited to HO-1 but was required for the dislocation and degradation of additional tail-anchored ER-resident proteins. Our study identifies tail-anchored proteins as novel SPP substrates and a specific requirement for SPP-mediated intramembrane cleavage in protein turnover

Murine gammaherpesvirus-68 glycoprotein B presents a difficult neutralization target to monoclonal antibodies derived from infected mice

Persistent viruses disseminate from immune hosts. They must therefore resist neutralization by antibody. Murine gammaherpesvirus-68 (MHV-68) represents an accessible model with which to address how resistance to neutralization is achieved and how overcoming it might improve infection control. The MHV-68 glycoprotein B (gB), like that of other herpesviruses, is a virion protein that is essential for infectivity. As such, it presents a potential neutralization target. In order to test whether virus-induced antibodies reduce virion infectivity by binding to gB, monoclonal antibodies (mAbs) were derived from MHV-68-infected mice. gB-specific mAbs were common, but only an IgM specific for the gB N terminus reduced virion infectivity significantly. It inhibited MHV-68 entry into BHK-21 cells at a post-binding step that was linked closely to membrane fusion. Reducing the mAb to IgM monomers compromised neutralization severely, suggesting that a pentameric structure was crucial to its function. Antibody treatment never blocked BHK-21 cell infection completely and blocked the infection of NMuMG epithelial cells hardly at all. Virions saturated with antibody also remained infectious to mice. Thus, the MHV-68 gB presents at best a very difficult target for antibody-mediated neutralization

Identification of a lysosomal pathway regulating degradation of the bone morphogenetic protein receptor type II.

Bone morphogenetic proteins (BMPs) are critically involved in early development and cell differentiation. In humans, dysfunction of the bone morphogenetic protein type II receptor (BMPR-II) is associated with pulmonary arterial hypertension (PAH) and neoplasia. The ability of Kaposi sarcoma-associated herpesvirus (KSHV), the etiologic agent of Kaposi sarcoma and primary effusion lymphoma, to down-regulate cell surface receptor expression is well documented. Here we show that KSHV infection reduces cell surface BMPR-II. We propose that this occurs through the expression of the viral lytic gene, K5, a ubiquitin E3 ligase. Ectopic expression of K5 leads to BMPR-II ubiquitination and lysosomal degradation with a consequent decrease in BMP signaling. The down-regulation by K5 is dependent on both its RING domain and a membrane-proximal lysine in the cytoplasmic domain of BMPR-II. We demonstrate that expression of BMPR-II protein is constitutively regulated by lysosomal degradation in vascular cells and provide preliminary evidence for the involvement of the mammalian E3 ligase, Itch, in the constitutive degradation of BMPR-II. Disruption of BMP signaling may therefore play a role in the pathobiology of diseases caused by KSHV infection, as well as KSHV-associated tumorigenesis and vascular disease

In vivo imaging of murid herpesvirus-4 infection

Luciferase-based imaging allows a global view of microbial pathogenesis. We applied this technique to gammaherpesvirus infection by inserting a luciferase expression cassette into the genome of murine herpesvirus-4 (MuHV-4). The recombinant virus strongly expressed luciferase in lytically infected cells without significant attenuation. We used it to compare different routes of virus inoculation. After intranasal infection of anaesthetized mice, luciferase was expressed in the nose and lungs for 7–10 days and in lymphoid tissue, most consistently the superficial cervical lymph nodes, for up to 30 days. Gastrointestinal infection was not observed. Intraperitoneal infection was very different to intranasal, with strong luciferase expression in the liver, kidneys, intestines, reproductive tract and spleen, but none in the nose or lungs. The nose has not previously been identified as a site of MuHV-4 infection. After intranasal infection of non-anaesthetized mice, it was the only site of non-lymphoid luciferase expression. Nevertheless, lymphoid colonization and persistence were still established, even at low inoculation doses. In contrast, virus delivered orally was very poorly infectious. Inoculation route therefore had a major impact on pathogenesis. Low dose intranasal infection without anaesthesia seems most likely to mimic natural transmission, and may therefore be particularly informative about normal viral gene functions

Glycosaminoglycan Interactions in Murine Gammaherpesvirus-68 Infection

Glycosaminoglycans (GAGs) commonly participate in herpesvirus entry. They are thought to provide a reversible attachment to cells that promotes subsequent receptor binding. Murine gamma-herpesvirus-68 (MHV-68) infection of fibroblasts and epithelial cells is highly GAG-dependent. This is a function of the viral gp150, in that gp150-deficient mutants are much less GAG-dependent than wild-type. Here we show that the major MHV-68 GAG-binding protein is not gp150 but gp70, a product of ORF4. Surprisingly, ORF4-deficient MHV-68 showed normal cell binding and was more sensitive than wild-type to inhibition by soluble heparin rather than less. Thus, the most obvious viral GAG interaction made little direct contribution to infection. Indeed, a large fraction of the virion gp70 had its GAG-binding domain removed by post-translational cleavage. ORF4 may therefore act mainly to absorb soluble GAGs and prevent them from engaging gp150 prematurely. In contrast to gp70, gp150 bound poorly to GAGs, implying that it provides little in the way of adhesion. We hypothesize that it acts instead as a GAG-sensitive switch that selectively activates MHV-68 entry at cell surfaces

Murid herpesvirus-4 lacking thymidine kinase reveals route-dependent requirements for host colonization

Gammaherpesviruses infect at least 90 % of the world's population. Infection control is difficult, in part because some fundamental features of host colonization remain unknown, for example whether normal latency establishment requires viral lytic functions. Since human gammaherpesviruses have narrow species tropisms, answering such questions requires animal models. Murid herpesvirus-4 (MuHV-4) provides one of the most tractable. MuHV-4 genomes delivered to the lung or peritoneum persist without lytic replication. However, they fail to disseminate systemically, suggesting that the outcome is inoculation route-dependent. After upper respiratory tract inoculation, MuHV-4 infects mice without involving the lungs or peritoneum. We examined whether host entry by this less invasive route requires the viral thymidine kinase (TK), a gene classically essential for lytic replication in terminally differentiated cells. MuHV-4 TK knockouts delivered to the lung or peritoneum were attenuated but still reached lymphoid tissue. In contrast, TK knockouts delivered to the upper respiratory tract largely failed to establish a detectable infection. Therefore TK, and by implication lytic replication, is required for MuHV-4 to establish a significant infection by a non-invasive route

Multiple Functions for ORF75c in Murid Herpesvirus-4 Infection

All gamma-herpesviruses encode at least one homolog of the cellular enzyme formyl-glycineamide-phosphoribosyl-amidotransferase. Murid herpesvirus-4 (MuHV-4) encodes 3 (ORFs 75a, 75b and 75c), suggesting that at least some copies have acquired new functions. Here we show that the corresponding proteins are all present in virions and localize to infected cell nuclei. Despite these common features, ORFs 75a and 75b did not substitute functionally for a lack of ORF75c, as ORF75c virus knockouts were severely impaired for lytic replication in vitro and for host colonization in vivo. They showed 2 defects: incoming capsids failed to migrate to the nuclear margin following membrane fusion, and genomes that did reach the nucleus failed to initiate normal gene expression. The latter defect was associated with a failure of in-coming virions to disassemble PML bodies. The capsid transport deficit seemed to be functionally more important, since ORF75c− MuHV-4 infected both PML+ and PML− cells poorly. The original host enzyme has therefore evolved into a set of distinct and multi-functional viral tegument proteins. One important function is moving incoming capsids to the nuclear margin for viral genome delivery