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 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

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

The murine gamma-herpesvirus-68 MK3 protein causes TAP degradation independent of MHC class I heavy chain degradation

The murine gamma-herpesvirus-68 MK3 protein has an intricate interaction with the peptide loading complex that involves MK3 stabilization, a rapid degradation of MHC class I heavy chains, and a slower degradation of TAP. Here we have used tapasin chimeras to distinguish functionally the different immune evasion mechanisms of MK3. Tapasin was cloned in two alternatively spliced forms that differed by a single transmembrane valine residue. Each restored antigen presentation and MK3 function in tapasin-deficient cells. The transmembrane/cytoplasmic portion of tapasin, linked to the extracellular domain of CD8, also restored TAP stability and MK3 stability in tapasin-deficient cells. MK3 did not associate with or degrade MHC class I in these cells, which lacked the endoplasmic reticulum domain of tapasin, but degraded TAP at least as efficiently as when full-length tapasin was present. The un-degraded MHC class I consequently showed impaired maturation. The fact that MK3 required intact tapasin to degrade MHC class I but only the transmembrane/cytoplasmic portion of tapasin to degrade TAP indicated that these two immune evasion functions operate independently

Protection against wild-type murine gammaherpesvirus-68 latency by a latency-deficient mutant

A murine gammaherpesvirus-68 (MHV-68) mutant with deregulated transcription of its ORF50 transactivator was severely impaired in latency establishment. The deregulated virus showed reduced immunogenicity, probably reflecting a lower antigen load. However, it still elicited effective immunity to a subsequent wild-type (WT) virus challenge. Infection was not completely prevented, but was very substantially reduced in extent and the long-term level of WT viral DNA in lungs and spleens remained low. Thus latency-deficient MHV-68 illustrates a possible general approach to creating attenuated gammaherpesvirus vaccines that can protect against pathogenic WT infections

Viral degradation of the MHC class I peptide loading complex

The murine γ-herpesvirus-68 MK3 protein inhibits CD8+ T cell recognition by ubiquitinating the cytoplasmic tails of classical MHC class I heavy chains. Here we show that MK3 also provides the first example of a protein that degrades tapasin and TAP. The degradation was MK3 RING finger dependent and primarily affected TAP. MK3 associated with TAP1 in the absence of tapasin or TAP2, suggesting that TAP1 was a primary binding partner in the peptide loading complex. TAP2 also played a major role in MK3 stability and function. By degrading TAP, therefore, MK3 limited its own expression. However, TAP degradation also broadened the MK3 inhibitory repertoire and achieved a remarkable resistance to MHC class I upregulation by interferon-γ, suggesting that it represents a specific adaptation to immune evasion in lymphoid tissue