Human cytomegalovirus (HCMV) evades T-cell recognition by down-regulating expression of major histocompatibility complex (MHC) class I and II molecules on the surfaces of infected cells. Contrary to the "missing-self" hypothesis, HCMV-infected cells are refractory to lysis by natural killer (NK) cells. Inhibition of NK cell function is mediated by a number of HCMV immune evasion molecules, which operate by delivering inhibitory signals to NK cells and preventing engagement of activating ligands. One such molecule is UL142, which is an MHC class I-related glycoprotein encoded by clinical isolates and low-passage-number strains of HCMV. UL142 is known to down-modulate surface expression of MHC class I-related chain A (MICA), which is a ligand of the activating NK receptor NKG2D. However, the mechanism by which UL142 interferes with MICA is unknown. Here, we show that UL142 localizes predominantly to the endoplasmic reticulum (ER) and cis-Golgi apparatus. The transmembrane domain of UL142 mediates its ER localization, while we propose that the UL142 luminal domain is involved in its cis-Golgi localization. We also confirm that UL142 downmodulates surface expression of full-length MICA alleles while having no effect on the truncated allele MICA*008. However, we demonstrate for the first time that UL142 retains full-length MICA alleles in the cis-Golgi apparatus. In addition, we propose that UL142 interacts with nascent MICA en route to the cell surface but not mature MICA at the cell surface. Our data also demonstrate that the UL142 luminal and transmembrane domains are involved in recognition and intracellular sequestration of full-length MICA alleles.Human cytomegalovirus (HCMV), a member of the Herpesviridae family, is a ubiquitous human pathogen with seroprevalence ranging between 50 to 100% worldwide (46). Severe morbidity and mortality are associated with HCMV infection in individuals that are immunocompromised or immunologically immature (25), and the severity of HCMV disease in immunocompromised individuals correlates with the level of immune suppression. However, HCMV infection in healthy immunocompetent individuals is usually asymptomatic/subclinical (25). Like all herpesviruses, HCMV establishes lifelong latent infection, HCMV residues, in hematopoietic cells of the myeloid lineage (45). Thus, following primary infection, HCMV persists in the host despite a robust humoral and cellmediated immune response.HCMV down-modulates surface expression of host major histocompatibility complex (MHC) class I molecules in order to evade T-cell recognition (5, 7, 24), but in doing so, the virus risks natural killer (NK) cell activation due to the lack of inhibitory receptor signaling (30). However, there is now ample evidence that HCMV evades NK cell-mediated lysis by a variety of different mechanisms (58). These include the expression of molecules that engage inhibitory NK receptors, as well as the down-modulation of ligands for activating NK receptors.The HCMV UL40 open reading frame encodes a nonameric peptide that ...

Poly(ADP-ribose) glycohydrolase (PARG) is the only enzyme known to catalyse hydrolysis of the O-glycosidic linkages of ADP-ribose polymers, thereby reversing the effects of poly(ADP-ribose) polymerases. PARG deficiency leads to cell death whilst PARG depletion causes sensitisation to certain DNA damaging agents, implicating PARG as a potential therapeutic target in several disease areas. Efforts to develop small molecule inhibitors of PARG activity have until recently been hampered by a lack of structural information on PARG. We have used a combination of bio-informatic and experimental approaches to engineer a crystallisable, catalytically active fragment of human PARG (hPARG). Here, we present high-resolution structures of the catalytic domain of hPARG in unliganded form and in complex with three inhibitors: ADP-ribose (ADPR), adenosine 5′-diphosphate (hydroxymethyl)pyrrolidinediol (ADP-HPD) and 8-n-octyl-amino-ADP-HPD. Our structures confirm conservation of overall fold amongst mammalian PARG glycohydrolase domains, whilst revealing additional flexible regions in the catalytic site. These new structures rationalise a body of published mutational data and the reported structure-activity relationship for ADP-HPD based PARG inhibitors. In addition, we have developed and used biochemical, isothermal titration calorimetry and surface plasmon resonance assays to characterise the binding of inhibitors to our PARG protein, thus providing a starting point for the design of new inhibitors.

The gamma-herpesviruses persist as latent episomes in a dynamic lymphocyte pool. Their consequent need to express a viral episome maintenance protein presents a potential immune target. The glycine–alanine repeat of the Epstein–Barr virus episome maintenance protein, EBNA-1, limits EBNA-1 epitope presentation to CD8+ T lymphocytes (CTLs). However, CTL recognition occurs in vitro, so the significance of such evasion for viral fitness is unclear. We used the murine gamma-herpesvirus-68 (MHV-68) to define the in vivo contribution of cis-acting CTL evasion to host colonisation. Although the ORF73 episome maintenance protein of MHV-68 lacks a glycine–alanine repeat, it was equivalent to EBNA-1 in conferring limited presentation on linked epitopes. This was associated with reduced protein synthesis and reduced protein degradation. We bypassed the cis-acting evasion of ORF73 by using an internal ribosome entry site to express in trans-a CTL target from the same mRNA. This led to a severe, MHC class I–restricted and CTL-dependent reduction in viral latency. Thus, despite MHV-68 encoding at least two trans-acting CTL evasion proteins, cis-acting evasion during episome maintenance was essential for normal host colonisation.

Human CMV (HCMV) encodes multiple genes that control NK cell activation and cytotoxicity. Some of these HCMV-encoded gene products modulate NK cell activity as ligands expressed at the cell surface that engage inhibitory NK cell receptors, whereas others prevent the infected cell from upregulating ligands that bind to activating NK cell receptors. A major activating NKR is the homodimeric NKG2D receptor, which has eight distinct natural ligands in humans. It was shown that HCMV is able to prevent the surface expression of five of these ligands (MIC A/B and ULBP1, 2, and 6). In this article, we show that the HCMV gene product UL142 can prevent cell surface expression of ULBP3 during infection. We further show that UL142 interacts with ULBP3 and mediates its intracellular retention in a compartment that colocalizes with markers of the cis-Golgi complex. In doing so, UL142 prevents ULBP3 trafficking to the surface and protects transfected cells from NK-mediated cytotoxicity. This is the first description of a viral gene able to mediate downregulation of ULBP3.

CD4+ T cells play a major role in containing herpesvirus infections. However, their cellular targets remain poorly defined. In vitro CD4+ T cells have been reported to kill B cells that harbor a latent gammaherpesvirus. We used the B cell‐tropic murine gammaherpesvirus‐68 (MHV‐68) to test whether this also occurred in vivo. MHV‐68 that expressed cytoplasmic ovalbumin (OVA) in tandem with its episome maintenance protein, ORF73, stimulated CD8+ T cells specific for the H2‐Kb‐restricted OVA epitope SIINFEKL and was rapidly eliminated from C57BL/6 (H2b) mice. However, the same virus failed to stimulate CD4+ T cells specific for the I‐Ad/I‐Ab‐restricted OVA323–339 epitope. We overcame any barrier to the MHC class II‐restricted presentation of an endogenous epitope by substituting OVA323–339 for the CLIP peptide of the invariant chain (ORF73‐IRES‐Ii‐OVA), again expressed in tandem with ORF73. This virus presented OVA323–339 but showed little or no latency deficit in either BALB/c (H2d) or C57BL/6 mice. Latent antigen‐specific CD4+ T cells therefore either failed to recognize key virus‐infected cell populations in vivo or lacked the effector functions required to control them.