The NK cell–activating receptor NKG2D interacts with three different cellular ligands, all of which are regulated by mouse cytomegalovirus (MCMV). We set out to define the viral gene product regulating murine UL16-binding protein-like transcript (MULT)-1, a newly described NKG2D ligand. We show that MCMV infection strongly induces MULT-1 gene expression, but surface expression of this glycoprotein is nevertheless completely abolished by the virus. Screening a panel of MCMV deletion mutants defined the gene m145 as the viral regulator of MULT-1. The MCMV m145-encoded glycoprotein turned out to be necessary and sufficient to regulate MULT-1 by preventing plasma membrane residence of MULT-1. The importance of MULT-1 in NK cell regulation in vivo was confirmed by the attenuating effect of the m145 deletion that was lifted after NK cell depletion. Our findings underline the significance of escaping MULT-1/NKG2D signaling for viral survival and maintenance.

Herpesviral capsids are assembled in the host cell nucleus before being translocated into the cytoplasm for further maturation. The crossing of the nuclear envelope represents a major event that requires the formation of the nuclear egress complex (NEC). Previous studies demonstrated that human cytomegalovirus (HCMV) proteins pUL50 and pUL53, as well as their homologs in all members of Herpesviridae, interact with each other at the nuclear envelope and form the heterodimeric core of the NEC. In order to characterize further the viral and cellular protein content of the multimeric NEC, the native complex was isolated from HCMV-infected human primary fibroblasts at various time points and analyzed using quantitative proteomics. Previously postulated components of the HCMVspecific NEC, as well as novel potential NEC-associated proteins such as emerin, were identified. In this regard, interaction and colocalization between emerin and pUL50 were confirmed by coimmunoprecipitation and confocal microscopy analyses, respectively. A functional validation of viral and cellular NEC constituents was achieved through siRNA-mediated knockdown experiments. The important role of emerin in NEC functionality was demonstrated by a reduction of viral replication when emerin expression was down-regulated. Moreover, under such conditions, reduced production of viral proteins and deregulation of viral late cytoplasmic maturation were observed. Combined, these data prove the functional importance of emerin as an NEC component, associated with pUL50, pUL53, pUL97, p32/ gC1qR, and further regulatory proteins. Summarized, our findings provide the first proteomics-based characterization and functional validation of the HCMV-specific multimeric NEC. Molecular & Cellular

Members of the α- and β-subfamily of herpesviridae encode glycoproteins that specifically bind to the Fc part of immunoglobulin (Ig)G. Plasma membrane resident herpesviral Fc receptors seem to prevent virus-specific IgG from activating antibody-dependent effector functions. We show that the mouse cytomegalovirus (MCMV) molecule fcr-1 promotes a rapid down-regulation of NKG2D ligands murine UL16-binding protein like transcript (MULT)-1 and H60 from the cell surface. Deletion of the m138/fcr-1 gene from the MCMV genome attenuates viral replication to natural killer (NK) cell response in an NKG2D-dependent manner in vivo. A distinct N-terminal module within the fcr-1 ectodomain in conjunction with the fcr-1 transmembrane domain was required to dispose MULT-1 to degradation in lysosomes. In contrast, down-modulation of H60 required the complete fcr-1 ectodomain, implying independent modes of fcr-1 interaction with the NKG2D ligands. The results establish a novel viral strategy for down-modulating NK cell responses and highlight the impressive diversity of Fc receptor functions.

Natural killer (NK) cells are an important defense mechanism against pathogens, particularly against viruses belonging to the herpesvirus family (45, 48). NK cell receptor genes do not undergo somatic recombination and clonal specification (38), and their activation is tightly regulated by a balance of signaling through inhibitory receptors specific for major histocompatibility complex (MHC) class I proteins and activating NK cell receptors with diverse specificities (28). Some activating NK receptors are specific for viral proteins, such as the m157 protein of murine cytomegalovirus (MCMV) and the hemagglutinins of Sendai virus and influenza virus, which are recognized by Ly49H, NKp44, and NKp46, respectively (4,5,31,46). NKG2D is a type II C-lectin-like activating NK cell receptor that was first identified as a member of the NKG2 family (21) and is expressed on all NK cells, as well as on CD8ϩ T cells, ␥␦ T cells, and macrophages (6,15,17,22). NKG2D is a promiscuous receptor that can recognize a broad spectrum of cell surface ligands that are distantly related to MHC class I molecules and are up-regulated on stressed, infected, or transformed cells (11). The known NKG2D ligands on human cells are the MHC class I chain-related molecules (MICA and MICB) (6, 47) and the UL-16 binding proteins (ULBP-1, ULBP-2, and ULBP-3) (12), whereas the mouse NKG2D ligands are H60 (15, 30), retinoic acid early inducible gene 1 (RAE-1␣, -␤, -␥, -␦, and -ε isoforms) (10), and the recently identified murine UL-16 binding protein-like transcript 1 (MULT-1) (9). H60 was originally described as a minor histocompatibility antigen recognized by T cells from C57BL/6 mice in response to BALB.B splenocytes (30), and RAE-1 has been shown to play a role during embryonic development (57).Cytomegaloviruses (CMVs) possess a remarkable range of mechanisms to escape or subvert the immune response (2). Both human CMV (HCMV) and MCMV have developed mechanisms for evading the control of NK cells by interfering with the expression of NKG2D ligands (36). The HCMV protein encoded by the UL16 gene binds ULBP-1, ULBP-2, and MICB (12), preventing these ligands from being expressed on the surfaces of HCMV-infected cells (16,55). Based on their early susceptibility to MCMV infection, mouse strains can be either resistant or sensitive to this virus (18,43). In resistant mouse strains, NK cells become activated via an interaction of Ly49H, an activating NK cell receptor, with the MCMV-encoded m157 protein (3,46). In contrast, Ly49H-negative mouse strains, including most wild mice, show very low NK activities against MCMV, rendering them susceptible to this virus (42). The puzzling fact that Ly49H-negative mice, although being capable of mounting an effective NK cell response against other pathogens (54), are unable to create effective NK cell control of MCMV, has recently been explained by the MCMV-driven down-regulation of cellular ligands for the NKG2D receptor (27). MCMV gp40, a viral glycoprotein encoded by the m152 gene, apart from down-regulating MHC c...

BackgroundThe use of checkpoint inhibitors has revolutionized cancer therapy. Unfortunately, these therapies often cause immune-related adverse effects, largely due to a lack of tumor specificity.MethodsWe stained human natural killer cells using fusion proteins composed of the extracellular portion of various tumor markers fused to the Fc portion of human IgG1, and identified Nectin4 as a novel TIGIT ligand. Next, we generated a novel Nectin4 blocking antibody and demonstrated its efficacy as a checkpoint inhibitor in killing assays and in vivo.ResultsWe identify Nectin4 to be a novel ligand of TIGIT. We showed that, as opposed to all other known TIGIT ligands, which bind also additional receptors, Nectin4 interacts only with TIGIT. We show that the TIGIT-Nectin4 interaction inhibits natural killer cell activity, a critical part of the innate immune response. Finally, we developed blocking Nectin4 antibodies and demonstrated that they enhance tumor killing in vitro and in vivo.ConclusionWe discovered that Nectin4 is a novel ligand for TIGIT and demonstrated that specific antibodies against it enhance tumor cell killing in vitro and in vivo. Since Nectin4 is expressed almost exclusively on tumor cells, our Nectin4-blocking antibodies represent a combination of cancer specificity and immune checkpoint activity, which may prove more effective and safe for cancer immunotherapy.