Two novel human cytomegalovirus NK cell evasion functions target MICA for lysosomal degradation

NKG2D plays a major role in controlling immune responses through the regulation of natural killer (NK) cells, αβ and γδ T-cell function. This activating receptor recognizes eight distinct ligands (the MHC Class I polypeptide-related sequences (MIC) A andB, and UL16-binding proteins (ULBP)1–6) induced by cellular stress to promote recognition cells perturbed by malignant transformation or microbial infection. Studies into human cytomegalovirus (HCMV) have aided both the identification and characterization of NKG2D ligands (NKG2DLs). HCMV immediate early (IE) gene up regulates NKGDLs, and we now describe the differential activation of ULBP2 and MICA/B by IE1 and IE2 respectively. Despite activation by IE functions, HCMV effectively suppressed cell surface expression of NKGDLs through both the early and late phases of infection. The immune evasion functions UL16, UL142, and microRNA(miR)-UL112 are known to target NKG2DLs. While infection with a UL16 deletion mutant caused the expected increase in MICB and ULBP2 cell surface expression, deletion of UL142 did not have a similar impact on its target, MICA. We therefore performed a systematic screen of the viral genome to search of addition functions that targeted MICA. US18 and US20 were identified as novel NK cell evasion functions capable of acting independently to promote MICA degradation by lysosomal degradation. The most dramatic effect on MICA expression was achieved when US18 and US20 acted in concert. US18 and US20 are the first members of the US12 gene family to have been assigned a function. The US12 family has 10 members encoded sequentially through US12–US21; a genetic arrangement, which is suggestive of an ‘accordion’ expansion of an ancestral gene in response to a selective pressure. This expansion must have be an ancient event as the whole family is conserved across simian cytomegaloviruses from old world monkeys. The evolutionary benefit bestowed by the combinatorial effect of US18 and US20 on MICA may have contributed to sustaining the US12 gene family

The Mouse Cytomegalovirus Gene m42 Targets Surface Expression of the Protein Tyrosine Phosphatase CD45 in Infected Macrophages

The receptor-like protein tyrosine phosphatase CD45 is expressed on the surface of cells of hematopoietic origin and has a pivotal role for the function of these cells in the immune response. Here we report that following infection of macrophages with mouse cytomegalovirus (MCMV) the cell surface expression of CD45 is drastically diminished. Screening of a set of MCMV deletion mutants allowed us to identify the viral gene m42 of being responsible for CD45 down-modulation. Moreover, expression of m42 independent of viral infection upon retroviral transduction of the RAW264.7 macrophage cell line led to comparable regulation of CD45 expression. In immunocompetent mice infected with an m42 deletion mutant lower viral titers were observed in all tissues examined when compared to wildtype MCMV, indicating an important role of m42 for viral replication in vivo. The m42 gene product was identified as an 18 kDa protein expressed with early kinetics and is predicted to be a tailanchored membrane protein. Tracking of surface-resident CD45 molecules revealed that m42 induces internalization and degradation of CD45. The observation that the amounts of the E3 ubiquitin ligases Itch and Nedd4 were diminished in cells expressing m42 and that disruption of a PY motif in the N-terminal part of m42 resulted in loss of function, suggest that m42 acts as an activator or adaptor for these Nedd4-like ubiquitin ligases, which mark CD45 for lysosomal degradation. In conclusion, the down-modulation of CD45 expression in MCMV-infected myeloid cells represents a novel pathway of virus-host interaction

The human cytomegalovirus ul11 protein interacts with the receptor tyrosine phosphatase cd45, resulting in functional paralysis of t cells

Human cytomegalovirus (CMV) exerts diverse and complex effects on the immune system, not all of which have been attributed to viral genes. Acute CMV infection results in transient restrictions in T cell proliferative ability, which can impair the control of the virus and increase the risk of secondary infections in patients with weakened or immature immune systems. In a search for new immunomodulatory proteins, we investigated the UL11 protein, a member of the CMV RL11 family. This protein family is defined by the RL11 domain, which has homology to immunoglobulin domains and adenoviral immunomodulatory proteins. We show that pUL11 is expressed on the cell surface and induces intercellular interactions with leukocytes. This was demonstrated to be due to the interaction of pUL11 with the receptor tyrosine phosphatase CD45, identified by mass spectrometry analysis of pUL11-associated proteins. CD45 expression is sufficient to mediate the interaction with pUL11 and is required for pUL11 binding to T cells, indicating that pUL11 is a specific CD45 ligand. CD45 has a pivotal function regulating T cell signaling thresholds; in its absence, the Src family kinase Lck is inactive and signaling through the T cell receptor (TCR) is therefore shut off. In the presence of pUL11, several CD45-mediated functions were inhibited. The induction of tyrosine phosphorylation of multiple signaling proteins upon TCR stimulation was reduced and T cell proliferation was impaired. We therefore conclude that pUL11 has immunosuppressive properties, and that disruption of T cell function via inhibition of CD45 is a previously unknown immunomodulatory strategy of CMV

Expression of the human cytomegalovirus UL11 glycoprotein in viral infection and evaluation of its effect on virus-epecific CD8 T cells.

The human cytomegalovirus (CMV) UL11 open reading frame (ORF) encodes a putative type I transmembrane glycoprotein, which displays remarkable amino acid sequence variability among different CMV isolates, suggesting that it represents an important virulence factor. In a previous study we have shown that UL11 can interact with the cellular receptor tyrosine phosphatase CD45, which has a central role for signal transduction in T cells, and treatment of T cells with high amounts of a soluble UL11 protein inhibited their proliferation. In order to analyze UL11 expression in CMV-infected cells, we constructed CMV recombinants that either encode tagged UL11 versions or carry a stop mutation in the UL11 ORF. Moreover, we examined whether UL11 affects the function of virus-specific cytotoxic T lymphocytes (CTL). We found that the UL11 ORF gives rise to several proteins due to both posttranslational modification and alternative translation initiation sites. Biotin labelling of surface proteins on infected cells indicated that only highly glycosylated UL11 forms are present at the plasma membrane, whereas low glycosylated UL11 forms were found in the endoplasmic reticulum. We did not find evidence of UL11 cleavage and secretion of a soluble UL11 version. Co-cultivation of CTLs recognizing different CMV epitopes with fibroblasts infected with a UL11 deletion mutant or the parental strain revealed that under the conditions applied UL11 did not influence the activation of CMV-specific CD8 T cells. For further studies we propose to investigate the interaction of UL11 with CD45 and the functional consequences in other immune cells expressing CD45. IMPORTANCE: Human cytomegalovirus belongs to those viruses that extensively interfere with the host immune response. Yet, the precise function of many putative immunomodulatory CMV proteins remains elusive. Previously, we have shown that the CMV UL11 protein interacts with the leukocyte common antigen CD45, a cellular receptor tyrosine phosphatase with a central role for signal transduction in T cells. Here, we examined the proteins expressed by the UL11 gene in CMV infected cells and found that at least one form of UL11 is present at the cell surface, enabling it to interact with CD45 on immune cells. Surprisingly, CMV-expressed UL11 did not affect the activity of virus-specific CD8 T cells. This finding warrants to investigate the impact of UL11 on CD45 functions in other leukocyte subpopulations

The human cytomegalovirus glycoprotein pUL11 acts via CD45 to induce T cell IL-10 secretion.

Human Cytomegalovirus (HCMV) is a widespread pathogen, infection with which can cause severe disease for immunocompromised individuals. The complex changes wrought on the host's immune system during both productive and latent HCMV infection are well known. Infected cells are masked and manipulated and uninfected immune cells are also affected; peripheral blood mononuclear cell (PBMC) proliferation is reduced and cytokine profiles altered. Levels increase of the anti-inflammatory cytokine IL-10, which may be important for the establishment of HCMV infections and is required for the development of high viral titres by murine cytomegalovirus. The mechanisms by which HCMV affects T cell IL-10 secretion are not understood. We show here that treatment of PBMC with purified pUL11 induces IL-10 producing T cells as a result of pUL11 binding to the CD45 phosphatase on T cells. IL-10 production induced by HCMV infection is also in part mediated by pUL11. Supernatants from pUL11 treated cells have anti-inflammatory effects on untreated PBMC. Considering the mechanism, CD45 can be a positive or negative regulator of TCR signalling, depending on its expression level, and we show that pUL11 also has concentration dependent activating or inhibitory effects on T cell proliferation and on the kinase function of the CD45 substrate Lck. pUL11 is therefore the first example of a viral protein that can target CD45 to induce T cells with anti-inflammatory properties. It is also the first HCMV protein shown to induce T cell IL-10 secretion. Understanding the mechanisms by which pUL11-induced changes in signal strength influence T cell development and function may provide the basis for the development of novel antiviral treatments and therapies against immune pathologies