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.

Characterization of Human Cytomegalovirus Genome Diversity in Immunocompromised Hosts by Whole-Genome Sequencing Directly From Clinical Specimens.

Background: Advances in next-generation sequencing (NGS) technologies allow comprehensive studies of genetic diversity over the entire genome of human cytomegalovirus (HCMV), a significant pathogen for immunocompromised individuals. Methods: Next-generation sequencing was performed on target enriched sequence libraries prepared directly from a variety of clinical specimens (blood, urine, breast milk, respiratory samples, biopsies, and vitreous humor) obtained longitudinally or from different anatomical compartments from 20 HCMV-infected patients (renal transplant recipients, stem cell transplant recipients, and congenitally infected children). Results: De novo-assembled HCMV genome sequences were obtained for 57 of 68 sequenced samples. Analysis of longitudinal or compartmental HCMV diversity revealed various patterns: no major differences were detected among longitudinal, intraindividual blood samples from 9 of 15 patients and in most of the patients with compartmental samples, whereas a switch of the major HCMV population was observed in 6 individuals with sequential blood samples and upon compartmental analysis of 1 patient with HCMV retinitis. Variant analysis revealed additional aspects of minor virus population dynamics and antiviral-resistance mutations. Conclusions: In immunosuppressed patients, HCMV can remain relatively stable or undergo drastic genomic changes that are suggestive of the emergence of minor resident strains or de novo infection.

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 viral CD45 ligand E3/49K on porcine cells reduces human anti-pig immune responses.

Transgenic expression of protective molecules in porcine cells and tissues is a promising approach to prevent xenograft rejection. Viruses have developed various strategies to escape the host's immune system. We generated porcine B cells (B cell line L23) expressing the human adenovirus protein E3/49K or the human cytomegalovirus protein pUL11 and investigated how human T, NK and B cell responses are affected by the expression of the viral proteins. Binding studies revealed that E3/49K and pUL11 interact with CD45 on human but not porcine peripheral blood mononuclear cells. T cell proliferation in response to L23-E3/49K cells was significantly reduced and accompanied by development of an anti-inflammatory cytokine milieu (low: TNF-alpha, IFN-gamma, IL-6; high: IL-4, IL-10). Human peripheral blood mononuclear cells which had been primed for four weeks by L23-E3/49K cells included an extended population of regulatory T cells. Cytotoxicity of effector T and natural killer cells against L23 cells was significantly reduced (40 to 50%) by E3/49K expression. B cell activation and antibody production to E3/49K expressing cells was also diminished. Surprisingly, pUL11 expression showed no effects. Reduction of human anti-pig immune responses by transgenic expression of selected viral genes may be a novel approach for protection of porcine xenografts.

Human Cytomegalovirus pUL11, a CD45 Ligand, Disrupts CD4 T Cell Control of Viral Spread in Epithelial Cells.

Human cytomegalovirus (HCMV) encodes numerous immunomodulatory genes that facilitate its persistence. Previously described mechanisms by which HCMV avoids T cell control typically involve evasion of detection by infected cells. Here, we show that the virus also inhibits T cells directly via an interaction between the pUL11 glycoprotein on infected cells and the CD45 phosphatase on T cells. The antiviral functions of CD4 T cells are impaired as a result of this interaction, largely via induced interleukin 10 (IL-10) secretion in the CD4 T cell central memory compartment, resulting in enhanced viral spread. This establishes CD45 as an inhibitory receptor that regulates antiviral T cell functions and has parallels with the manipulation of natural killer (NK) cells by HCMV. By coculturing donor T cells with HCMV-infected epithelial cells, we observed that CD4 T cells can respond to epithelial cell antigen presentation and can control HCMV spread via cytolytic and cytokine-dependent mechanisms. pUL11 impairs both mechanisms. We showed that pUL11-induced IL-10 secretion requires IL-2, mTOR, and T cell receptor signaling. This characterization of the effects of the pUL11-CD45 interaction may allow for the development of new antiviral therapies and treatments for inflammatory disorders. IMPORTANCE Human cytomegalovirus (HCMV) is adept at avoiding its host's immune defenses, both by evading detection and by directly inhibiting immune cells. This can lead to a loss of control of the infection, and dangerous disease can result, particularly in cases in which an individual's immune system is immature, weak, or suppressed. T cells form a crucial part of the response to HCMV and are used in cellular HCMV therapies. We show that an interaction between a viral glycoprotein (pUL11) and a T cell surface receptor (CD45) impairs T cell memory functions and allows for increased viral spread. This defines a new immunomodulatory strategy for the virus as well as a new T cell regulatory mechanism. These results are important, as they increase our understanding of how T cells function and how HCMV disrupts them. This will allow for the development of new antiviral therapies that restore T cell functions and indicates a new target for controlling pathological T cell disorders.