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

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          Abstract

          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 tail-anchored 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.

          Author Summary

          Human cytomegalovirus (HCMV) is a tenacious pathogen, which can be life-threatening for immunocompromised patients and immunologically immature newborns. The pathogenicity of HCMV is owed to a plethora of immunomodulatory functions that interfere with host defense mechanisms. Such viral functions can teach us about viral pathogenesis mechanisms, and also about the functioning of immune cells. In this study we report that the mouse cytomegalovirus (MCMV)–a close relative of HCMV–influences surface expression of the cellular protein CD45 on macrophages and we identified the viral gene m42 mediating this effect. CD45 has long been known to be essential for the functioning of lymphocytes, however, its role in macrophages is less well understood. Growth analysis of a viral mutant indicated that the m42 gene confers a replication advantage to MCMV in vivo. We found that the m42 protein induces internalization of CD45 from the plasma membrane and degradation in lysosomes—most likely triggered by interaction of m42 with a ubiquitin ligase. In our study we detected a new element in the complex interaction of cytomegaloviruses with host cells, and further investigation into this mechanism may provide us with new insights into the functions of CD45 in myeloid cells.

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          Physiological functions of the HECT family of ubiquitin ligases.

          The ubiquitylation of proteins is carried out by E1, E2 and E3 (ubiquitin ligase) enzymes, and targets them for degradation or for other cellular fates. The HECT enzymes, including Nedd4 family members, are a major group of E3 enzymes that dictate the specificity of ubiquitylation. In addition to ubiquitylating proteins for degradation by the 26S proteasome, HECT E3 enzymes regulate the trafficking of many receptors, channels, transporters and viral proteins. The physiological functions of the yeast HECT E3 ligase Rsp5 are the best known, but the functions of HECT E3 enyzmes in metazoans are now becoming clearer from in vivo studies.
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            The ins and outs of leukocyte integrin signaling.

            Integrins are the principal cell adhesion receptors that mediate leukocyte migration and activation in the immune system. These receptors signal bidirectionally through the plasma membrane in pathways referred to as inside-out and outside-in signaling. Each of these pathways is mediated by conformational changes in the integrin structure. Such changes allow high-affinity binding of the receptor with counter-adhesion molecules on the vascular endothelium or extracellular matrix and lead to association of the cytoplasmic tails of the integrins with intracellular signaling molecules. Leukocyte functional responses resulting from outside-in signaling include migration, proliferation, cytokine secretion, and degranulation. Here, we review the key signaling events that occur in the inside-out versus outside-in pathways, highlighting recent advances in our understanding of how integrins are activated by a variety of stimuli and how they mediate a diverse array of cellular responses.
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              MHC class I antigen presentation: learning from viral evasion strategies.

              The cell surface display of peptides by MHC class I molecules to lymphocytes provides the host with an important surveillance mechanism to protect against invading pathogens. However, in turn, viruses have evolved elegant strategies to inhibit various stages of the MHC class I antigen presentation pathway and prevent the display of viral peptides. This Review highlights how the elucidation of mechanisms of viral immune evasion is important for advancing our understanding of virus-host interactions and can further our knowledge of the MHC class I presentation pathway as well as other cellular pathways.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                7 December 2016
                December 2016
                : 12
                : 12
                : e1006057
                Affiliations
                [1 ]Institute of Virology, Hannover Medical School, Hannover, Germany
                [2 ]Institute of Virology, University Medical Center of the Johannes Gutenberg University, Mainz, Germany
                [3 ]Helmholtz Centre for Infection Research, Braunschweig, Germany
                [4 ]Institute of Virology, Medical Center, University of Freiburg, Freiburg, Germany
                [5 ]Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia
                University of Wisconsin-Madison, UNITED STATES
                Author notes

                The authors have declared that no competing interests exist. The current affiliation of JDO indicates that she is now employed by this company. Her employment at preclinics GmbH started after the work for this study was performed. preclinics GmbH had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

                • Conceptualization: NT MM.

                • Formal analysis: NT NAWL.

                • Funding acquisition: AH MMB SJ LCS MM.

                • Investigation: NT KAK NAWL JDO KW CE TLR.

                • Project administration: AH TLR MMB SJ LCS MM.

                • Resources: TLR SJ.

                • Supervision: AH MMB SJ LCS MM.

                • Validation: NAWL JDO.

                • Visualization: NT NAWL.

                • Writing – original draft: NT NAWL MM.

                • Writing – review & editing: NT NAWL JDO AH TLR MMB SJ LCS MM.

                [¤a]

                Current address: preclinics GmbH, Potsdam, Germany

                Author information
                http://orcid.org/0000-0001-9190-6497
                http://orcid.org/0000-0001-6590-8413
                http://orcid.org/0000-0002-1227-3933
                Article
                PPATHOGENS-D-16-01509
                10.1371/journal.ppat.1006057
                5142792
                27926943
                19f62f5d-1e65-4337-9db3-1685f30d1c9e
                © 2016 Thiel et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 4 July 2016
                : 11 November 2016
                Page count
                Figures: 7, Tables: 0, Pages: 26
                Funding
                Funded by: funder-id http://dx.doi.org/10.13039/501100001656, Helmholtz-Gemeinschaft;
                Award ID: VH-VI-424
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001659, Deutsche Forschungsgemeinschaft;
                Award ID: SFB 900
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001656, Helmholtz-Gemeinschaft;
                Award ID: VH-VI-424
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001656, Helmholtz-Gemeinschaft;
                Award ID: VH-VI-424
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001656, Helmholtz-Gemeinschaft;
                Award ID: VH-VI-424
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100001656, Helmholtz-Gemeinschaft;
                Award ID: VH-VI-424
                Award Recipient :
                Funded by: funder-id http://dx.doi.org/10.13039/501100000781, European Research Council;
                Award ID: ERC StG 260934
                Award Recipient :
                Funded by: Hannover Biomedical Research School, Center of Infection Biology
                Award Recipient :
                Funded by: Hannover Biomedical Research School, Center of Infection Biology
                Award Recipient :
                This work was funded by the Helmholtz Association ( https://www.helmholtz.de/) through grant VI-VH-424 (Viral Strategies of Immune Evasion, VISTRIE) to AH, MMB, SJ, LCS, and MM. In part the work was also supported by a grant of the Sonderforschungsbereich 900 (to MM) and an ERC Starting Grant (No. 260934; to LCS). The work of SJ was funded by the EC | European Research Council (ERC) (grant No. 322693) and by the Ministry of Science, Education and Sports of Croatia (CERVir-Vac). NT and JDO were supported by stipends of the Hannover Biomedical Research School (HBRS; Center for Infection Biology [ZIB]; https://www.mh-hannover.de/hbrs.html). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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