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      The Viral Chemokine MCK-2 of Murine Cytomegalovirus Promotes Infection as Part of a gH/gL/MCK-2 Complex

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          Abstract

          Human cytomegalovirus (HCMV) forms two gH/gL glycoprotein complexes, gH/gL/gO and gH/gL/pUL(128,130,131A), which determine the tropism, the entry pathways and the mode of spread of the virus. For murine cytomegalovirus (MCMV), which serves as a model for HCMV, a gH/gL/gO complex functionally homologous to the HCMV gH/gL/gO complex has been described. Knock-out of MCMV gO does impair, but not abolish, virus spread indicating that also MCMV might form an alternative gH/gL complex. Here, we show that the MCMV CC chemokine MCK-2 forms a complex with the glycoprotein gH, a complex which is incorporated into the virion. We could additionally show that mutants lacking both, gO and MCK-2 are not able to produce infectious virus. Trans-complementation of these double mutants with either gO or MCK-2 showed that both proteins can promote infection of host cells, although through different entry pathways. MCK-2 has been extensively studied in vivo by others. It has been shown to be involved in attracting cells for virus dissemination and in regulating antiviral host responses. We now show that MCK-2, by forming a complex with gH, strongly promotes infection of macrophages in vitro and in vivo. Thus, MCK-2 may play a dual role in MCMV infection, as a chemokine regulating the host response and attracting specific target cells and as part of a glycoprotein complex promoting entry into cells crucial for virus dissemination.

          Author Summary

          Several human herpesviruses form alternative gH/gL complexes which determine the tropism for different cell types. For murine cytomegalovirus (MCMV), a gH/gL/gO complex has recently been characterized. Here, we present the identification and characterization of an alternative gH/gL/MCK-2 complex which promotes MCMV spread and is important for efficient infection of macrophages in vitro and in vivo. Association of the MCMV CC chemokine MCK-2 with a glycoprotein complex promoting virus entry is a novel function for the well-characterized MCK-2. Virus mutants lacking MCK-2 have been shown to exhibit a reduced capacity to attract leukocytes and a disregulated T cell control of the MCMV infection in vivo. These defects can be attributed to the chemokine function of MCK-2. Yet, the observation that MCK-2 knock-out mutants additionally are impaired in infecting leukocytes in vivo is consistent with our new finding that MCK-2 forms a glycoprotein complex promoting entry into monocytic cells. gH/gL complexes associating with multifunctional proteins add a new level of complexity to the interpretation of infection phenotypes of the respective knock-out herpesviruses.

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          Most cited references48

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          Two physically, functionally, and developmentally distinct peritoneal macrophage subsets.

          The peritoneal cavity (PerC) is a unique compartment within which a variety of immune cells reside, and from which macrophages (MØ) are commonly drawn for functional studies. Here we define two MØ subsets that coexist in PerC in adult mice. One, provisionally called the large peritoneal MØ (LPM), contains approximately 90% of the PerC MØ in unstimulated animals but disappears rapidly from PerC following lipopolysaccharide (LPS) or thioglycolate stimulation. These cells express high levels of the canonical MØ surface markers, CD11b and F4/80. The second subset, referred to as small peritoneal MØ (SPM), expresses substantially lower levels of CD11b and F4/80 but expresses high levels of MHC-II, which is not expressed on LPM. SPM, which predominates in PerC after LPS or thioglycolate stimulation, does not derive from LPM. Instead, it derives from blood monocytes that rapidly enter the PerC after stimulation and differentiate to mature SPM within 2 to 4 d. Both subsets show clear phagocytic activity and both produce nitric oxide (NO) in response to LPS stimulation in vivo. However, their responses to LPS show key differences: in vitro, LPS stimulates LPM, but not SPM, to produce NO; in vivo, LPS stimulates both subsets to produce NO, albeit with different response patterns. These findings extend current models of MØ heterogeneity and shed new light on PerC MØ diversity, development, and function. Thus, they introduce a new context for interpreting (and reinterpreting) data from ex vivo studies with PerC MØ.
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            Fusing structure and function: a structural view of the herpesvirus entry machinery.

            Herpesviruses are double-stranded DNA, enveloped viruses that infect host cells through fusion with either the host cell plasma membrane or endocytic vesicle membranes. Efficient infection of host cells by herpesviruses is remarkably more complex than infection by other viruses, as it requires the concerted effort of multiple glycoproteins and involves multiple host receptors. The structures of the major viral glycoproteins and a number of host receptors involved in the entry of the prototypical herpesviruses, the herpes simplex viruses (HSVs) and Epstein-Barr virus (EBV), are now known. These structural studies have accelerated our understanding of HSV and EBV binding and fusion by revealing the conformational changes that occur on virus-receptor binding, depicting potential sites of functional protein and lipid interactions, and identifying the probable viral fusogen.
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              Human cytomegalovirus virion protein complex required for epithelial and endothelial cell tropism.

              Human cytomegalovirus replicates in many different cell types, including epithelial cells, endothelial cells, and fibroblasts. However, laboratory strains of the virus, many of which were developed as attenuated vaccine candidates by serial passage in fibroblasts, have lost the ability to infect epithelial and endothelial cells. Their growth is restricted primarily to fibroblasts, due to mutations in the UL131-UL128 locus. We now demonstrate that two products of this locus, pUL130 and pUL128, form a complex with gH and gL, but not gO. The AD169 laboratory strain, which lacks a functional UL131 protein, produces virions containing only the gH-gL-gO complex. An epithelial and endothelial cell tropic AD169 variant in which the UL131 ORF has been repaired, termed BADrUL131, produces virions that carry both gH-gL-gO and gH-gL-pUL128-pUL130 complexes. Antibodies against pUL130 and pUL128 block infection of epithelial and endothelial cells by BADrUL131 and the fusion-inducing factor X clinical human cytomegalovirus isolate but do not affect the efficiency with which fibroblasts are infected.
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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, USA )
                1553-7366
                1553-7374
                July 2013
                July 2013
                25 July 2013
                : 9
                : 7
                : e1003493
                Affiliations
                [1 ]Max von Pettenkofer-Institute for Virology, Ludwig-Maximilians-University Munich, Munich, Germany
                [2 ]Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia
                [3 ]Institute for Virology and Research Center for Immunology (FZI), University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany
                [4 ]Laboratory of Virology and Infectious Disease, Rockefeller University, New York, New York, United States of America
                [5 ]Research Unit Gene Vectors, German Research Center for Environmental Health (GmbH), Munich, Germany
                Oregon Health and Science University, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: BA. Performed the experiments: FMW IB AP FL JBB MG LM HA BA TT MA NAWL JP. Analyzed the data: BA UHK FMW IB MJR. Contributed reagents/materials/analysis tools: MM. Wrote the paper: BA.

                Article
                PPATHOGENS-D-12-01826
                10.1371/journal.ppat.1003493
                3723581
                23935483
                29262701-1866-4fc0-afed-f198963f04df
                Copyright @ 2013

                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
                : 29 July 2012
                : 22 May 2013
                Page count
                Pages: 15
                Funding
                BA was supported by the Deutsche Forschungsgemeinschaft through grants AD131/3-1 and AD131/3-2. NAWL was supported by the Young Investigators Program MAIFOR at the University Medical Center of the Johannes Gutenberg-University Mainz, and MJR by the Deutsche Forschungsgemeinschaft, Clinical Research Group KFO 183. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Microbiology
                Virology
                Medicine
                Infectious Diseases
                Viral Diseases

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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