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      The HCMV gH/gL/UL128-131 Complex Triggers the Specific Cellular Activation Required for Efficient Viral Internalization into Target Monocytes

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          Abstract

          We have established that HCMV acts as a specific ligand engaging and activating cellular integrins on monocytes. As a result, integrin signaling via Src activation leads to the functional activation of paxillin required for efficient viral entry and for the biological changes in monocytes needed for viral dissemination. These biological/molecular changes allow HCMV to use monocytes as “vehicles” for systemic spread and the establishment of lifelong persistence. However, it remains unresolved how HCMV specifically induces this observed monocyte activation. It was previously demonstrated that the HCMV gH/gL/UL128-131 glycoprotein complex facilitates viral entry into biologically relevant cell types. Nevertheless, the mechanism by which the gH/gL/UL128-131 complex promotes this process is unknown. We now show that only HCMV virions possessing the gH/gL/UL128-131 complex are capable of activating integrin/Src/paxillin-signaling in monocytes. In fibroblasts, this signaling is reversed, such that virus lacking the gH/gL/UL128-131 complex is the only virus able to induce the paxillin activation cascade. The presence of the gH/gL/UL128-131 complex also may have an inhibitory effect on integrin-mediated signaling pathway in fibroblasts. Furthermore, we demonstrate that the presence of the gH/gL/UL128-131 complex on the viral envelope, through its activation of the integrin/Src/paxillin pathway, is necessary for efficient HCMV internalization into monocytes and that appropriate actin and dynamin regulation is critical for this entry process. Importantly, productive infection in monocyte-derived macrophages was seen only in cells exposed to HCMV expressing the gH/gL/UL128-131 complex. From our data, the HCMV gH/gL/U128-131 complex emerges as the specific ligand driving the activation of the receptor-mediated signaling required for the regulation of the actin cytoskeleton and, consequently, for efficient and productive internalization of HCMV into monocytes. To our knowledge, our studies demonstrate a possible molecular mechanism for why the gH/gL/UL128-131 complex dictates HCMV tropism and why the complex is lost as clinical isolates are passaged in the laboratory.

          Author Summary

          We previously demonstrated that HCMV, by engaging cellular receptors, changes the biology of blood monocytes, allowing for efficient viral entry into these cells and their use as virus-carriers in HCMV systemic spread. However, it was unclear how HCMV induces receptor-mediated signaling in infected cells. Here we report that HCMV by expressing a specific complex of five glycoproteins, present on HCMV clinical isolates, engages cellular integrin receptors and subsequently triggers integrin-mediated signaling leading to efficient viral entry into monocytes and productive infection of monocyte-derived macrophages. We also demonstrate that the HCMV pentameric complex has an inhibitory effect on integrin-mediated signaling in fibroblasts, an in vitro model system of HCMV infection, suggesting that the presence of the pentameric complex is not advantageous for HCMV infection of fibroblasts. Together, our results argue that HCMV uses distinct mechanisms to enter monocytes and fibroblasts. In support, our findings indicate that HCMV utilizes an endocytic-like route of entry into monocytes that is in contrast to viral fusion at the cell surface seen in fibroblasts. Our studies provide a molecular explanation for a previously observed critical role of the HCMV pentameric complex during infection of clinically relevant cell types, which in the future may lead to the development of better targets for antiviral therapy.

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          Most cited references101

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          Virus entry by endocytosis.

          Although viruses are simple in structure and composition, their interactions with host cells are complex. Merely to gain entry, animal viruses make use of a repertoire of cellular processes that involve hundreds of cellular proteins. Although some viruses have the capacity to penetrate into the cytosol directly through the plasma membrane, most depend on endocytic uptake, vesicular transport through the cytoplasm, and delivery to endosomes and other intracellular organelles. The internalization may involve clathrin-mediated endocytosis (CME), macropinocytosis, caveolar/lipid raft-mediated endocytosis, or a variety of other still poorly characterized mechanisms. This review focuses on the cell biology of virus entry and the different strategies and endocytic mechanisms used by animal viruses.
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            Genetic content of wild-type human cytomegalovirus.

            The genetic content of wild-type human cytomegalovirus was investigated by sequencing the 235 645 bp genome of a low passage strain (Merlin). Substantial regions of the genome (genes RL1-UL11, UL105-UL112 and UL120-UL150) were also sequenced in several other strains, including two that had not been passaged in cell culture. Comparative analyses, which employed the published genome sequence of a high passage strain (AD169), indicated that Merlin accurately reflects the wild-type complement of 165 genes, containing no obvious mutations other than a single nucleotide substitution that truncates gene UL128. A sizeable subset of genes exhibits unusually high variation between strains, and comprises many, but not all, of those that encode proteins known or predicted to be secreted or membrane-associated. In contrast to unpassaged strains, all of the passaged strains analysed have visibly disabling mutations in one or both of two groups of genes that may influence cell tropism. One comprises UL128, UL130 and UL131A, which putatively encode secreted proteins, and the other contains RL5A, RL13 and UL9, which are members of the RL11 glycoprotein gene family. The case in support of a lack of protein-coding potential in the region between UL105 and UL111A was also strengthened.
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              Local actin polymerization and dynamin recruitment in SV40-induced internalization of caveolae.

              Simian virus 40 (SV40) utilizes endocytosis through caveolae for infectious entry into host cells. We found that after binding to caveolae, virus particles induced transient breakdown of actin stress fibers. Actin was then recruited to virus-loaded caveolae as actin patches that served as sites for actin "tail" formation. Dynamin II was also transiently recruited. These events depended on the presence of cholesterol and on the activation of tyrosine kinases that phosphorylated proteins in caveolae. They were necessary for formation of caveolae-derived endocytic vesicles and for infection of the cell. Thus, caveolar endocytosis is ligand-triggered and involves extensive rearrangement of the actin cytoskeleton.
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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
                11 July 2013
                : 9
                : 7
                : e1003463
                Affiliations
                [1 ]Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, Shreveport, Louisiana, United States of America
                [2 ]Feist-Weiller Cancer Center, Louisiana State University Health Sciences Center, Shreveport, Louisiana, United States of America
                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: MTN GCTC ADY. Performed the experiments: MTN GCTC EVS DKCM. Analyzed the data: MTN GCTC EVS DKCM ADY. Contributed reagents/materials/analysis tools: MTN GCTC EVS DKCM ADY. Wrote the paper: MTN GCTC ADY.

                [¤]

                Current address: Department of Microbiology and Immunology, State University of New York Upstate Medical University, Syracuse, New York, United States of America.

                Article
                PPATHOGENS-D-12-00173
                10.1371/journal.ppat.1003463
                3708883
                23853586
                7d4562b4-4f53-4f8d-9dea-83b8236217ae
                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
                : 18 January 2012
                : 13 May 2013
                Page count
                Pages: 20
                Funding
                This work was supported by grants from the National Institutes of Health (AI050677, HD-051998, and P20-RR018724), a Malcolm Feist cardiovascular research fellowship, and an American Heart Association predoctoral fellowship (10PRE4200007). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Medicine

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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