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      A Differential Role for Macropinocytosis in Mediating Entry of the Two Forms of Vaccinia Virus into Dendritic Cells

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          Abstract

          Vaccinia virus (VACV) is being developed as a recombinant viral vaccine vector for several key pathogens. Dendritic cells (DCs) are specialised antigen presenting cells that are crucial for the initiation of primary immune responses; however, the mechanisms of uptake of VACV by these cells are unclear. Therefore we examined the binding and entry of both the intracellular mature virus (MV) and extracellular enveloped virus (EV) forms of VACV into vesicular compartments of monocyte-derived DCs. Using a panel of inhibitors, flow cytometry and confocal microscopy we have shown that neither MV nor EV binds to the highly expressed C-type lectin receptors on DCs that are responsible for capturing many other viruses. We also found that both forms of VACV enter DCs via a clathrin-, caveolin-, flotillin- and dynamin-independent pathway that is dependent on actin, intracellular calcium and host-cell cholesterol. Both MV and EV entry were inhibited by the macropinocytosis inhibitors rottlerin and dimethyl amiloride and depended on phosphotidylinositol-3-kinase (PI(3)K), and both colocalised with dextran but not transferrin. VACV was not delivered to the classical endolysosomal pathway, failing to colocalise with EEA1 or Lamp2. Finally, expression of early viral genes was not affected by bafilomycin A, indicating that the virus does not depend on low pH to deliver cores to the cytoplasm. From these collective results we conclude that VACV enters DCs via macropinocytosis. However, MV was consistently less sensitive to inhibition and is likely to utilise at least one other entry pathway. Definition and future manipulation of these pathways may assist in enhancing the activity of recombinant vaccinia vectors through effects on antigen presentation.

          Author Summary

          Vaccinia virus (VACV) is a relative of the smallpox virus and was used for many decades as a successful vaccine that contributed to the eradication of smallpox. Today, through genetic recombination technology, VACV shows potential as a modern vaccine for many unconquered diseases including HIV and cancer. Dendritic cells (DCs) are a specialised subset of immune cells that initiate adaptive immune responses and exploiting the interaction between VACV and DCs, which has not been well studied, may be a key to improving the efficacy of these vaccines. In this study we investigated the mechanisms by which VACV binds to and enters DCs. Here, we examined both the abundant mature virus form of VACV as well as the less common, poorly studied extracellular form. We found that VACV does not bind to the common pathogen-uptake C-type lectin receptors expressed on DCs and that the virus enters DCs via macropinocytosis—a fluid-phase uptake process. Furthermore, the virus is not delivered to the conventional endolysosomal antigen processing pathway in these cells. Our study provides new insights into VACV biology and into possible mechanisms of action of VACV as a recombinant viral vaccine vector which may assist in their rational design in the future.

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          Most cited references64

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          Specificity and mechanism of action of some commonly used protein kinase inhibitors.

          The specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays.
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            Virus entry by macropinocytosis.

            As obligatory intracellular parasites, viruses rely on host-cell functions for most aspects of their replication cycle. This is born out during entry, when most viruses that infect vertebrate and insect cells exploit the endocytic activities of the host cell to move into the cytoplasm. Viruses belonging to vaccinia, adeno, picorna and other virus families have been reported to take advantage of macropinocytosis, an endocytic mechanism normally involved in fluid uptake. The virus particles first activate signalling pathways that trigger actin-mediated membrane ruffling and blebbing. Usually, this is followed by the formation of large vacuoles (macropinosomes) at the plasma membrane, internalization of virus particles and penetration by the viruses or their capsids into the cytosol through the limiting membrane of the macropinosomes. We review the molecular machinery involved in macropinocytosis and describe what is known about its role in virus entry.
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              The formation and function of extracellular enveloped vaccinia virus.

              Vaccinia virus produces four different types of virion from each infected cell called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). These virions have different abundance, structure, location and roles in the virus life-cycle. Here, the formation and function of these virions are considered with emphasis on the EEV form and its precursors, IEV and CEV. IMV is the most abundant form of virus and is retained in cells until lysis; it is a robust, stable virion and is well suited to transmit infection between hosts. IEV is formed by wrapping of IMV with intracellular membranes, and is an intermediate between IMV and CEV/EEV that enables efficient virus dissemination to the cell surface on microtubules. CEV induces the formation of actin tails that drive CEV particles away from the cell and is important for cell-to-cell spread. Lastly, EEV mediates the long-range dissemination of virus in cell culture and, probably, in vivo. Seven virus-encoded proteins have been identified that are components of IEV, and five of them are present in CEV or EEV. The roles of these proteins in virus morphogenesis and dissemination, and as targets for neutralizing antibody are reviewed. The production of several different virus particles in the VV replication cycle represents a coordinated strategy to exploit cell biology to promote virus spread and to aid virus evasion of antibody and complement.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                April 2010
                April 2010
                22 April 2010
                : 6
                : 4
                : e1000866
                Affiliations
                [1 ]Centre for Virus Research, Westmead Millennium Institute, Sydney, New South Wales, Australia
                [2 ]Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia
                [3 ]Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden
                [4 ]Children's Medical Research Institute, Westmead, Sydney, New South Wales, Australia
                University of Florida, United States of America
                Author notes
                [¤]

                Current address: Center For Infectious Medicine, Department of Medicine, Karolinska Institutet, Stockholm, Sweden

                Conceived and designed the experiments: KJS JW ALC. Performed the experiments: KJS JW MMS GMM. Analyzed the data: KJS JW KBW. Contributed reagents/materials/analysis tools: PJR. Wrote the paper: KJS.

                Article
                09-PLPA-RA-1584R3
                10.1371/journal.ppat.1000866
                2858709
                20421949
                8020500c-64d2-41c9-9f7f-90a8e3b96b8c
                Sandgren 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
                : 8 September 2009
                : 22 March 2010
                Page count
                Pages: 16
                Categories
                Research Article
                Immunology/Innate Immunity
                Infectious Diseases/Viral Infections
                Virology/Host Invasion and Cell Entry

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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