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

PLoS Pathogens

Kerrie J. Sandgren¹  Phillip J. Robinson¹  Monica Miranda-Saksena¹  Gerald M. McInerney¹  Anthony L. Cunningham²  Karen Byth-Wilson³  John Wilkinson⁴ 

[1] Centre for Virus Research, Westmead Millennium Institute, Sydney, New South Wales, Australia;Children's Medical Research Institute, Westmead, Sydney, New South Wales, Australia;Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden;Faculty of Medicine, University of New South Wales, Sydney, New South Wales, Australia

关键词: Vaccinia virus;    Cholesterol;    Viral entry;    Cell membranes;    Endocytosis;    Flow cytometry;    Confocal microscopy;    Cell binding;   

DOI  :  10.1371/journal.ppat.1000866

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY