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

Kerrie J Sandgren,Gerald M Mcinerney,Karen Byth-Wilson,Phillip J Robinson,Monica Miranda-Saksena,Anthony L Cunningham,John Wilkinson,Grant Mcfadden

doi:10.1371/journal.ppat.1000866

Kerrie J Sandgren, Gerald M Mcinerney + Show 6 more

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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.

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Vaccinia virus (VACV) is best known for its role as a vaccine in the global eradication of smallpox
The fragility of the outer enveloped virus (EV) envelope makes purification of this virus difficult we were able to use gentle centrifugal filtration to produce a concentrated stock of GFP-labelled EV in which contaminating mature virus (MV) or damaged EV particles were subsequently neutralised with an MV-neutralising antibody (Fig. S2)
The presence of intact EV in these preparations was confirmed by plaque assay in the presence of the neutralising antibody and by immunofluorescence microscopy where intact EV was identified by direct detection with an EV-specific antibody or GFP-fluorescence as well as exclusion of MV-specific antibody staining (Fig. S3)

Summary

Introduction

Vaccinia virus (VACV) is best known for its role as a vaccine in the global eradication of smallpox. Dendritic cells (DCs) are key players in the initiation of adaptive immune responses and as such are attractive targets for vaccination [1,2]. They are specialised at antigen uptake and highly express C-type lectin receptors (CLRs), a family of Ca2+dependent carbohydrate recognition receptors that bind to an array of microbial pathogens [3]. Further information about the mechanisms of DC binding and uptake of VACV could be employed to better target VACV-vectored vaccines to DCs, either directly or via uptake of bystander infected cells and influence recombinant antigen processing to enhance immune responses

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