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

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.

Vaccinia Virus Infection Requires Maturation of Macropinosomes

“…We and others have previously shown that the vacuoles containing MVs also contain fluid markers such as dextran, and that they are devoid of transferrin, a cellular cargo molecule present in early and recycling endosomes 34, 35, 37, 38. This is consistent with reports indicating that classical endosomes and macropinosomes give rise to distinct transport vesicle populations 28.…”

“…VACV MVs and EVs initiate entry into host cells by triggering their own macropinocytic uptake 34, 35, 37, 38. About 30 min later, the viral core is delivered into the cytosol after fusion of the viral envelope with the limiting membrane of endocytic vacuoles 39, 46.…”

“…VACV EVs also enter cells by macropinocytosis but the process of penetration is more complicated due to the additional envelope membrane 35, 37. That the hits were largely overlapping with VACV MVs strongly suggested that EVs also require macropinosome maturation for productive infection.…”

“…During infection, VACV produces two types of infectious particles: mature virions (MVs) with one envelope membrane and extracellular virions (EVs) with two membranes. For internalization and infection, both trigger their own macropinocytosis 33, 34, 35, 36, 37, 38.…”

Rho’ing in and out of cells

“…PAK activation contributes to the initial stages of actin remodeling and is later also involved in the macropinosome closure by activating C-terminal-binding protein-1/brefeldin A-ADP ribosylated substrate (CtBP-1 also known as BARS). 31,32,37,[76][77][78][79] Macropinocytic uptake has been reported for many viruses including, among others, poxviruses, Ebola virus, influenza virus, adenoviruses, herpesviruses and retroviruses. For example, several labs have reported on the role of Rho GTPases and macropinocytosis during vaccinia virus (VV) entry 27,37,77,78,80 For an extensive overview of the (subtle) differences in macropinocytic uptake between the different viruses we refer to a review by Mercer and Helenius.…”