Vaccinia Virus Uses Macropinocytosis and Apoptotic Mimicry to Enter Host Cells

Abstract

Viruses employ many different strategies to enter host cells. Vaccinia virus, a prototype poxvirus, enters cells in a pH-dependent fashion. Live cell imaging showed that fluorescent virus particles associated with and moved along filopodia to the cell body, where they were internalized after inducing the extrusion of large transient membrane blebs. p21-activated kinase 1 (PAK1) was activated by the virus, and the endocytic process had the general characteristics of macropinocytosis. The induction of blebs, the endocytic event, and infection were all critically dependent on the presence of exposed phosphatidylserine in the viral membrane, which suggests that vaccinia virus uses apoptotic mimicry to enter cells.

Vaccinia Virus Infection Requires Maturation of Macropinosomes

“…Consistent with this, when release of cores into the cytosol was observed, it occurred from LAMP1‐containing vacuoles. We have previously shown that virus‐containing vacuoles positive for the endosomal content marker dextran do not contain transferrin at any time 34, 35 confirming that they are distinct from classical endosomes.…”

“…We have previously employed this strategy to test the specificity of entry inhibitors 34, 55. To determine whether YM201636 inhibited MV entry, WR E EGFP MVs were bound to cells in the presence or absence of YM201636 and subsequently treated with pH 5.0 media (Figure 4E).…”

“…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.…”

“…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.…”

“…The cellular factors required for macropinocytic uptake of VACV MVs and EVs include epidermal growth factor receptor (EGFR) and receptor tyrosine kinase (RTK) and RTK signaling, actin and myosin dynamics, small Rho GTPases and protein kinase C (PKC) as well as PI(3)kinase activity 33, 34, 35, 36, 38, 39. However, the macropinocytic trafficking requirements for VACV infection remain undefined.…”