Vaccinia virus strains use distinct forms of macropinocytosis for host-cell entry

Abstract

To enter host cells, vaccinia virus, a prototype poxvirus, can induce transient macropinocytosis followed by endocytic internalization and penetration through the limiting membrane of pinosomes by membrane fusion. Although mature virions (MVs) of the Western reserve (WR) strain do this in HeLa cells by activating transient plasma membrane blebbing, MVs from the International Health Department-J strain were found to induce rapid formation (and lengthening) of filopodia. When the signaling pathways underlying these responses were compared, differences were observed at the level of Rho GTPases. Key to the filopodial formation was the virus-induced activation of Cdc42, and for the blebbing response the activation of Rac1. In addition, unlike WR, International Health Department-J MVs did not rely on genistein-sensitive tyrosine kinase and PI(3)K activities. Only WR MVs had membrane fusion activity at low pH. Inhibitor profiling showed that MVs from both strains entered cells by macropinocytosis and that this was induced by virion-exposed phosphatidylserine. Both MVs relied on the activation of epidermal growth factor receptor, on serine/ threonine kinases, protein kinase C, and p21-activated kinase 1. The results showed that different strains of the same virus can elicit dramatically different responses in host cells during entry, and that different macropinocytic mechanisms are possible in the same cell line through subtle differences in the activating ligand.epidermal growth factor receptor | endocytosis | poxvirus | Rho GTPases | virus entry

Vaccinia Virus Infection Requires Maturation of Macropinosomes

“…At later time points, VACV was found within EGFP‐Rab7a and LAMP1‐EGFP vesicles (Movies S3–S6), both of which were capable of homotypic fusion events (Movies S4 and S6). In agreement with the low pH requirement of VACV penetration by fusion out of vacuoles 33, 44, 45, 46, 47, we occasionally observed virus cores escaping from LAMP1‐positive late macropinosomes (Figure 1F and Movie S5).…”

“…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). The epidermal growth factor inhibitor Iressa, previously shown to inhibit VACV infection at the level of entry 33, was used as a positive control for bypass. For both, exposure to pH 7.4 media served as a bypass control.…”

“…The VACV strain used here, WR, has been shown to require low pH (pH = 4.5–5.0) for envelope fusion 46, 61, 62. However, inhibition of WR MV infection by lysosomotropic agents varies within the range of 40–60%, and other VACV strains, such as IHD‐J, fuse from macropinosomes in a pH‐independent fashion 33, 44. This suggests that other macropinosome factors, in addition to low pH, may be required for VACV fusion.…”

“…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.…”