Entry of the Two Infectious Forms of Vaccinia Virus at the Plasma Membane Is Signaling-Dependent for the IMV but Not the EEV

Abstract

The simpler of the two infectious forms of vaccinia virus, the intracellular mature virus (IMV) is known to infect cells less efficiently than the extracellular enveloped virus (EEV), which is surrounded by an additional, TGN-derived membrane. We show here that when the IMV binds HeLa cells, it activates a signaling cascade that is regulated by the GTPase rac1 and rhoA, ezrin, and both tyrosine and protein kinase C phosphorylation. These cascades are linked to the formation of actin and ezrin containing protrusions at the plasma membrane that seem to be essential for the entry of IMV cores. The identical cores of the EEV also appear to enter at the cell surface, but surprisingly, without the need for signaling and actin/membrane rearrangements. Thus, in addition to its known role in wrapping the IMV and the formation of intracellular actin comets, the membrane of the EEV seems to have evolved the capacity to enter cells silently, without a need for signaling.

Vaccinia Virus Infection Requires Maturation of Macropinosomes

“…The results indicated, in accordance with previous reports 39, 46, that VACV MV endocytosis in HeLa cells is rapid and relatively efficient. Vacuoles that contained the internalized virus recruited Rab5a and, in some cases, EEA1 immediately after formation.…”

“…However, as previously reported 39, neither depolymerization of MTs with nocodazole or colchicine, nor stabilization with taxol had any effect on VACV early infection events (Figure S6). Although in agreement with the co‐existence of MT‐dependent and ‐independent macropinocytic pathways 66, in light of these findings we suspect that MT‐based transport of VACV‐containing macropinosomes is not necessary for VACV infection, but rather serves to increase the likelihood of macropinosome fusion with LEs or LYs, which could serve to speed entry kinetics.…”

“…Penetration and disassembly of the viral cores in the cytosol are known to begin between 20 and 30 min after endocytosis 39, 41. Thus, the subsequent decline in detectable viral spots observed between 60 and 120 min was consistent with the known kinetics of uncoating of the MV genome and core degradation 39, 41, 42.…”

“…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. In this study, we followed the viruses during the time of passage within the endocytic network, and analyzed the maturation of the virus‐containing macropinosomes.…”

“…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.…”