A Novel Cellular Protein, VPEF, Facilitates Vaccinia Virus Penetration into HeLa Cells through Fluid Phase Endocytosis

Abstract

Vaccinia virus is a large DNA virus that infects many cell cultures in vitro and animal species in vivo.Although it has been used widely as a vaccine, its cell entry pathway remains unclear. In this study, we showed that vaccinia virus intracellular mature virions bound to the filopodia of HeLa cells and moved toward the cell body and entered the cell through an endocytic route that required a dynamin-mediated pathway but not a clathrin-or caveola-mediated pathway. Moreover, virus penetration required a novel cellular protein, vaccinia virus penetration factor (VPEF). VPEF was detected on cell surface lipid rafts and on vesicle-like structures in the cytoplasm. Both vaccinia virus and dextran transiently colocalized with VPEF, and, importantly, knockdown of VPEF expression blocked vaccinia virus penetration as well as intracellular transport of dextran, suggesting that VPEF mediates vaccinia virus entry through a fluid uptake endocytosis process in HeLa cells. Intracellular VPEF-containing vesicles did not colocalize with Rab5a or caveolin but partially colocalized with Rab11, supporting the idea that VPEF plays a role in vesicle trafficking and recycling in HeLa cells. In summary, this study characterized the mechanism by which vaccinia virus enters HeLa cells and identified a cellular factor, VPEF, that is exploited by vaccinia virus for cell entry through fluid phase endocytosis.The poxviruses form a group of large DNA viruses that includes variola virus, the causative agent of smallpox disease. Though smallpox itself has been eradicated, the fear of biological warfare and the recent occurrence of accidental monkeypox virus transmission between species (22) and of eczema vaccinatum (33) have alerted us to the potential danger of new emerging diseases. In addition, the potential applications of poxviruses as improved vaccines (34) and as oncolytic agents for cancer therapy (57) have raised new interest in poxvirus biology.Vaccinia virus, the well-studied prototype of the Orthopoxvirus genus in the family Poxviridae, has a wide range of infectivity in many cell lines and animals (20). It produces several forms of infectious particles, of which the vaccinia intracellular mature virus (IMV) is the most abundant in cells (see reference 14 and references therein). An IMV is enclosed by a single envelope and contains more than 70 viral proteins (11,45,65).The molecular mechanism of vaccinia IMV entry remains largely unknown. IMV binds to ubiquitous cellular attachment factors, such as glycosaminoglycans (12, 27) and the extracellular matrix protein laminin (10). It is not known whether IMV recognizes additional cellular coreceptors to trigger the postbinding fusion step, although virus entry through fusion with the plasma membrane (3, 9, 19, 37) or intracellular compartments (16, 58) has been reported. Interestingly, IMV has been shown to trigger cellular signaling during virus entry (2, 37, 44), but the molecular pathway of virus uptake has not been characterized.In this study, we characterized the mechanism by whi...

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.…”

“…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.…”

Vaccinia Viral Protein A27 Is Anchored to the Viral Membrane via a Cooperative Interaction with Viral Membrane Protein A17

“…The viral L1 protein also mediates nonglycosaminoglycan binding to cells (11). Subsequently, the MV particles cluster at membrane lipid rafts (12), interact with integrin (13) and CD98 (14), and then undergo endocytosis (15)(16)(17). After uptake into cellular vesicles, the acidic environment triggers membrane fusion mediated by the viral entry fusion complex (18).…”