We characterized vaccinia IMV entry into HeLa cells and showed that the virus infection process has several interesting features. First, vaccinia IMV required actin polymerization prior to cell entry. That virus “surfing” requires normal actin dynamics has been reported for some retroviruses and vesicular stomatitis virus (36
). Surfing may turn out to be a common transport mechanism shared by many viruses if vaccinia virus is added to the list of those using it. Plasma membrane ruffling and actin protrusions regulated by actin dynamics were also involved in vaccinia IMV recruitment. Next, we showed that IMV particles were endocytosed into HeLa cells in a manner independent of clathrin- and caveola-mediated pathways but dependent on dynamin, suggesting a virus entry pathway through fluid phase endocytosis (7
). The role of endogenous dynamin 2 in vaccinia IMV entry into HeLa cells was demonstrated using three experimental approaches, first with an expressed DN-Dyn1 isoform, next with a dynamin-specific inhibitor, dynasore, and finally with a siRNA approach. In our hands, DN-Dyn2 blocked transferrin uptake but not IMV entry; we suspect that a higher level of DN-Dyn2 is needed to block virus entry than to block ligand uptake. Alternatively, endogenous dynamin in HeLa cells may contain splicing variants that interacted with DN-Dyn1 and DN-Dyn2 with different affinities. It is worth noting that, despite a clear function of dynamin in clathrin- and caveola-mediated coated-vesicle formation, its role in coat-independent endocytosis processes such as fluid uptake has been controversial (1
). Four splicing variants of dynamin 2 have been reported (8
), and the DN GTPase mutants of all four splicing variants blocked transferrin uptake well; however, only two splicing variants blocked dextran uptake, suggesting differential functions of the distinct splicing variants in fluid uptake (7
). Furthermore, we identified and characterized VPEF, a novel cellular protein that is important for vaccinia IMV penetration into HeLa cells. Our data also suggested that VPEF and dynamin mediate IMV penetration through different mechanisms. How VPEF interacts with viral components to allow penetration remains to be clarified in the future. VPEF mRNA was detected in many mammalian cell lines (data not shown), suggesting a conserved function for VPEF. This would not be too surprising, given the wide host infectivity of vaccinia virus; while we demonstrated the importance of VPEF in HeLa cells, it remains to be determined whether the same conclusion will apply to other cell types. It also awaits more experiments to determine whether the extracellular form of vaccinia virus requires VPEF for cell entry or not.
Because vaccinia virus attachment to cells at 4°C was relatively inefficient and only 1 to 2% of the input viral particles bound to cells after a 60-min incubation (37
), we had to use relatively high-MOI (MOI = 40 to 100) infections in the EM and confocal analyses. Using viral early gene expression assays, we observed that vaccinia IMV entry into HeLa cells remained sensitive to BFLA in low-MOI (MOI = 0.1 to 5) infections, supporting the idea that MOI per se did not dictate the cell entry pathways employed by viruses. It is also worth noting that endocytosis of the WR strain of vaccinia IMV into HeLa cells, as described here, is not simply an artifact of high-MOI infections since infections of cells with the IHD-J strain of vaccinia IMV at a high MOI of 200 to 300 did not result in obvious endocytosis into BSC-1 (58
) and HeLa cells (37
In summary, these results demonstrate that vaccinia IMV is able to pirate cellular actin to get closer to cell bodies and manages to enter cells by hijacking a ubiquitous VPEF-dependent fluid transport pathway that is part of these cells' normal repertoire. We hypothesize that VPEF represents a new cargo or component of the earliest acceptor compartment of the clathrin- and caveolin-independent vesicle trafficking system (41
) for the uptake of bulk phase fluid. The verification of this hypothesis will require further study.