Abstract
Virus attachment and entry is a complex interplay of viral and cellular interaction partners. Employing bovine viral diarrhea virus (BVDV) encoding an mCherry-E2 fusion protein (BVDVE2-mCherry), being the first genetically labelled member of the family Flaviviridae applicable for the analysis of virus particles, the early events of infection—attachment, particle surface transport, and endocytosis—were monitored to better understand the mechanisms underlying virus entry and their dependence on the virus receptor, bovine CD46. The analysis of 801 tracks on the surface of SK6 cells inducibly expressing fluorophore labelled bovine CD46 (CD46fluo) demonstrated the presence of directed, diffusive, and confined motion. 26 entry events could be identified, with the majority being associated with a CD46fluo positive structure during endocytosis and occurring more than 20 min after virus addition. Deletion of the CD46fluo E2 binding domain (CD46fluo∆E2bind) did not affect the types of motions observed on the cell surface but resulted in a decreased number of observable entry events (2 out of 1081 tracks). Mean squared displacement analysis revealed a significantly increased velocity of particle transport for directed motions on CD46fluo∆E2bind expressing cells in comparison to CD46fluo. These results indicate that the presence of bovine CD46 is only affecting the speed of directed transport, but otherwise not influencing BVDV cell surface motility. Instead, bovine CD46 seems to be an important factor during uptake, suggesting the presence of additional cellular proteins interacting with the virus which are able to support its transport on the virus surface.
Highlights
Before entering a host cell, viruses have to establish contact with the cell and travel to sites that are suitable for entry
SK6 cells inducibly expressing the fluorophore labelled, cellular surface receptor, bovine CD46 were chosen as system to study the infection cycle, as these cells demonstrated a high susceptibility to infection with bovine viral diarrhea virus (BVDV) after induction
SK6 cells not expressing bovine CD46 display a low susceptibility to BVDV infection, which is not mediated by porcine CD46, as porcine CD46 has previously been shown not to be involved in BVDV invasion [16]
Summary
Before entering a host cell, viruses have to establish contact with the cell and travel to sites that are suitable for entry This process of attachment can involve different receptor molecules and transport mechanisms, which can be classified into diffusion, drifts, and confinement [1]. Virus surfing—the directed, actin-dependent transport of virus particles on the outside of filopodia, cytonemes, and retraction fibres—was discovered employing a member of the Retroviridae, namely murine leukemia virus (MLV) [2]. This mode of extracellular, directed transport has since been described for members of the Adenoviridae [1], Herpesviridae [3], and Papillomaviridae [4]
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