Viral and cellular determinants of vesicular stomatitis virus entry

Viral pathogens are obligate intracellular parasites. To establish a productive infection, virus particles must breach the protective cellular membrane and deliver their genetic material into a permissive intracellular compartment. As inert objects, virions cannot actively navigate their way into host cells. Instead, viruses have evolved to engage and respond to cellular factors and processes that ideally position them to trigger membrane penetration at the proper time and location. Herein, we investigated the complex interplay between vesicular stomatitis virus (VSV) particles and host cells during the process of viral entry. Using a combination of novel reporter assays that detect specific entry steps and high-resolution confocal fluorescence microscopy to visualize individual virions as they enter live cells, we discovered several novel aspects of the entry process that extend and clarify the current model of VSV entry. Specifically, we found that VSV induces its own uptake into cells through unconventional clathrin-coated structures that require actin polymerization for efficient internalization. Furthermore, we provide evidence that VSV releases its genetic material early in the endosomal pathway and that forcing virus to penetrate cells at more acidic pHs diminishes the efficiency of entry. Our findings show that VSV has evolved to utilize the most prominent and highly-conserved endocytic mechanism that operates in eukaryotic cells, in part, explaining its extremely broad host range in cell culture.