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Adenoviruses are the most commonly used viral vectors for clinical applications, mainly for the treatment of cancer and for the prevention of infectious diseases. A large number of adenoviruses—over 100 types have been isolated from humans—have evolved to infect different cells and tissues and to cause a range of diseases. As can be expected, several distinct entry mechanisms have been identified and characterized, which contribute to, or even determine cell and tissue tropism. Knowledge about adenovirus-host factors interactions is important for efficient and specific transduction of adenovirus vectors to cells and tissues of interest. Here we describe the state-of-the-art of human adenovirus cell entry, and discuss with perspectives outstanding questions in the field.

Enteric adenovirus types F40 and 41 (EAdVs) are a leading cause of diarrhea and diarrhea-associated death in young children and have recently been proposed to cause acute hepatitis in children. EAdVs have a unique capsid architecture and exhibit — unlike other human adenoviruses — a relatively strict tropism for gastrointestinal tissues with, to date, understudied infection mechanism and unknown target cells. In this study, we turn to potentially limiting host factors by comparing EAdV entry in cell lines with respiratory and intestinal origin by cellular perturbation, virus particle tracking, and transmission electron microscopy. Our analyses highlight kinetic advantages for EAdVs in duodenal HuTu80 cell infection and reveal a larger fraction of mobile particles, faster virus uptake, and infectious particle entry in intestinal cells. Moreover, EAdVs display a dependence on clathrin- and dynamin-dependent pathways in intestinal cells. Detailed knowledge of virus entry routes and host factor requirements is essential to understanding pathogenesis and developing new countermeasures. Hence, this study provides novel insights into the entry mechanisms of a medically important virus with emerging tropism in a cell line originating from a relevant tissue. IMPORTANCE Enteric adenoviruses have historically been difficult to grow in cell culture, which has resulted in lack of knowledge of host factors and pathways required for infection of these medically relevant viruses. Previous studies in non-intestinal cell lines showed slow infection kinetics and generated comparatively low virus yields compared to other adenovirus types. We suggest duodenum-derived HuTu80 cells as a superior cell line for studies to complement efforts using complex intestinal tissue models. We show that viral host cell factors required for virus entry differ between cell lines from distinct origins and demonstrate the importance of clathrin-mediated endocytosis.

Human adenovirus (HAdV)-F40 and -F41 are leading causes of diarrhea and diarrhea-associated mortality in children under the age of five, but the mechanisms by which they infect host cells are poorly understood. HAdVs initiate infection through interactions between the knob domain of the fiber capsid protein and host cell receptors. Unlike most other HAdVs, HAdV-F40 and -F41 possess two different fiber proteins-a long fiber and a short fiber. Whereas the long fiber binds to the Coxsackievirus and adenovirus receptor (CAR), no binding partners have been identified for the short fiber. In this study, we identified heparan sulfate (HS) as an interaction partner for the short fiber of enteric HAdVs. We demonstrate that exposure to acidic pH, which mimics the environment of the stomach, inactivates the interaction of enteric adenovirus with CAR. However, the short fiber:HS interaction is resistant to and even enhanced by acidic pH, which allows attachment to host cells. Our results suggest a switch in receptor usage of enteric HAdVs after exposure to acidic pH and add to the understanding of the function of the short fibers. These results may also be useful for antiviral drug development and the utilization of enteric HAdVs for clinical applications such as vaccine development.