Characterization of human astrovirus cell entry

Area of Science:

• Virology
• Cell Biology

Background:

• Human astroviruses (HAstV) are a common cause of gastroenteritis, particularly in young children and immunocompromised individuals.
• Understanding the initial stages of HAstV infection is crucial for developing effective antiviral strategies.

Purpose of the Study:

• To elucidate the binding and entry mechanisms of human astroviruses in Caco-2 cells.
• To identify the cellular pathways utilized by HAstV for host cell invasion.

Main Methods:

• Characterization of virus-cell surface binding kinetics and decapsidation time.
• Assessment of drug effects on HAstV infectivity, targeting endocytosis, endosome acidification, and membrane cholesterol.
• Genetic manipulation including RNA interference for clathrin heavy chain (CHC) silencing and overexpression of dominant-negative dynamin 2 and Eps15 mutants.
• Investigating the role of Rab7 in endosome maturation via gene silencing.

Main Results:

• Virus binding to Caco-2 cells occurred with a half-time of approximately 10 minutes.
• Virus decapsidation was a slower process, taking around 130 minutes.
• Infectivity was significantly reduced by inhibitors of clathrin-mediated endocytosis, endosome acidification, actin polymerization, and cholesterol depletion.
• Silencing CHC or overexpressing dominant-negative dynamin 2 and Eps15 impaired viral entry.
• Rab7 silencing demonstrated that HAstV entry is dependent on endosome maturation to the late endosome stage.

Conclusions:

• Human astroviruses utilize a clathrin-dependent endocytic pathway for entry into Caco-2 cells.
• The viral entry process is dependent on endosome acidification and maturation, requiring progression to late endosomes.
• HAstV infection involves a complex cellular mechanism requiring specific endocytic machinery for host cell invasion.