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ICAM-1 Binding Rhinoviruses Enter HeLa Cells via Multiple Pathways and Travel to Distinct Intracellular Compartments
Haleh Ganjian1, Christin Zietz2, Diana Mechtcheriakova3
1Department of Pathophysiology and Allergy Research, Medical University of Vienna, Währinger Gürtel 18-20, A-1090 Wien, Austria. halehganjian85@gmail.com.
Abstract:
Of the more than 150 human rhinovirus (RV) serotypes, 89 utilize intercellular adhesion molecule-1 (ICAM-1) for cell entry. These belong either to species A or B. We recently demonstrated that RV-B14 and RV-A89, despite binding this same receptor, are routed into distinct endosomal compartments for release of their RNA into the cytosol. To gain insight into the underlying mechanism we now comparatively investigate the port of entry, temperature-dependence of uncoating, and intracellular routing of RV-B3, RV-B14, RV-A16, and RV-A89 in HeLa cells. The effect of various drugs blocking distinct stages on the individual pathways was determined via comparing the number of infected cells in a TissueFaxs instrument. We found that RV-B14 and RV-A89 enter via clathrin-, dynamin-, and cholesterol-dependent pathways, as well as by macropinocytosis. Drugs interfering with actin function similarly blocked entry of all four viruses, indicating their dependence on a dynamic actin network. However, uniquely, RV-A89 was able to produce progeny when internalized at 20 °C followed by neutralizing the endosomal pH and further incubation at 37 °C. Blocking dynein-dependent endosomal transport prevented uncoating of RV-A16 and RV-A89, but not of RV-B3 and RV-B14, indicative for routing of RV-A16 and RV-A89 into the endocytic recycling compartment for uncoating. Our results call for caution when developing drugs aimed at targeting entry or intracellular trafficking of all rhinovirus serotypes.
Insights
Human rhinoviruses (RV) use ICAM-1 for cell entry but follow different intracellular pathways. Understanding these distinct routes is crucial for developing effective antiviral therapies against RV infections.
Area of Science:
- Virology
- Cell Biology
- Molecular Biology
Background:
- Over 150 human rhinovirus (RV) serotypes exist, with 89 using intercellular adhesion molecule-1 (ICAM-1) for cellular entry.
- RV serotypes are classified into species A and B.
- Previous research showed RV-B14 and RV-A89, despite sharing ICAM-1, utilize distinct endosomal compartments for RNA release.
Purpose of the Study:
- To comparatively investigate the entry mechanisms, temperature-dependent uncoating, and intracellular trafficking of specific RV serotypes (RV-B3, RV-B14, RV-A16, RV-A89).
- To elucidate the underlying molecular mechanisms governing differential intracellular routing of RV species A and B.
Main Methods:
- Utilized HeLa cells for infection studies.
- Investigated viral entry pathways using drugs that target specific cellular processes (clathrin, dynamin, cholesterol, actin, dynein).
- Quantified viral infection levels using a TissueFaxs instrument to assess drug effects on viral pathways.
Main Results:
- RV-B14 and RV-A89 utilize clathrin-, dynamin-, cholesterol-dependent pathways and macropinocytosis for entry.
- All four investigated RV serotypes depend on a dynamic actin network for entry.
- RV-A89 uniquely produced progeny at 20°C with subsequent pH neutralization and 37°C incubation.
- Dynein inhibition blocked uncoating for RV-A16 and RV-A89, suggesting their routing to the endocytic recycling compartment, unlike RV-B3 and RV-B14.
Conclusions:
- Rhinoviruses exhibit diverse entry and intracellular trafficking mechanisms, even when sharing the same receptor (ICAM-1).
- Specific RV serotypes are routed through distinct endosomal compartments, influencing their uncoating and replication.
- Findings underscore the complexity of RV intracellular pathways, necessitating caution in developing broad-spectrum antiviral drugs targeting entry or trafficking.