ICAM-1 Binding Rhinoviruses Enter HeLa Cells via Multiple Pathways and Travel to Distinct Intracellular Compartments for Uncoating

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.

Catching Common Cold Virus with a Net: Pyridostatin Forms Filaments in Tris Buffer That Trap Viruses—A Novel Antiviral Strategy?

“…For detection, secondary antibodies labeled with Alexa fluor (Life Technologies, Carlsbad, CA, USA) were used at 1 μg/mL, followed by extensive washing. The slides were mounted, and fluorescence microscopy images were recorded on a TissueFAXS automated microscope (TissueGnostics, Vienna, Austria) as in Ganjian et al [ 14 ]. The presence of PDS diminished the number of virus-positive cells in all cases by about 70% compared with cells identically infected with the respective untreated virus.…”

Virus Entry: Looking Back and Moving Forward

“…Sometimes, the activation of penetration by alternative cues, such as low pH or proteolytic cleavage, depend on cell line or virus strain [37]. The pathway by which the virus negotiates the endocytic network in the cell may involve alternative routes and penetration compartments as shown by the rhinovirus example in Table 2 [79].…”

“…Serotypes that use ICAM-1 as receptor enter via clathrin-mediated endocytosis and micropinocytosis. Among these, members of the A serotype are routed for acid-activated uncoating in recycling endosomes and members of the B serotype in maturing endosomes [79].…”

Dynein Engages and Disassembles Cytosol-Localized Simian Virus 40 To Promote Infection

“…In agreement with our study, dynein was shown to be important during cell entry of the murine PyV (40), although it is not known whether this motor acts to disassemble the virus. Beyond the PyV family, other viruses, including human immunodeficiency virus type 1 (41,42), influenza A virus (43), rhinoviruses (44), herpes simplex virus (45), and murine leukemia virus (46), exploit dynein's function to support some aspect of their entry processes. Thus, our study further underscores the concept that exploiting cellular motors is a common strategy used by viruses during infection.…”