Comparative Analysis of the Mechanism of Entry and Uncoating of Three Different Picornaviruses

The early events in picornavirus infection such as entry into the host cell, penetration into the cytoplasm and uncoating of the viral genome are poorly understood. Therefore, we have studied the initial events in the infection of three different picornaviruses: HRV14, a major receptor group human rhinovirus, HRV2, a minor receptor group HRV, and of poliovirus type 2 (PV2). Our data as well as results from other laboratories suggest the following mechanisms for infection: 1.After binding and internalization, HRV14 undergoes a receptor and temperature dependent modification of its capsid proteins resulting in endosome rupture and delivery of modified subviral particles into the cytoplasm. Although low endosomal pH is not an absolute requirement for infectivity, it nevertheless facilitates HRV14 uncoating. 2. HRV2 appears to be internalized preferentially by a clathrin independent pathway. Uncoating of the viral genome occurs by pore formation in the endosome solely dependent on pH < 5.6 3. Uncoating of PV2 is known to require a receptor and temperature dependent but low pH independent structural modification of the viral capsid. PV2 is internalized via clathrin coated pits and vesicles into early and late endosomes. However, the majority of the virus remains attached to the plasma membrane up to 2h after infection. Since modified RNA-free subviral particles are mainly recovered from the cell supernatant, this let suggest that uncoating of PV2 takes place at the plasma membrane by pore formation. Taken together, three different picornaviruses appear to differ in their mechanism and cellular location of uncoating. Therefore, the goal of the proposed project is to further characterize the early events - (infectious) entry pathway and mechanism of uncoating - of the three picornaviruses. The specific aims of this proposal are: 1. Infectious entry route of picornaviruses: This will be achieved by the use of mutant HeLa cells defective in clathrin dependent endocytosis. The binding, cellular accumulation and recycling of HRV 14 and PV2 will be determined. Most importantly, the rate of endocytosis of the respective virus will be correlated with the production of infectious virions to gain insight into the infectious entry pathway. Since HRV2 recycling is inhibited under conditions where clathrin dependent endcytosis is blocked, the role of clathrin in HRV2 recycling will be analyzed biochemically and morphologically. 2. Endosomal pH regulation and endosomal penetration of HRV 14 and PV2: First, endocytic pathways in HeLa cells will be characterized morphologically using route-specific ligands. pH-sensitive ligands will then be applied to study pH regulation in endosome subcompartments using FACS and single organelle flow analysis (SOFA). The influence of virus infection on the amount of a given marker in endosomes will be determined. This should reveal whether penetration/uncoating occurs by endosome disruption or pore formation. Finally, the influence of virus infection on the pH in (residual) early endosomes and late endosomes will be determined. 3. Electrophysiology of picornavirus uncoating: This will be achieved by patch-clamp techniques using whole-cell and on-cell recordings. Since receptor and temperature dependent uncoating of PV2 might take place at the plasma membrane, the influence of the virus on the conductance of the plasma membrane as a function of temperature will be assessed. pH independent uncoating of HRV 14 could also occur at the plasma membrane. Consequently, similar experiments will be carried out with HRV14, where receptor and temperature induced uncoating has also been demonstrated. In contrast, HRV2 is uncoated in endosomes by pore formation solely dependent on low pH. Nevertheless, we will investigate whether low pH dependent rise in membrane conductances is induced by HRV2. These experiments should reveal whether pores/channels by the viruses can be induced at the plasma membrane. The relevance of these channels in the infectious entry can be determined by the use of uncoating inhibitors.