Capillary Electrophoresis of Cellular Endocytic Compartments

Membrane bounded endocytic compartments exhibit an electrically charged outer surface enabling migration in an electric field. This property will be explored and utilized for their separation and characterization by capillary electrophoresis (CE). To trace coated vesicles, early endosomes, endosomal carrier vesicles, late endosomes, and lysosomes, respectively, suitable fluorescent dyes and/or recombinant marker proteins fused to green fluorescent protein (or derivatives thereof) and expressed in transfected cells will employed. The kinetic evolution of the various compartments will also be followed by pulse chase experiments employing conventional fluorescent fluid phase markers and well known endocytic markers such as transferrin or low-density lipoprotein whose pathways are well characterized. Human rhinoviruses (HRVs) are small RNA containing non-enveloped particles which are the main cause of common cold infections. They gain acces to the cell by receptor-mediated endocytosis via intercellular adhesion molecule 1 (major group HRVs) and the low-density lipoprotein receptor (minor group HRVs). A methodology will be developed enabling to follow the fate of representatives of the two receptor groups during the infection pathway. The viruses will be traced from their attachment to the cellular receptors at the plasma membrane through the various vesicular compartments. To this end methods will be established to differentially label the viral capsid proteins and the viral genomic RNA either in vitro or in vivo. The analytes will be resolved by CE and further identified by their different spectral properties. In this way the fate of the intact virus upon internalization, and that of the empty capsid and the RNA, respectively, after the uncoating process will be explored in detail. Co- localization of the components with the endocytic markers will thus allow to define the vesicular compartment in which the RNA is released from the viral shell and subsequently gains access to the cytosol. The combination of the high resolution power of CE and the high selectivity and sensitivity of fluorescence detection will allow for the analysis of the viral components in the cellular compartments during their kinetic evolution. Knowledge of the detailed mechanism of internalization of the virions and of the site of genome release is essential for the development of novel antiviral stategies.

A better understanding of the different steps of the infectious pathway of a virus into the host cell is important for the development of rational antiviral strategies. Human Rhinoviruses (HRVs) are small, RNA containing lipid-free particles, which are the main cause of common cold. They gain access into the cell by receptor-mediated endocytosis. The virus capsid consists of proteins, and is thus electrically charged under appropriate conditions. For this reason virus particles, and also their reaction and degradation products can be separated under the influence of an electric field. In capillary electrophoresis separation is carried out at high voltage in an aqueous solution in narrow tubes which. The components of virus sample are introduced at the one end of the capillary in a very narrow plug, differentially migrate in the electric field and are detected at the other capillary end by fluorescence, exited by the aid of a laser beam. Prerequisite for this high performance method is the labelling of the viral constituents (genomic RNA and capsid proteins) by fluorescence dyes. Labelling with different dyes was possible, enabling in this way the independent detection of the different viral constituents, in intact virus and after its induced decay as well. This method allows to monitor interactions between viruses and cell receptors, and to investigate the attachment of virus particles onto artificial cell membranes. It enables to assess the first steps of endocytosis of viruses with defined model systems by capillary electrophoresis.