Protein quality control in the endoplasmic reticulum (472.3)

Abstract

In the context of our studies on host‐pathogen interactions we have developed a series of tools that are likely to be of broad utility. These tools include the development of a sortase transacylation‐based system for protein modification, which allows the stoichiometric and site‐specific installation of a wide variety of functionalities that cannot be provided in a template‐encoded manner. The method is particularly useful for site‐specific modification of type II membrane proteins on living cells. We have applied this method to labeling of the flu virus glycoproteins HA and N, both of which are refractory to tagging with GFP in the context of an infectious virus. We have developed a semi‐intact cell system, based on selective perforation of the plasma membrane with perfringolysin‐O. This preparation allows us to track the biogenesis of flu particles in detail. In this analysis we make use of single domain antibody fragments, derived from heavy chain‐only antibodies produced in camelids, to interfere with select steps in the virus assembly process. This analysis has revealed the participation of host factors that were not uncovered in any of the published genome‐wide shRNA screens. Finally, on the host side, we have used somatic cell nuclear transfer to generate new mouse models with which to study the interactions of flu with flu‐specific B cells. This approach uncovered the requirement for an internalizing receptor, in addition to surface‐disposed sialic acids to mediate entry of the virus.