22] Analysis of membrane association in vivo and targeting of viral proteins in polarized epithelial cells

Abstract

This chapter describes the morphological and biochemical methods used for studying the posttranslational membrane association of viral proteins and for targeting viral proteins to either the apical or basolateral plasma membrane. There are two types of membrane proteins: (1) the first consists of proteins that are cotranslationally translocated into the endoplasmic reticulum (ER) and then vectorially transported sequentially through the exocytic pathways from the ER, pre-Golgi, cis -Golgi, medial Golgi, trans -Golgi, and trans -Golgi network (TGN) to the plasma membrane and (2) proteins of the second type associate posttranslationally with membranes either via a direct interaction with the lipid bilayer or by an indirect interaction with the cytoplasmic tail/transmembrane domain of another transmembrane protein or by a combination of both. Membrane association and targeting of viral proteins during the assembly process are important steps in the biology of enveloped viruses because of the following reasons: (1) when the specific interaction between membrane components is inhibited, the budding of mature virus particles does not take place, and therefore virus infection becomes abortive, (2) the elucidation of the steps in the assembly process would explain why some viruses bud selectively from the ER, the Golgi apparatus, or the plasma membrane, and (3) an understanding of the steps involved in the membrane association of viral proteins may facilitate the development of antiviral agents that may interfere with one or more steps involved in virus assembly and budding. Similarly, the targeting of viral proteins to specific domains of the plasma membrane plays an important role in viral pathogenesis. Viruses such as influenza, Sendai, and parainfluenza viruses bud from the apical membrane, whereas viruses such as vesicular stomatitis virus (VSV) or retroviruses bud from the basolateral membrane in polarized epithelial cells.