Fluorescent fatty acid analogs as a tool to study development of the picornavirus replication organelles

Abstract

Genome replication of positive strand RNA viruses of eukaryotes is universally associated with specialized membranous structures referred to as replication organelles. Accumulating evidence show that new membrane synthesis is important for the development of the replication organelles of diverse picornaviruses and likely for other positive strand RNA viruses as well. The hydrophobic part of the structural phospholipid molecules defining the barrier properties of biological membranes consists of two long chain fatty acid moieties attached to the glycerol backbone. Fluorescent long chain fatty acid analogs represent a very convenient tool to monitor membrane synthesis in infected cells offering significant advantages over conventional radioactively labeled compounds. Bodipy-containing fatty acid analogs are readily imported from the extracellular media and utilized in lipid synthesis by cellular machinery. The strong fluorescence of the Bodipy group allows monitoring the molecules in situ by fluorescent microscopy as well as provides an opportunity for quantitative assessment of fatty acid import in a multi-well plate format. Moreover lipids with incorporated fluorescent fatty acid chain can be resolved by thin layer chromatography and easily identified using conventional UV imaging systems thus providing a simple and convenient way of monitoring the perturbation of the lipid synthesis pathways upon infection.