Effects of Commons Sialic Acid Modifications on Virus Binding and Infection
Principal Investigator: Colin Parrish
DESCRIPTION (provided by applicant):
Sialic acids (Sia) come in many modified forms and linkages, and are expressed at high levels as components of glycoproteins, glycolipids, and polysaccharides on cells, at mucosal surfaces, on erythrocytes, and within tissues or in the circulation. Sia also determine the properties of cell, tissue and mucosal surfaces, and control many different physiological functions, as well as cell-cell and cell-ligand interactions. Sia are bound by many different pathogens, including viruses, bacteria and parasites (and their products such as toxins). Many different viruses use Sia as primary receptors to attach to and enter cells, and some of those viruses also express enzymes (neuramindases, sialidases, or esterases) that can specifically remove or modify the Sia to change their binding properties, and thereby modulate the viral/host interactions. Those binding and cleavage processes are often structure-specific, and would be altered by modifications of the Sia structure, or by changing the Sia linkage to the oligosaccharide. Despite their central roles in so many normal functions and pathogen interactions, we lack a clear understanding of where the different modified Sia are expressed or how they regulate virus:host interactions, or control viral host ranges and tropisms. The goals of these studies are therefore to better define the effects of several modified Sia that differ in expression within the tissues of humans and other animals that are common viral hosts or animal models. We will specifically define the effects of the modifications on binding of four different viruses or viral proteins as models, on the fusion of influenza A and C viruses, and on the viral infectivity for cells that express modified Sia forms. In this exploratory and developmental project we will use a variety of tools to specifically express or regulate these Sia, and then examine their effects on virus binding, fusion, and infection. Prepare samples containing the different modified forms of Sia, and use glycoengineering to prepare cells with altered expression for functional testing. We would use probes recognizing modified Sia variants (including Neu5Gc, Neu5Ac, Neu4,5Ac2 or Neu5,9Ac2) to test for their presence on a variety of cells, mucin, and erythocytes. Cell lines engineered to express different forms and linkages of the Sia would be characterized, altering the proportions of Neu5Gc, 9-O- and 7,9-O-acetyl Sia in HEK293 cells, as well as some of those modifications and α2,3, α2,6 linkages in MDCK and A549 cells. Define the effects of the modified Sia on binding, fusion, or infection of four viruses. Examine the binding of influenza A and C viruses, polyomaviruses, and reovirus to the modified Sia in vitro on mucins or glycoproteins, or expressed on cells and tissues. We would use microscopic analysis to follow the binding, release and fusion of single viruses or viral proteins on the variant Sia. We would correlate the binding and analysis with the results of studies of infection, replication and growth in the modified cells.