Academic Background

Viral pathogenesis and virulence; viral entry and the mechanism of virus-induced membrane fusion; viral glycoproteins and receptors

Paramyxoviruses are enveloped, negative-stranded RNA viruses, which include clinically important human pathogens such as measles, mumps, parainfluenza, and respiratory syncytial viruses; important animal pathogens, such as Newcastle disease (NDV), and Sendai viruses; and, emerging viruses such as Nipah and Hendra. Though primarily respiratory pathogens, several members of the group are capable of causing central nervous system disease. We are interested in understanding the molecular basis for the pathogenesis and virulence of this group of viruses.

Paramyxoviruses gain entry into the cell by mediating direct fusion of the virus and cell membranes. Fusion involves a virus-specific interaction between the two viral surface glycoprotein spikes, the attachment protein and the fusion protein. Mediated through this interaction, receptor binding by the attachment protein is thought to trigger a conformational change in the fusion protein from a metastable, pre-fusion form to a fusion-active form. We are exploring various aspects of the fusion process for several paramyxoviruses. We have developed a sensitive co-immunoprecipitation assay to detect the attachment-fusion protein complex at the cell surface and have identified mutations in several paramyxovirus attachment proteins that block fusion by modulating the interaction between the two surface glycoproteins. Our evidence is consistent with the idea that the attachment protein - fusion protein interaction regulates fusion by a different mechanism in viruses such as NDV, which recognize the ubiquitous sugar sialic acid as receptor, and viruses such as measles, which recognize specific proteins as receptors.

NDV virulence involves contributions from at least three viral proteins. The site of cleavage activation of the fusion protein is a major determinant of virulence. However, recent evidence has revealed that the attachment protein also plays a role. In addition, the V protein, produced by RNA editing from the P gene, is an interferon antagonist and thereby also plays a role in virulence. We are interested in understanding the mechanisms by which all three proteins exert their effects on NDV virulence, as well as the basis for the differences in virulence observed among members of the NDV serotype.

Rotation Projects

Rotation projects will involve construction of site-directed mutants or chimeras of either the attachment, fusion or V proteins and evaluation of their ability to promote the functions attributed to that protein. Depending on the target protein, the structure and function of each mutated or chimeric protein will be evaluated using several functional assays. These include flow cytometry (cell surface expression and antibody recognition), hemadsorption of red blood cells (receptor recognition activity), neuraminidase (receptor destroying activity), content mixing reporter gene assay (fusion), immunoprecipitation and SDS-PAGE (protein structure) and co-immunoprecipitation (determination of the ability of the mutated proteins to interact with a co-expressed protein). For the V protein, we will be evaluating various aspects of its interferon inhibitory activity.