Reoviruses provide a well-established experimental model for studies of viral neuropathogenesis. Following primary infection in the intestine of newborn mice, serotype 3 reoviruses spread through nerves and infect neurons, causing lethal encephalitis. Viral attachment protein sigma1 plays a crucial role in the progression of these pathologic events. The sigma1 protein is a filamentous trimer with head and tail morphology. The sigma1 tail binds sialic acid (SA), whereas the sigma1 head binds junctional adhesion molecule A (JAM-A), a component of intercellular tight junctions. The major objective of the proposed research is to determine how interactions between sigma1 and cell-surface receptors lead to organ-specific disease. Four integrated specific aims are proposed to study structural and functional properties of sigma1-receptor interactions. In Specific Aim 1, sequence determinants of sigma1- JAM-A interactions will be identified. Structure-guided mutations will be introduced into putative binding surfaces of each molecule, and affinities of purified wild-type and mutant proteins will be determined by surface plasmon resonance. In complementary experiments, adenoviruses expressing mutant forms of sigma1 will be tested for the capacity to infect JAM-A-expressing cells. In Specific Aim 2, the physiological consequences of reovirus infection of polarized cells will be determined. Intracellular distribution of JAM-A and JAM-A-associated proteins and integrity of tight junctions will be monitored during reovirus internalization. The significance of JAM-A phosphorylation in reovirus attachment and infection will be determined. In Specific Aim 3, the role of SA in reovirus pathogenesis will be defined. Reovirus strains differing in the capacity to bind SA due to a single amino acid polymorphism in sigma1 will be studied for pathways of spread and neural tropism in mice. The importance of SA in reovirus infection of neurons will be defined by testing reovirus strains that differ in SA-binding capacity for growth in primary neuronal cultures. In Specific Aim 4, the role of JAM-A in reovirus pathogenesis will be determined. Mice deficient in expression of JAM-A will be inoculated with reovirus, and viral growth and histopathology will be assessed at primary and secondary sites of replication. These studies will yield a precise understanding of reovirus cell-attachment and provide novel insights into mechanisms by which reoviruses and other neurotropic viruses select cellular targets and produce disease.