Mammalian orthoreoviruses (reoviruses) are highly tractable experimental models for studies of double-stranded (ds) RNA virus replication and pathogenesis. Reoviruses infect respiratory and intestinal epithelium and disseminate systemically in newborn animals. Until now, a strategy to rescue infectious virus from cloned cDNA has not been available for any member of the Reoviridae family of dsRNA viruses. We report the generation of viable reovirus following plasmid transfection of murine L929 (L) cells using a strategy free of helper virus and independent of selection. We used the reovirus reverse genetics system to introduce mutations into viral capsid proteins sigma1 and sigma3 and to rescue a virus that expresses a green fluorescent protein (GFP) transgene, thus demonstrating the tractability of this technology. The plasmid-based reverse genetics approach described here can be exploited for studies of reovirus replication and pathogenesis and used to develop reovirus as a vaccine vector.

Viral attachment to specific host receptors is the first step in viral infection and serves an essential function in the selection of target cells. Mammalian reoviruses are highly useful experimental models for studies of viral pathogenesis and show promise as vectors for oncolytics and vaccines. Reoviruses engage cells by binding to carbohydrates and the immunoglobulin superfamily member, junctional adhesion molecule-A (JAM-A). JAM-A exists at the cell surface as a homodimer formed by extensive contacts between its N-terminal immunoglobulin-like domains. We report the crystal structure of reovirus attachment protein σ1 in complex with a soluble form of JAM-A. The σ1 protein disrupts the JAM-A dimer, engaging a single JAM-A molecule via virtually the same interface that is used for JAM-A homodimerization. Thus, reovirus takes advantage of the adhesive nature of an immunoglobulin-superfamily receptor by usurping the ligand-binding site of this molecule to attach to the cell surface. The dissociation constant (KD) of the interaction between σ1 and JAM-A is 1,000-fold lower than that of the homophilic interaction between JAM-A molecules, indicating that JAM-A strongly prefers σ1 as a ligand. Analysis of reovirus mutants engineered by plasmid-based reverse genetics revealed residues in σ1 required for binding to JAM-A and infectivity of cultured cells. These studies define biophysical mechanisms of reovirus cell attachment and provide a platform for manipulating reovirus tropism to enhance vector targeting.

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Serotype-specific patterns of reovirus disease in the CNS of newborn mice segregate with the viral S1 gene segment, which encodes attachment protein 1 and nonstructural protein 1s. The importance of receptor recognition in target cell selection by reovirus implicates the 1 protein as the primary effector of disease outcome. However, the contribution of 1s to reovirus disease is unknown. To define the function of 1s in reovirus pathogenesis, we generated a 1s-deficient virus by altering a single nucleotide to disrupt the 1s translational start site. Viruses were recovered that contain nine gene segments from strain type 3 Dearing and either the wild-type or 1s-null S1 gene segment from strain type 1 Lang. Following peroral inoculation of newborn mice, both viruses replicated in the intestine, although the wildtype virus achieved higher yields than the 1s-null virus. However, unlike the wild-type virus, the 1s-deficient virus failed to disseminate to sites of secondary viral replication, including the brain, heart, and liver. Within the small intestine, both viruses were detected in Peyer's patches, but only the wild-type virus reached the mesenteric lymph node. Concordantly, wild-type virus, but not 1s-deficient virus, was detected in the blood of infected animals. Wild-type and 1s-null viruses produced equivalent titers following intracranial inoculation, indicating that 1s is dispensable for viral growth in the murine CNS. These results suggest a key role for 1s in virus spread from intestinal lymphatics to the bloodstream, thereby allowing the establishment of viremia and dissemination to sites of secondary replication within the infected host.mice ͉ pathogenesis ͉ tropism ͉ vector design