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

Junctional adhesion molecule-A (JAM-A

Rotaviruses perform the remarkable tasks of transcribing and replicating 11 distinct double-stranded RNA genome segments within the confines of a subviral particle. Multiple viral polymerases are tethered to the interior of a particle, each dedicated to a solitary genome segment but acting in synchrony to synthesize RNA. Although the rotavirus polymerase specifically recognizes RNA templates in the absence of other proteins, its enzymatic activity is contingent upon interaction with the viral capsid. This intraparticle strategy of RNA synthesis helps orchestrate the concerted packaging and replication of the viral genome. Here, we review our current understanding of rotavirus RNA synthetic mechanisms.A distinguishing feature of viruses with segmented doublestranded RNA (dsRNA) 2 genomes is that they perform all stages of RNA synthesis inside icosahedral subviral particles (1-4). Each genome segment is dedicated to a single polymerase complex (PC) anchored to the interior of the particle. Particle-associated PCs act in synchrony to transcribe and replicate the viral dsRNA segments. Thus, subviral particles of dsRNA viruses can be viewed as nanoscale factories containing multiple highly coordinated PC machines. During transcription, each genome segment serves as a template for the generation of plus-strand RNAs (ϩRNAs). In addition to directing viral protein expression, ϩRNAs also serve as templates for minus-strand synthesis to recreate dsRNA duplexes. The intraparticle mechanism of RNA synthesis not only protects the genome from recognition by antiviral dsRNA sensors but also provides a mechanism for coordinating ϩRNA packaging with genome replication (4).As the primary cause of life-threatening gastroenteritis in young children, rotaviruses (RVs) have long been the subject of basic research (5). Specifically, biochemical studies have probed the functions of viral proteins involved in RNA synthesis and have elucidated critical features of viral RNA templates. Moreover, the recent determination of a high resolution structure of the RV RNA-dependent RNA polymerase (RdRp) in complex with RNA has greatly enhanced our understanding of intraparticle RNA synthesis (6).

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Mammalian orthoreoviruses (reoviruses) are prototype members of the Reoviridae family of nonenveloped viruses. Reoviruses contain ten double-stranded RNA gene segments enclosed in two concentric protein shells, outer capsid and core. These viruses serve as a versatile experimental system for studies of virus cell entry, innate immunity, and organ-specific disease. Reoviruses engage cells by binding to cell-surface carbohydrates and the immunoglobulin superfamily member, junctional adhesion molecule-A (JAM-A). JAM-A is a homodimer formed by extensive contacts between its N-terminal immunoglobulin-like domains. Reo-virus attachment protein σ1 disrupts the JAM-A dimer, engaging a single JAM-A molecule by virtually the same interface used for JAM-A homodimerization. Following attachment to JAM-A and carbohydrate, reovirus internalization is promoted by β1 integrins, most likely via clathrin-dependent endocytosis. In the endocytic compartment, reovirus outer-capsid protein σ3 is removed by cathepsin proteases, which exposes the viral membrane-penetration protein, μ1. Proteolytic processing and conformational rearrangements of μ1 mediate endosomal membrane rupture and delivery of transcriptionally active reovirus core particles into the host cell cytoplasm. These events also allow the ϕ cleavage fragment of μ1 to escape into the cytoplasm where it activates NF-κB and elicits apoptosis. This review will focus on mechanisms of reovirus cell entry and activation of innate immune response signaling pathways.