Reoviruses are nonenveloped viruses that contain a genome of 10 segments of double-stranded RNA (Fig. 1). These viruses display a broad host range, but only young hosts develop the disease. After infection of newborn mice, reoviruses cause injury to a variety of organs, including the central nervous system, heart, and liver, depending on the viral strain. By virtue of the ability to genetically manipulate both the virus and its host, reoviruses are tractable models for studies of viral replication and pathogenesis. Moreover, reovirus efficiently kills transformed cells and is being evaluated in clinical trials as an oncolytic agent. The central objective of our work is to determine the role of each step in the reovirus infectious cycle in the pathogenesis of reovirus-induced disease.
Figure 1: The reovirus virion. Left: Schematic of a reovirus virion. Reovirus virions are composed of two concentric protein shells, outer capsid, and core. The core contains the viral genome, which consists of 10 segments of double-stranded RNA. Viral gene segments are classified as L, M, or S (large, medium, or small) based on size. S1, the largest of the S-class gene segments, encodes the σ1 protein. Right: Cryo-EM image reconstruction of a reovirus virion at 23 Å resolution. Note the finger-like projections of σ3 (blue) that sit atop a layer of μ1 (green). The λ2 protein (yellow) forms a pentamer at each of the virion fivefold symmetry axes. The σ1 protein is too flexible to be visualized using this technique.
Reovirus-receptor interactions. Reovirus-receptor interactions. Different reovirus serotypes cause distinct patterns of disease in the central nervous system of newborn mice. Type 1 reoviruses infect ependymal cells, causing hydrocephalus, whereas type 3 reoviruses infect neurons, resulting in lethal encephalitis. The capacity of the reovirus attachment protein, Sigma1 (Fig. 2), to bind to either ependymal cells or neurons is a major determinant of the serotype-specific patterns of neurologic disease in infected mice. The goal of our work on reovirus-receptor interactions is to understand the molecular basis of target cell selection by reovirus. We are collaborating with the laboratory of Dr. Thilo Stehle's laboratory at the University of Tübingen in Germany to determine atomic-resolution structures of s1 (alone and in complex with its receptors) and define how the independent s1 receptor-binding domains function in tropism of reovirus in vivo.
Figure 2. Structural and functional domains of reovirus attachment protein σ1. The σ1 protein forms a filamentous trimer at each of the twelve icosahedral vertices of the viral outer capsid. The σ1 molecule is composed of an N-terminal tail and a C-terminal globular head. Shown is a full-length model of T3D σ1. The three monomers of the crystallized fragment are shown in red, yellow, and blue; modeled regions are shown in grey. Receptor-binding domains contained within σ1 are indicated. The sialic acid-binding site in T1L σ1 is located in the head domain, and the sialic acid-binding site in T3D σ1 is located in the body domain. The head domain binds junctional adhesion molecule-A (JAM-A) with high affinity. The glycan-binding sites do not overlap with the JAM-A-binding site, suggesting that both receptors can be engaged independently.
Cell entry of reovirus. Reoviruses enter cells by receptor-mediated endocytosis, which is most likely dependent on clathrin. In the endocytic compartment, virions undergo stepwise disassembly forming sequential disassembly intermediates, leading to penetration of the endosomal membrane and release of transcriptionally active core particles into the cytoplasm. Our current studies employ molecular genetics, biochemical analyses, electron cryomicroscopy (Fig. 3), confocal microscopy (Fig. 4), and X-ray crystallography (in collaboration with Dr. B. V. Prasad’s laboratory at Baylor College of Medicine) to define the key intramolecular and intermolecular interactions at each step in the reovirus entry pathway. Additional experiments are focused on defining cellular determinants of reovirus entry using pharmacologic inhibitors of various cellular uptake pathways and RNA interference to diminish expression of specific components of the internalization machinery. A final series of experiments seeks to determine whether alterations in capsid stability influence reovirus oncolytic activity. Together, these studies may reveal general mechanisms used by nonenveloped viruses to enter cells and accelerate development of reovirus as a cancer therapeutic.
Figure 3. Electron micrograph of reovirus entry into a cell.
Figure 4. Uptake of reovirus (red) into Rab5A-labeled early endosomes (green). Endosomal uptake of labeled particle visible at 6-7 seconds.
Mechanisms of reovirus replication. Like most RNA viruses, reovirus remodels host membranes to form neoorganelles that serve as sites of viral genome replication and particle assembly. These highly specialized structures concentrate viral replication enzymes and prevent replication intermediates from activating cell-intrinsic defenses. Both viral and host proteins are required for replication complex formation and, in some cases, identical host components are used by diverse viruses. The replication organelles formed by reovirus are called inclusions and grow to occupy a substantial portion of the cytoplasm of infected cells (Fig. 5). Despite the importance of inclusion formation in reovirus replication, it is not well understood how these novel machines form and function, nor is it precisely known how reoviruses migrate from inclusions and exit infected cells.
Figure 5. Reovirus infection is initiated by binding of the virion to cell surface receptors. Following internalization of virions by receptor-mediated endocytosis, the viral outer capsid undergoes acid-dependent proteolysis within endosomes to generate core particles containing all components of the viral transcriptional machinery. Transcriptionally active cores are released into the cytoplasm and synthesize full-length, message-sense, single-stranded (ss) RNAs corresponding to each viral gene segment. These ssRNAs can be translated and serve as templates for minus-strand synthesis to generate nascent genomic dsRNA. The viral replication cycle is completed by condensation of outer-capsid proteins onto newly formed dsRNA-containing particles, producing fully assembled infectious progeny.
Mechanisms of reovirus egress. After replication and particle assembly, some nonenveloped viruses are released by cell lysis, while others can exit infected cells without compromising cell viability. Reovirus infection of some cell types leads to apoptotic or necrotic cell death, whereas in polarized endothelial and epithelial cells, reovirus infection leads to noncytolytic release of viral progeny. Cellular pathways used for noncytolytic reovirus egress are not known. In collaboration with Dr. Cristina Risco's laboratory at the Centro Nacional de Biotecnología in Madrid, we are imaging reovirus cell exit from polarized endothelial cells using electron tomography. In addition, we are searching for new host mediators of reovirus exit using RNAi screening. Findings from these experiments will enhance an understanding of egress mechanisms for macromolecular cargo and may lead to the development of antivirals that target nonenveloped virus release.
Function and mechanisms of reovirus-induced apoptosis. Mechanisms by which viruses injure and kill their host cells are of critical importance to viral pathogenesis. Many viruses are capable of inducing the genetically programmed cell-death pathway that leads to apoptosis. In some cases, apoptosis triggered by virus infection may serve as a host defense mechanism to limit viral replication. In other cases, apoptosis may enhance viral infection by facilitating virus spread or allowing virus to evade host inflammatory or immune responses. We are conducting experiments to define the biochemical pathways used by reovirus to produce apoptotic cell death and determine the relationship between apoptosis and reovirus virulence. These studies will contribute important information about how viruses use host-cell signaling machinery to cause cell death and disease. Such knowledge is of critical importance to an understanding of viral pathogenesis and may lead to the development of new antiviral therapeutics capable of apoptosis blockade.
Reovirus vaccine vectors. We developed a fully plasmid-based reverse genetics technology for reovirus that permits selective introduction of mutations into each of the 10 viral gene segments. Neither helper virus nor co-expression of viral replication proteins is required. We have used this powerful new strategy to introduce changes into viral outer-capsid and replication proteins to define the roles of individual amino acids, functional domains, and structural motifs in receptor utilization, virion disassembly, and viral replication and spread in vivo. We are now using this core technology to harness reovirus as a replication-competent vector for oral immunization of mucosal surfaces against HIV-1 and influenza virus (Fig. 6).
Figure 6. The long filamentous σ1 attachment protein of reovirus is ideally shaped to display epitopes of pathogenic viruses such a HIV and influenza. We have designed recombinant viruses that contain small portions of these pathogenic viruses (green) that are recognized by human neutralizing antibodies (purple), effectively disguising the harmless reovirus as the pathogen. These vectors are now being tested for immunogenicity in mice and rabbits.
Viral Triggers of Celiac Disease
Celiac disease is an immune-mediated intestinal disorder that occurs in genetically predisposed individuals exposed to dietary gluten. Although approximately 30-45% of the U.S. population carries the risk genes, only 1% of the population develops the disease. This finding suggests that other genetic and environmental factors are required for celiac disease development. Viral infections are associated with the induction of many autoimmune and inflammatory diseases. Although several viruses have been implicated in celiac disease, little is known about the mechanisms by which viruses evoke the disease. Recent studies from our lab indicate inoculation of mice with mammalian orthoreovirus (reovirus) antagonizes the host response to food antigen. This disruption to intestinal immune homeostasis is similar to that observed with celiac disease, suggesting viral infections of the gastrointestinal tract may be an environmental trigger for celiac disease. We plan to define the viral components required to trigger disease onset using two different reovirus strains. Knowledge obtained from this research will aid in the development of targeted therapeutics and vaccines to prevent the induction of celiac disease.
Two reoviruses similarly infect the intestine and induce protective immunity, but differ in their immunopathological outcomes. Under homeostatic conditions, regulatory T cell responses to dietary antigen are induced in the small intestine (oral tolerance). T1L and T3D-RV reoviruses induce immune responses in Peyer’s patches, the site where protective immunity to reovirus is induced. In contrast, in mesenteric lymph nodes, the site of oral tolerance induction, T1L but not T3D-RV upregulates type-1 IFN signaling (Mx1; type-1 IFN induced gene; proxy for type-1 IFN) as well as IL-12 and IL-27 production by dendritic cells. Consequently, T1L but not T3D-RV infection inhibits the conversion of peripheral regulatory T cells (pTreg) and promotes the development of TH1 immunity to dietary antigen (Loss of oral tolerance). While type-1 IFN signaling (Mx1) in antigen-presenting cells plays a role in blocking pTreg conversion, IRF1 is required for the induction of IL-12 and IL-27, and the subsequent development of TH1 immunity to dietary antigen.