Members of the Reoviridae
family are non-enveloped viruses containing genomes of 10-12 segments of double-stranded (ds) RNA (Fig. 1). This family includes mammalian orthoreoviruses (reoviruses), orbiviruses, and rotaviruses. Rotaviruses are the most common cause of severe diarrheal illness in children, causing over 50,000 hospitalizations in the U.S. and over 600,000 deaths worldwide each year. In contrast, reoviruses infect many mammalian species, including humans, but are rarely associated with disease. Accordingly, they serve as a useful model for studies of virus replication, virus-cell interactions, and viral pathogenesis.
Like other members of the Reoviridae, reovirus particles are formed from concentric protein shells. Two such shells exist for reoviruses, termed outer capsid and core. For reoviruses, the viral proteins are designated with a Greek letter corresponding to the size of the encoding genome segment: sigma (σ) for proteins encoded by small (S) genome segments, mu (μ) by medium (M) segments, and lambda (λ) by large (L) segments. Each of the 10 genome segments encodes a single protein with the exception of the S1 gene, which encodes the viral attachment protein, σ1, and a small nonstructural protein, σ1s.
Three reovirus serotypes have been recognized based on neutralization and hemagglutination profiles. Each is represented by a prototype strain, type 1 Lang (T1L), type 2 Jones (T2J), and type 3 Dearing (T3D), which differ primarily in sigma1 sequence. The pathogenesis of reovirus infections has been most extensively studied using newborn mice, in which serotype-specific patterns of disease have been identified. Serotype 1 strains spread hematogenously and cause hydrocephalus. Serotype 3 strains spread to the central nervous system (CNS) by neural routes and cause lethal encephalitis. Reoviruses also replicate in the heart and liver and cause myocarditis and biliary atresia. Viruses and the innate immune response
Cells possess a wide array of sensors to detect the presence of invading pathogens, including cell-surface Toll-like receptors (TLRs) and the cytoplasmic RNA helicases RIG-I and Mda-5 (Fig. 2). These sensors recognize viral “pathogen-associated molecular patterns,” such as conserved viral proteins or dsRNA, and initiate signaling cascades that activate the cellular transcriptional factors IRF-3 and NF-kB. These transcription factors induce production of antiviral cytokines including the type 1 interferons (IFNs). IFNs act through autocrine or paracrine receptor-mediated signaling pathways to induce a secondary transcriptional response leading to the expression interferon-stimulated genes (ISGs). ISGs limit viral dissemination by interfering with many steps of the virus replication cycle or by inducing programmed cell death (apoptosis) in infected cells to prevent further viral replication.
Figure 2. Schematic diagram of innate immune responses to viral infection. Virus-associated patterns, including double-stranded RNA, are recognized by the cytoplasmic proteins RIG-I and Mda-5. These sensors signal through the downstream adaptor protein IPS-1 to activate kinase complexes that phosphorylate the transcription factors IRF-3 and NF-κB. Upon phosphorylation, these transcription factors translocate to the nucleus, where they induce a broad transcriptional response, including IFN-β. IFN-β, in turn, signals through its receptor to activate a second signaling cascade, leading to the activation of the STAT1/STAT2/IRF-9 transcription factor complex. This complex induces interferon-stimulated genes (ISGs) that inhibit virus replication.
Research in my lab seeks to answer a number of fundamental questions centered on the innate immune response to mammalian reovirus:
What are the molecular mechanisms by which cells detect and respond to reovirus infection?
How do some strains of reovirus overcome the cellular antiviral response, and how does this influence reovirus pathogenesis?
What cellular genes function to limit reovirus replication, and what is their mechanism of action?
1) Previously, both we and others demonstrated that reovirus dsRNA is recognized by both RIG-I and Mda-5 (see Holm, et al, J. Biol. Chem., 2007, 282: 21953). However, the molecular mechanisms that lead to cytoplasmic exposure of dsRNA following infection are not well characterized. We will utilize biochemical treatments of virions and known particle-destabilizing mutations to assess the role of capsid stability in dsRNA exposure.
2) Genetic characterization of reovirus serotype reassortant viruses has identified the reovirus S2, M1, and L2 genes as key determinants of reovirus induction of type 1 IFNs (see Sherry et al., J. Virol., 1998, 72: 1314). However, the molecular bases of these phenotypes are not known. Using a recently-developed reverse genetic system for mammalian reoviruses (see Kobayashi, Holm, et al., Cell Host & Microbe, 1: 147), we aim to generate reovirus mutants altered in these determinants in order to further elucidate the mechanism of strain-specific differences in IFN induction in infected cells.
3) Through microarray analysis, we determined genes that are upregulated in reovirus-infected cells in an NF-κB-dependent manner (see O’Donnell, Holm, et al., J. Virol., 2006, 80: 1077). This analysis identified multiple candidate genes to examine for their role in reovirus replication and pathogenesis. Using genetically altered cell lines, dominant-negative proteins, and siRNA knockdown, we will examine promising cellular targets for their effect on reovirus replication and apoptotic signaling cascades induced by infection. We will also use a similar microarray approach to identify reovirus strain-specific IFN-dependent and -independent transcriptional networks.