Reovirus Induction of and Sensitivity to Beta Interferon in Cardiac Myocyte Cultures Correlate with Induction of Myocarditis and Are Determined by Viral Core Proteins

Reovirus-induced acute myocarditis in mice serves as a model to investigate non-immune-mediated mechanisms of viral myocarditis. We have used primary cardiac myocyte cultures infected with a large panel of myocarditic and nonmyocarditic reassortant reoviruses to identify determinants of viral myocarditic potential. Here, we report that while both myocarditic and nonmyocarditic reoviruses kill cardiac myocytes, viral myocarditic potential correlates with viral spread through cardiac myocyte cultures and with cumulative cell death. To address the role of secreted interferon (IFN), we added anti-IFN-α/β antibody to infected cardiac myocyte cultures. Antibody benefited nonmyocarditic more than myocarditic virus spread (P < 0.001), and this benefit was associated with the reovirus M1 and L2 genes. There was no benefit for a differentiated skeletal muscle cell line culture (C2C12 cells), suggesting cell type specificity. IFN-β induction in reovirus-infected cardiac myocyte cultures correlated with viral myocarditic potential (P = 0.006) and was associated with the reovirus M1, S2, and L2 genes. Sensitivity to the antiviral effects of IFN-α/β added to cardiac myocyte cultures also correlated with viral myocarditic potential (P = 0.004) and was associated with the same reovirus genes. Several reoviruses induced IFN-β levels discordant with their myocarditic phenotypes, and for those tested, sensitivity to IFN-α/β compensated for the anomalous induction levels. Thus, the combination of induction of and sensitivity to IFN-α/β is a determinant of reovirus myocarditic potential. Finally, a nonmyocarditic reovirus induced cardiac lesions in mice depleted of IFN-α/β, demonstrating that IFN-α/β is a determinant of reovirus-induced myocarditis. This provides the first identification of reovirus genes associated with IFN induction and sensitivity and provides the first evidence that IFN-β can be a determinant of viral myocarditis and reovirus disease.     

Reovirus Growth in Cell Culture Does Not Require the Full Complement of Viral Proteins: Identification of a ς1s-Null Mutant

The reovirus ς1s protein is a 14-kDa nonstructural protein encoded by the S1 gene segment. The S1 gene has been linked to many properties of reovirus, including virulence and induction of apoptosis. Although the function of ς1s is not known, the ς1s open reading frame is conserved in all S1 gene sequences determined to date. In this study, we identified and characterized a variant of type 3 reovirus, T3C84-MA, which does not express ς1s. To facilitate these experiments, we generated two monoclonal antibodies (MAbs) that bind different epitopes of the ς1s protein. Using these MAbs in immunoblot and immunofluorescence assays, we found that L929 (L) cells infected with T3C84-MA do not contain ς1s. To determine whether ς1s is required for reovirus infection of cultured cells, we compared the growth of T3C84-MA and its parental strain, T3C84, in L cells and Madin-Darby canine kidney (MDCK) cells. After 48 h of growth, yields of T3C84-MA were equivalent to yields of T3C84 in L cells and were fivefold lower than yields of T3C84 in MDCK cells. After 7 days of growth following adsorption at a low multiplicity of infection, yields of T3C84-MA and T3C84 did not differ significantly in either L cells or MDCK cells. To determine whether ς1s is required for apoptosis induced by reovirus infection, T3C84-MA and T3C84 were tested for their capacity to induce apoptosis, using an acridine orange staining assay. In these experiments, the percentages of apoptotic cells following infection with T3C84-MA and T3C84 were equivalent. These findings indicate that nonstructural protein ς1s is not required for reovirus growth in cell culture and does not influence the capacity of reovirus to induce apoptosis. Therefore, reovirus replication does not require the full complement of virally encoded proteins.     

Utilization of Sialylated Glycans as Coreceptors Enhances the Neurovirulence of Serotype 3 Reovirus

Mammalian reoviruses display serotype-specific patterns of tropism and disease in the murine central nervous system (CNS) attributable to polymorphisms in viral attachment protein σ1. While all reovirus serotypes use junctional adhesion molecule-A as a cellular receptor, they differ in their utilization of carbohydrate coreceptors. This observation raises the possibility that carbohydrate binding by σ1 influences reovirus pathology in the CNS. In this study, we sought to define the function of carbohydrate binding in reovirus neuropathogenesis. Newborn mice were inoculated intramuscularly with wild-type strain type 3 Dearing (T3D) and T3D-σ1R202W, a point mutant T3D derivative that does not bind sialic acid (SA). Infected mice were monitored for survival, and viral loads at the sites of primary and secondary replication were quantified. Fewer mice inoculated with the wild-type virus survived in comparison to those inoculated with the mutant virus. The wild-type virus also produced higher titers in the spinal cord and brain at late times postinoculation but lower titers in the liver in comparison to those produced by the mutant virus. In addition, the wild-type virus was more virulent and produced higher titers in the brain than the mutant following intracranial inoculation. These animal infectivity studies suggest that T3D-σ1R202W harbors a defect in neural growth. Concordantly, compared with the wild-type virus, the mutant virus displayed a decreased capacity to infect and replicate in primary cultures of cortical neurons, a property dependent on cell surface SA. These results suggest that SA binding enhances the kinetics of reovirus replication in neural tissues and highlight a functional role for sialylated glycans as reovirus coreceptors in the CNS.     

Structure of Reovirus σ1 in Complex with Its Receptor Junctional Adhesion Molecule-A

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 (K_D) 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.     

Cleavage Susceptibility of Reovirus Attachment Protein ς1 during Proteolytic Disassembly of Virions Is Determined by a Sequence Polymorphism in the ς1 Neck

A requisite step in reovirus infection of the murine intestine is proteolysis of outer-capsid proteins to yield infectious subvirion particles (ISVPs). When converted to ISVPs by intestinal proteases, virions of reovirus strain type 3 Dearing (T3D) lose 90% of their original infectivity due to cleavage of viral attachment protein ς1. In an analysis of eight field isolate strains of type 3 reovirus, we identified one additional strain, type 3 clone 31 (T3C31), that loses infectivity and undergoes ς1 cleavage upon conversion of virions to ISVPs. We examined the ς1 deduced amino acid sequences of T3D and the eight field isolate strains for a correlation between sequence variability and ς1 cleavage. The ς1 proteins of T3D and T3C31 contain a threonine at amino acid position 249, whereas an isoleucine occurs at this position in the ς1 proteins of the remaining strains. Thr²⁴⁹ occupies the d position of a heptad repeat motif predicted to stabilize ς1 oligomers through α-helical coiled-coil interactions. This region of sequence comprises a portion of the fibrous tail domain of ς1 known as the neck. Substitution of Thr²⁴⁹ with isoleucine or leucine resulted in resistance to cleavage by trypsin, whereas replacement with asparagine did not affect cleavage susceptibility. These results demonstrate that amino acid position 249 is an independent determinant of T3D ς1 cleavage susceptibility and that an intact heptad repeat is required to confer cleavage resistance. We performed amino-terminal sequence analysis on the ς1 cleavage product released during trypsin treatment of T3D virions to generate ISVPs and found that trypsin cleaves ς1 after Arg²⁴⁵. Thus, the sequence polymorphism at position 249 controls cleavage at a nearby site in the neck region. The relevance of these results to reovirus infection in vivo was assessed by treating virions with the contents of a murine intestinal wash under conditions that result in generation of ISVPs. The pattern of ς1 cleavage susceptibility generated by using purified protease was reproduced in assays using the intestinal wash. These results provide a mechanistic explanation for ς1 cleavage during exposure of virions to intestinal proteases and may account for certain strain-dependent patterns of reovirus pathogenesis.     

JAM-A-independent, antibody-mediated uptake of reovirus into cells leads to apoptosis

Apoptosis plays a major role in the cytopathic effect induced by reovirus following infection of cultured cells and newborn mice. Strain-specific differences in the capacity of reovirus to induce apoptosis segregate with the S1 and M2 gene segments, which encode attachment protein σ1 and membrane penetration protein μ1, respectively. Virus strains that bind to both junctional adhesion molecule-A (JAM-A) and sialic acid are the most potent inducers of apoptosis. In addition to receptor binding, events in reovirus replication that occur during or after viral disassembly but prior to initiation of viral RNA synthesis also are required for reovirus-induced apoptosis. To determine whether reovirus infection initiated in the absence of JAM-A and sialic acid results in apoptosis, Chinese hamster ovary (CHO) cells engineered to express Fc receptors were infected with reovirus using antibodies directed against viral outer-capsid proteins. Fc-mediated infection of CHO cells induced apoptosis in a σ1-independent manner. Apoptosis following this uptake mechanism requires acid-dependent proteolytic disassembly, since treatment of cells with the weak base ammonium chloride diminished the apoptotic response. Analysis of T1L × T3D reassortant viruses revealed that the μ1-encoding M2 gene segment is the only viral determinant of the apoptosis-inducing capacity of reovirus when infection is initiated via Fc receptors. Additionally, a temperature-sensitive, membrane penetration-defective M2 mutant, tsA279.64, is an inefficient inducer of apoptosis. These data suggest that signaling pathways activated by binding of σ1 to JAM-A and sialic acid are dispensable for reovirus-mediated apoptosis and that the μ1 protein plays an essential role in stimulating proapoptotic signaling.     

Reovirus serotypes elicit distinctive patterns of recall immunity in humans

Mammalian orthoreoviruses (reoviruses) are ubiquitous viral agents that infect cells in respiratory and enteric tracts. The frequency and nature of human cellular immunoregulatory responses against reovirus are unknown. Here we establish systems to detect and quantify reovirus-induced cytokine and chemokine recall responses using primary cultures of virus-infected peripheral blood mononuclear cells (PBMC) and two widely used reovirus serotypes, type 1 Lang (T1L) and type 3 Dearing (T3D) reexposure in vitro. In cultures from 44 healthy adults, reovirus induced exceptionally strong CD4 and CD8 T-cell-dependent gamma interferon (IFN-γ) recall responses concomitant with intense interleukin 10 (IL-10) production. These responses were elicited independently of viral replication. Surprisingly, paired analyses of subject responses to these two common serotypes revealed that while both elicit intense Th1-dominated immunity, median T3D-driven responses were 2.2-fold weaker (P = 0.0004) than those elicited by T1L. Recall responses evoked by these viral serotypes differed markedly in their mechanism of regulation. T3D IL-10 and IFN-γ responses were CD4 and CD8 dependent and blocked by interfering with CD86 costimulation but were CD80 independent. T1L responses were consistently CD28 and CD80/86 independent. Thus, despite extensive genetic and morphological similarities between reovirus serotypes, the nature and intensity of the human recall responses as well as the control mechanisms regulating them are clearly distinct.     

Reovirus infection in rat lungs as a model to study the pathogenesis of viral pneumonia.

We undertook the present study to elucidate the pathogenesis of the pathologic response to reovirus infection in the lungs and further understand the interactions of reoviruses with pulmonary cells. We found that reoviruses were capable of causing acute pneumonia in 25- to 28-day-old Sprague-Dawley rats following intratracheal inoculation with the reoviruses type 1 Lang (T1L) and type 3 Dearing (T3D). The onset of the pneumonia was rapid, marked by type I alveolar epithelial cell degeneration, type II alveolar epithelial cell hyperplasia, and the infiltration of leukocytes into the alveolar spaces. More neutrophils were recruited into the lungs during T3D infection than during T1L infection, and the serotype difference in the neutrophil response was mapped to the S1 gene of reovirus. Viral replication in the lungs was required for the development of pneumonia due to T1L and T3D infections, and replication occurred in type I alveolar epithelial cells. T1L grew to higher titers in the lungs than did either T3D or type 3 clone 9, and the S1 gene was found to play a role in determining the level of viral replication. We propose that experimental reovirus infection in the lungs can serve as a model for the pathogenesis of viral pneumonia in which pulmonary inflammation results following direct infection of lung epithelial cells.     

Genetic mapping of reovirus virulence and organ tropism in severe combined immunodeficient mice: organ-specific virulence genes.

We used reovirus reassortant genetics and severe combined immunodeficient (SCID) mice to define viral genes important for organ tropism and virulence in the absence of antigen-specific immunity. Adult SCID mice infected with reovirus serotype 1 strain Lang (T1L) died after 20 +/- 6 days, while infection with serotype 3 strain Dearing (T3D) was lethal after 77 +/- 22 days. One hundred forty-five adult SCID mice were infected with T1L, T3D, and 25 different T1L x T3D reassortant reoviruses, and gene segments associated with the increased virulence of T1L were identified. Gene segments S1, L2, M1, and L1 accounted for > 90% of the genetically determined increase in T1L virulence. Gene segment M1 was independently important for virulence, with S1, L2, and L1 alone or in combination also playing a role. T1L grew to higher titers in multiple organs and caused more severe hepatitis than T3D. Seventy adult SCID mice, T1L, T3D, and 15 T1L x T3D reassortant viruses were used to map genetic determinants of viral titers in the brain, intestines, and liver, as well as the severity of hepatitis. Different sets of gene segments were important for determining viral titers in different organs. Gene segments L1 (encoding a core protein) and L2 (encoding the core spike of the virion) were important in all of the organs analyzed. The M1 gene segment (encoding a core protein), but not the S1 gene segment, was a critical determinant of reovirus titer in the liver and severity of hepatitis. The S1 gene segment (encoding the viral cell attachment protein and a nonstructural protein), but not the M1 gene segment, was a critical determinant of titers in intestines and brains. These studies demonstrate that viral growth in different organs is dependent on different subsets of the genes important for virulence. The virion-associated protein products of the four gene segments (L1, L2, M1, and S1) important for virulence and organ tropism in SCID mice likely form a structural unit, the reovirus vertex. Organs (the brain and intestines versus the liver) differ in properties that determine which virulence genes, and thus which parts of this structural unit, are important.     

Genetic Determinants of Reovirus Pathogenesis in a Murine Model of Respiratory Infection

Many viruses invade mucosal surfaces to establish infection in the host. Some viruses are restricted to mucosal surfaces, whereas others disseminate to sites of secondary replication. Studies of strain-specific differences in reovirus mucosal infection and systemic dissemination have enhanced an understanding of viral determinants and molecular mechanisms that regulate viral pathogenesis. After peroral inoculation, reovirus strain type 1 Lang replicates to high titers in the intestine and spreads systemically, whereas strain type 3 Dearing (T3D) does not. These differences segregate with the viral S1 gene segment, which encodes attachment protein σ1 and nonstructural protein σ1s. In this study, we define genetic determinants that regulate reovirus-induced pathology following intranasal inoculation and respiratory infection. We report that two laboratory isolates of T3D, T3D^C and T3D^F, differ in the capacity to replicate in the respiratory tract and spread systemically; the T3D^C isolate replicates to higher titers in the lungs and disseminates, while T3D^F does not. Two nucleotide polymorphisms in the S1 gene influence these differences, and both S1 gene products are involved. T3D^C amino acid polymorphisms in the tail and head domains of σ1 protein influence the sensitivity of virions to protease-mediated loss of infectivity. The T3D^C polymorphism at nucleotide 77, which leads to coding changes in both S1 gene products, promotes systemic dissemination from the respiratory tract. A σ1s-null virus produces lower titers in the lung after intranasal inoculation and disseminates less efficiently to sites of secondary replication. These findings provide new insights into mechanisms underlying reovirus replication in the respiratory tract and systemic spread from the lung.     

Nonrandom segregation of parental alleles in reovirus reassortants.

To test for nonrandom segregations among their 10 genomic RNA segments, we examined a set of 83 reassortants derived from mammalian reovirus type 1 Lang and type 3 Dearing. After confirming the genotypes of the reassortants, we performed statistical analyses on the distributions of parental alleles for each of the 10 gene segments, as well as for the 45 possible pairings of the 10 segments. The analyses revealed nonrandom associations of parental alleles in the L1-L2, L1-M1, L1-S1, and L3-S1 segment pairs, at levels indicating high statistical significance (P < 0.005). Such associations may reflect specific interactions between viral components (protein-protein, protein-RNA, or RNA-RNA) and may influence both the evolution of reoviruses in nature and their genetic analysis in the laboratory. The data may also support an hypothesis that reovirus reassortants commonly contain mutations that improve their fitness for independent replication.     

Nonstructural Protein σ1s Mediates Reovirus-Induced Cell Cycle Arrest and Apoptosis

Reovirus nonstructural protein σ1s is implicated in cell cycle arrest at the G₂/M boundary and induction of apoptosis. However, the contribution of σ1s to these effects in an otherwise isogenic viral background has not been defined. To evaluate the role of σ1s in cell cycle arrest and apoptosis, we used reverse genetics to generate a σ1s-null reovirus. Following infection with wild-type virus, we observed an increase in the percentage of cells in G₂/M, whereas the proportion of cells in G₂/M following infection with the σ1s-null mutant was unaffected. Similarly, we found that the wild-type virus induced substantially greater levels of apoptosis than the σ1s-null mutant. These data indicate that σ1s is required for both reovirus-induced cell cycle arrest and apoptosis. To define sequences in σ1s that mediate these effects, we engineered viruses encoding C-terminal σ1s truncations by introducing stop codons in the σ1s open reading frame. We also generated viruses in which charged residues near the σ1s amino terminus were replaced individually or as a cluster with nonpolar residues. Analysis of these mutants revealed that amino acids 1 to 59 and the amino-terminal basic cluster are required for induction of both cell cycle arrest and apoptosis. Remarkably, viruses that fail to induce cell cycle arrest and apoptosis also are attenuated in vivo. Thus, identical sequences in σ1s are required for reovirus-induced cell cycle arrest, apoptosis, and pathogenesis. Collectively, these findings provide evidence that the σ1s-mediated properties are genetically linked and suggest that these effects are mechanistically related.     

Suppression of PPARγ through MKRN1-mediated ubiquitination and degradation prevents adipocyte differentiation

The central regulator of adipogenesis, PPARγ, is a nuclear receptor that is linked to obesity and metabolic diseases. Here we report that MKRN1 is an E3 ligase of PPARγ that induces its ubiquitination, followed by proteasome-dependent degradation. Furthermore, we identified two lysine sites at 184 and 185 that appear to be targeted for ubiquitination by MKRN1. Stable overexpression of MKRN1 reduced PPARγ protein levels and suppressed adipocyte differentiation in 3T3-L1 and C3H10T1/2 cells. In contrast, MKRN1 depletion stimulated adipocyte differentiation in these cells. Finally, MKRN1 knockout MEFs showed an increased capacity for adipocyte differentiation compared with wild-type MEFs, with a concomitant increase of PPARγ and adipogenic markers. Together, these data indicate that MKRN1 is an elusive PPARγ E3 ligase that targets PPARγ for proteasomal degradation by ubiquitin-dependent pathways, and further depict MKRN1 as a novel target for diseases involving PPARγ.     

The Reovirus Mutant tsA279 L2 Gene Is Associated with Generation of a Spikeless Core Particle: Implications for Capsid Assembly

Previous studies which used intertypic reassortants of the wild-type reovirus serotype 1 Lang and the temperature-sensitive (ts) serotype 3 mutant clone tsA279 identified two ts lesions; one lesion, in the M2 gene segment, was associated with defective transmembrane transport of restrictively assembled virions (P. R. Hazelton and K. M. Coombs, Virology 207:46–58, 1995). In the present study we show that the second lesion, in the L2 gene segment, which encodes the λ2 protein, is associated with the accumulation of a core-like particle defective for the λ2 pentameric spike. Physicochemical, biochemical, and immunological studies showed that these structures were deficient for genomic double-stranded RNA, the core spike protein λ2, and the minor core protein μ2. Core particles with the λ2 spike structure accumulated after temperature shift-down from a restrictive to a permissive temperature in the presence of cycloheximide. These data suggest the spike-deficient, core-like particle is an assembly intermediate in reovirus morphogenesis. The existence of this naturally occurring primary core structure suggests that the core proteins λ1, λ3, and ς2 interact to initiate the process of virion capsid assembly through a dodecahedral mechanism. The next step in the proposed capsid assembly model would be the association of the minor core protein μ2, either preceding or collateral to the condensation of the λ2 pentameric spike at the apices of the primary core structure. The assembly pathway of the reovirus double capsid is further elaborated when these observations are combined with structures identified in other studies.     

The Na+/H+ exchanger NHE1 is required for directional migration stimulated via PDGFR-α in the primary cilium

We previously demonstrated that the primary cilium coordinates platelet-derived growth factor (PDGF) receptor (PDGFR) α–mediated migration in growth-arrested fibroblasts. In this study, we investigate the functional relationship between ciliary PDGFR-α and the Na⁺/H⁺ exchanger NHE1 in directional cell migration. NHE1 messenger RNA and protein levels are up-regulated in NIH3T3 cells and mouse embryonic fibroblasts (MEFs) during growth arrest, which is concomitant with cilium formation. NHE1 up-regulation is unaffected in Tg737^orpk MEFs, which have no or very short primary cilia. In growth-arrested NIH3T3 cells, NHE1 is activated by the specific PDGFR-α ligand PDGF-AA. In wound-healing assays on growth-arrested NIH3T3 cells and wild-type MEFs, NHE1 inhibition by 5′-(N-ethyl-N-isopropyl) amiloride potently reduces PDGF-AA–mediated directional migration. These effects are strongly attenuated in interphase NIH3T3 cells, which are devoid of primary cilia, and in Tg737^orpk MEFs. PDGF-AA failed to stimulate migration in NHE1-null fibroblasts. In conclusion, stimulation of directional migration in response to ciliary PDGFR-α signals is specifically dependent on NHE1 activity, indicating that NHE1 activation is a critical event in the physiological response to PDGFR-α stimulation.     

IFN-γ-Inducible Irga6 Mediates Host Resistance against Chlamydia trachomatis via Autophagy

Chlamydial infection of the host cell induces Gamma interferon (IFNγ), a central immunoprotector for humans and mice. The primary defense against Chlamydia infection in the mouse involves the IFNγ-inducible family of IRG proteins; however, the precise mechanisms mediating the pathogen''s elimination are unknown. In this study, we identify Irga6 as an important resistance factor against C. trachomatis, but not C. muridarum, infection in IFNγ-stimulated mouse embryonic fibroblasts (MEFs). We show that Irga6, Irgd, Irgm2 and Irgm3 accumulate at bacterial inclusions in MEFs upon stimulation with IFNγ, whereas Irgb6 colocalized in the presence or absence of the cytokine. This accumulation triggers a rerouting of bacterial inclusions to autophagosomes that subsequently fuse to lysosomes for elimination. Autophagy-deficient Atg5−/− MEFs and lysosomal acidification impaired cells surrender to infection. Irgm2, Irgm3 and Irgd still localize to inclusions in IFNγ-induced Atg5−/− cells, but Irga6 localization is disrupted indicating its pivotal role in pathogen resistance. Irga6-deficient (Irga6−/−) MEFs, in which chlamydial growth is enhanced, do not respond to IFNγ even though Irgb6, Irgd, Irgm2 and Irgm3 still localize to inclusions. Taken together, we identify Irga6 as a necessary factor in conferring host resistance by remodelling a classically nonfusogenic intracellular pathogen to stimulate fusion with autophagosomes, thereby rerouting the intruder to the lysosomal compartment for destruction.