Borna disease virus nucleoprotein interacts with the CDC2-cyclin B1 complex

Transition from G(2) to M phase, a cell cycle checkpoint, is regulated by the Cdc2-cyclin B1 complex. Here, we report that persistent infection with Borna disease virus (BDV), a noncytolytic RNA virus infecting the central nervous system, results in decelerated proliferation of infected host cells due to a delayed G(2)-to-M transition. Persistent BDV-infected rat fibroblast cells showed reduced proliferation compared to uninfected cells. In pull-down assays we observed an interaction of the viral nucleoprotein with the Cdc2-cyclin B1 complex. Transfection of the viral nucleoprotein but not of the phosphoprotein also results in decelerated proliferation. This phenomenon was found in BDV-susceptible primary rat fibroblast cells and also in primary mouse cells, which are not susceptible to BDV infection. This is the first evidence that the noncytolytic Borna disease virus can manipulate host cell functions via interaction of the viral nucleoprotein with mitotic entry regulators. BDV preferentially infects and persists in nondividing neurons. The present report could give an explanation for this selective choice of host cell by BDV.     

Activation of microglia by borna disease virus infection: in vitro study

Neonatal Borna disease virus (BDV) infection of the rat brain is associated with microglial activation and damage to the certain neuronal populations. Since persistent BDV infection of neurons in vitro is noncytolytic and noncytopathic, activated microglia have been suggested to be responsible for neuronal cell death in vivo. However, the mechanisms of activation of microglia in neonatally BDV-infected rat brain have not been investigated. To address these issues, activation of primary rat microglial cells was studied following exposure to purified BDV or to persistently BDV-infected primary cortical neurons or after BDV infection of primary mixed neuron-glial cultures. Neither purified virus nor BDV-infected neurons alone activated primary microglia as assessed by the changes in cell shape or production of the proinflammatory cytokines. In contrast, in the BDV-infected primary mixed cultures, we observed proliferation of microglia cells that acquired the round morphology and expressed major histocompatibility complex molecules of classes I and II. These manifestations of microglia activation were observed in the absence of direct BDV infection of microglia or overt neuronal toxicity. In addition, compared to uninfected mixed cultures, activation of microglia in BDV-infected mixed cultures was associated with a significantly greater lipopolysaccharide-induced release of tumor necrosis factor alpha, interleukin 1beta, and interleukin 10. Taken together, the present data are the first in vitro evidence that persistent BDV infection of neurons and astrocytes rather than direct exposure to the virus or dying neurons is critical for activating microglia.     

Viral interference with neuronal integrity: what can we learn from the Borna disease virus?

The neurotropic Borna disease virus (BDV) is unusual in that it can persistently infect neurons of the central nervous system (CNS) without causing general cell death, reflecting its favourable adaptation to the brain. The activity-dependent enhancement of neuronal network activity is however disturbed after BDV infection, possibly by its effect on the protein kinase C signalling pathway. The best model for studying BDV, which has a non-cytolytic replication strategy in primary neurons, is the rat. Infection of adult rats results in a fatal immune-mediated disease, whereas BDV establishes persistent infection of the brain in newborn rats resulting in progressive neuronal cell loss in defined regions of the CNS. Our recently developed system of BDV-infected hippocampal slice cultures has clearly shown that the onset of granule cell loss begins after the formation of the mossy fibre projection. Quantitative analysis has revealed a significant increase in synaptic density on identified remaining granule cell dendrites at 6?weeks after infection, followed by a decline. Granule cells are the major target of entorhinal afferents. However, despite an almost complete loss of dentate granule cells during BDV infection, entorhinal axons persist in their correct layer, both in vivo and in slice cultures, possibly exploiting rewiring capabilities and thereby allowing new synapse formation with available targets. These morphological observations, together with electrophysiological and biochemical data, indicate that BDV is a suitable model virus for studying virus-induced morphological and functional changes of neurons and connectivity patterns.     

Downregulation of an astrocyte-derived inflammatory protein, S100B, reduces vascular inflammatory responses in brains persistently infected with Borna disease virus

Borna disease virus (BDV) is a neurotropic virus that causes a persistent infection in the central nervous system (CNS) of many vertebrate species. Although a severe reactive gliosis is observed in experimentally BDV-infected rat brains, little is known about the glial reactions contributing to the viral persistence and immune modulation in the CNS. In this regard, we examined the expression of an astrocyte-derived factor, S100B, in the brains of Lewis rats persistently infected with BDV. S100B is a Ca(2+)-binding protein produced mainly by astrocytes. A prominent role of this protein appears to be the promotion of vascular inflammatory responses through interaction with the receptor for advanced glycation end products (RAGE). Here we show that the expression of S100B is significantly reduced in BDV-infected brains despite severe astrocytosis with increased glial fibrillary acidic protein immunoreactivity. Interestingly, no upregulation of the expression of S100B, or RAGE, was observed in the persistently infected brains even when incited with several inflammatory stimuli, including lipopolysaccharide. In addition, expression of the vascular cell adhesion molecule 1 (VCAM-1), as well as the infiltration of encephalitogenic T cells, was significantly reduced in persistently infected brains in which an experimental autoimmune encephalomyelitis was induced by immunization with myelin-basic protein. Furthermore, we demonstrated that the continuous activation of S100B in the brain may be necessary for the progression of vascular immune responses in neonatally infected rat brains. Our results suggested that BDV infection may impair astrocyte functions via a downregulation of S100B expression, leading to the maintenance of a persistent infection.     

Characterization of a glial cell line persistently infected with borna disease virus (BDV): influence of neurotrophic factors on BDV protein and RNA expression.

Borna disease virus (BDV) infects cells of the nervous system in a wide range of species. Previous work suggests that there are differences in BDV replication in neuronal cells and glial cells. Many neurons are lysed by the immunopathologic response to BDV; lysis of dentate gyrus neurons in the absence of encephalitis is seen in rats inoculated with BDV as neonates. In contrast, persistently BDV-infected astrocytes increase over the course of BDV infection. Therefore, we compared BDV replication in neuronal (SK-N-SH and SK-N-SHEP) and astrocytic (C6) cell lines. While SK-N-SH cells produced more infectious virions per cell, the C6 cells contained more BDV proteins and RNA. BDV sequences in the supernatants of both cell types were identified, despite low titers of infectious virus, suggesting the release of incomplete virions into the medium. C6 cells secreted a factor or factors into the medium that enhanced the production of BDV proteins and RNA in other cell lines. In addition, nerve growth factor treatment produced the same enhancement. Thus, BDV replication in certain neural cells in vitro may be linked to the production of cell-specific factors which affect viral replication.     

Severity of neurological signs and degree of inflammatory lesions in the brains of rats with Borna disease correlate with the induction of nitric oxide synthase.

The putative role of nitric oxide in the neuropathogenesis of Borna disease was investigated by determining changes in the expression of inducible nitric oxide synthase (iNOS) mRNA and constitutively expressed NOS (cNOS) mRNA in brains of Borna disease virus (BDV)-infected rats. iNOS mRNA was not detected in normal rat brain but was identified in BDV-infected brain at 14 days postinfection (p.i.), reaching maximum levels at 21 days p.i., when neurological signs and inflammatory reactions in the brain were also at a peak. cNOS mRNA was expressed in both normal brain and infected brain, increasing markedly at 17 days p.i. and reaching a peak at 21 days p.i. In situ hybridization analysis revealed iNOS mRNA in some, but not all, BDV-infected regions of the brain, particularly in the basolateral cortex and the hippocampus. iNOS-positive cells, as identified immunohistologically, were preferentially localized in perivascular areas of the hippocampus and in outer cortical layers. These iNOS-positive cells resembled monocytes/macrophages in morphology and distribution pattern but were significantly fewer. The correlation of iNOS and cNOS mRNA expression with the development of neurological disease, as well as the enhanced expression of iNOS within brain regions with inflammatory lesions, strongly suggests that NO may contribute to pathogenesis of Borna disease.     

A Functional Role for Neutralizing Antibodies in Borna Disease: Influence on Virus Tropism outside the Central Nervous System

Borna disease virus (BDV) is a negative-strand RNA virus that infects the central nervous systems (CNS) of warm-blooded animals and causes disturbances of movement and behavior. The basis for neurotropism remains poorly understood; however, the observation that the distribution of infectious virus in immunocompetent rats is different from that in immunoincompetent rats indicates a role for the immune system in BDV tropism: whereas in immunocompetent rats virus is restricted to the central, peripheral, and autonomic nervous systems, immunoincompetent rats also have virus in nonneural tissues. In an effort to examine the influence of the humoral immune response on BDV pathogenesis, we examined the effects of passive immunization with neutralizing antiserum in immunoincompetent rats. Serum transfer into immunoincompetent rats did not prevent persistent CNS infection but did result in restriction of virus to neural tissues. These results indicate that neutralizing antibodies may play a role in preventing generalized infection with BDV.     

Persistent borna disease virus infection confers instability of HSP70 mRNA in glial cells during heat stress

Borna disease virus (BDV) is a highly neurotropic RNA virus that causes neurological disorders in many vertebrate species. Although BDV readily establishes lasting persistence, persistently infected cells maintain an apparently normal cell phenotype in terms of morphology, viability, and proliferation. In this study, to understand the regulation of stress responses in BDV infection, we investigated the expression of heat shock proteins (HSPs) in glial cells persistently infected with BDV. Interestingly, we found that BDV persistence did not upregulate HSP70 expression even in cells treated with heat stress. Furthermore, BDV-infected glial cells exhibited rapid rounding and detachment from the culture plate under various stressful conditions. Immunofluorescence analysis demonstrated that heat stress rapidly disrupts the cell cytoskeleton only in persistently infected cells, suggesting a lack of thermotolerance. Intriguingly, we found that although persistently infected glial cells expressed HSP70 mRNA after heat stress, its expression rapidly disappeared during the recovery period. These observations indicated that persistent BDV infection may affect the stability of HSP70 mRNA. Finally, we found that the double-stranded RNA-dependent protein kinase (PKR) is expressed at a constant level in persistently infected cells with or without heat shock. Considering the interrelationship between HSP70 and PKR production, our data suggest that BDV infection disturbs the cellular stress responses to abolish antiviral activities and maintain persistence.     

The central nervous system inflammatory response to neurotropic virus infection is peroxynitrite dependent.

We have recently demonstrated that increased blood-CNS barrier permeability and CNS inflammation in a conventional mouse model of experimental allergic encephalomyelitis are dependent upon the production of peroxynitrite (ONOO(-)), a product of the free radicals NO* and superoxide (O2*(-)). To determine whether this is a reflection of the physiological contribution of ONOO(-) to an immune response against a neurotropic pathogen, we have assessed the effects on adult rats acutely infected with Borna disease virus (BDV) of administration of uric acid (UA), an inhibitor of select chemical reactions associated with ONOO(-). The pathogenesis of acute Borna disease in immunocompetent adult rats results from the immune response to the neurotropic BDV, rather than the direct effects of BDV infection of neurons. An important stage in the BDV-specific neuroimmune response is the invasion of inflammatory cells into the CNS. UA treatment inhibited the onset of clinical disease, and prevented the elevated blood-brain barrier permeability as well as CNS inflammation seen in control-treated BDV-infected rats. The replication and spread of BDV in the CNS were unchanged by the administration of UA, and only minimal effects on the immune response to BDV Ags were observed. These results indicate that the CNS inflammatory response to neurotropic virus infection is likely to be dependent upon the activity of ONOO(-) or its products on the blood-brain barrier.     

High-Dose Borna Disease Virus Infection Induces a Nucleoprotein-Specific Cytotoxic T-Lymphocyte Response and Prevention of Immunopathology

Experimental Borna disease virus (BDV) infection of rats and natural infection of horses and sheep leads to severe central nervous system disease based on immunopathological pathways. The virus replicates slowly, and the cellular immune response results in immunopathology. CD8(+) T cells exert effector cell functions, and their activity results in the destruction of virus-infected cells. Previously, Oldach and colleagues (D. Oldach, M. C. Zink, J. M. Pyper, S. Herzog, R. Rott, O. Narayan, and J. E. Clements, Virology 206:426-434, 1995) have reported protection against Borna disease after inoculation of high-dose cell-adapted BDV. Here we show that the outcome of the infection, i.e., immunopathology versus protection, is simply dependent on the amount of virus used for infection. High-dose BDV (10(6) FFU) triggers an early virus-specific reaction of the immune system, as demonstrated by strong cellular and humoral responses. In particular, the early presence and function of nucleoprotein-specific CD8(+) T cells could be demonstrated in the brain. We present evidence that in a noncytolytic and usually persistent virus infection, high-dose input virus mediates early control of the pathogen due to an efficient induction of an antiviral immune mechanism. From these data, we conclude that immune reactivity, in particular the cytotoxic T-cell response, determines whether the virus is controlled with prevention of the ensuing immunopathological disease or whether a persistent infection is established.     

Neutralizing antibodies in persistent borna disease virus infection: prophylactic effect of gp94-specific monoclonal antibodies in preventing encephalitis

Borna disease virus (BDV) infection triggers an immune-mediated encephalomyelitis and results in a persistent infection. The immune response in the acute phase of the disease is characterized by a cellular response in which CD8(+) T cells are responsible for the destruction of virus-infected brain cells. CD4(+) T cells function as helper cells and support the production of antiviral antibodies. Antibodies generated in the acute phase of the disease against the nucleoprotein and the phosphoprotein are nonneutralizing. In the chronic phase of the disease, neutralizing antibodies directed against the matrix protein and glycoprotein are synthesized. In the present work, the biological role of the neutralizing-antibody response to BDV was further investigated. By analyzing the blood of rats infected intracerebrally with BDV, a highly neurotropic virus, nucleic acid could be detected between 30 and 50 days after infection. Neutralizing antibodies were found between 60 and 100 days after infection. Furthermore, we produced hybridomas secreting BDV-specific neutralizing monoclonal antibodies. These antibodies, directed against the major glycoprotein (gp94) of BDV, were able to prevent Borna disease if given prophylactically. These data suggest that the late appearance of BDV-specific neutralizing antibodies is due to the presence of BDV in the blood of chronically infected rats. Furthermore, these antibodies have the potential to neutralize the infectious virus when given early, which is an important finding with respect to the development of a vaccine.     

The Bicolored White-Toothed Shrew Crocidura leucodon (HERMANN 1780) Is an Indigenous Host of Mammalian Borna Disease Virus

Borna disease (BD) is a sporadic neurologic disease of horses and sheep caused by mammalian Borna disease virus (BDV). Its unique epidemiological features include: limited occurrence in certain endemic regions of central Europe, yearly varying disease peaks, and a seasonal pattern with higher disease frequencies in spring and a disease nadir in autumn. It is most probably not directly transmitted between horses and sheep. All these features led to the assumption that an indigenous virus reservoir of BDV other than horses and sheep may exist. The search for such a reservoir had been unsuccessful until a few years ago five BDV-infected shrews were found in a BD-endemic area in Switzerland. So far, these data lacked further confirmation. We therefore initiated a study in shrews in endemic areas of Germany. Within five years 107 shrews of five different species were collected. BDV infections were identified in 14 individuals of the species bicolored white-toothed shrew (Crocidura leucodon, HERMANN 1780), all originating from BD-endemic territories. Immunohistological analysis showed widespread distribution of BDV antigen both in the nervous system and in epithelial and mesenchymal tissues without pathological alterations. Large amounts of virus, demonstrated by presence of viral antigen in epithelial cells of the oral cavity and in keratinocytes of the skin, may be a source of infection for natural and spill-over hosts. Genetic analyses reflected a close relationship of the BDV sequences obtained from the shrews with the regional BDV cluster. At one location a high percentage of BDV-positive shrews was identified in four consecutive years, which points towards a self-sustaining infection cycle in bicolored white-toothed shrews. Analyses of behavioral and population features of this shrew species revealed that the bicolored white-toothed shrew may indeed play an important role as an indigenous host of BDV.     

Borna disease virus: a unique pathogen and its interaction with intracellular signalling pathways

Borna disease virus (BDV) is a neurotropic RNA virus that establishes non-cytolytic persistent infection in the central nervous system of warm-blooded animals. Depending on the host species and the route of infection, BDV persistence can modulate neuronal plasticity and animal behaviour and/or may provoke a T cell-mediated immunopathological reaction with high mortality. Therefore, BDV functions as a model pathogen to study persistent virus infection in the central nervous system. Here, we review recent evidence showing that BDV interferes with a spectrum of intracellular signalling pathways, which may be involved in viral spread, maintenance of persistence and modulation of neurotransmitter pathways.     

Metabolomic profiling of three brain regions from a postnatal infected Borna disease virus Hu-H1 rat model

Neonatal rat infection with Borna disease virus (BDV), termed neonatal Borna disease, is an established model for investigating the BDV-associated pathogenesis of neurodevelopmental abnormalities. BDV produces a persistent noncytolytic infection in all culture cell systems assayed to date, while persistent infection in neonatal rats results in a progressive loss of hippocampal granule cells, cerebellar Purkinje cells, and cortical GABA-ergic neurons. Persistent infection also results in behavioral deficits including hyperactivity, cognitive impairment, and abnormal social behavior. However, the molecular mechanisms underlying the neuronal degeneration and behavioral abnormalities remain unclear. Using a metabolomic approach based on gas chromatography coupled with mass spectrometry in conjunction with statistical pattern recognition, the metabolic changes in response to BDV Hu-H1 infection were characterized in the rat hippocampus, cerebellum, and cortex. Metabonomic profiling revealed significant perturbations in nucleotide (e.g., adenosine, uracil, inosine, adenosine-5′-monophosphate, uridine-5′-monophosphate, d-ribose 5-phosphate, and sedoheptulose 7-phosphate), amino acid (e.g., lysine, glycine, phenylalanine, tyrosine, proline, serine, cysteine, aspartic acid, pyroglutamic acid, and γ-aminobutyric acid), lipid (e.g., cholesterol, myristic acid, stearic acid, palmitic acid, 1-monopalmitoylglycerol, and arachidonic acid), and energy (e.g., glucose, lactose, 3-phosphoglyceric acid, and pyruvic acid) metabolites. These metabolites participate in pathways crucial to viral proliferation and neurotransmitter homeostasis. This metabolomic profiling study provides insight into the pathogenic mechanisms of BDV and new directions with which to investigate the in vivo effects of persistent BDV infection.     

Cell-to-cell spread of Borna disease virus proceeds in the absence of the virus primary receptor and furin-mediated processing of the virus surface glycoprotein

Borna disease virus (BDV) is an enveloped virus with a nonsegmented negative-strand RNA genome whose organization is characteristic of Mononegavirales. BDV cell entry follows a receptor-mediated endocytosis pathway, which is initiated by the recognition of an as-yet-unidentified receptor at the cell surface by the virus glycoprotein G. BDV G is synthesized as a precursor (GPC) that is cleaved by the cellular protease furin to produce the mature glycoproteins GP1 and GP2, which have been implicated in receptor recognition and pH-dependent fusion events, respectively. BDV is highly neurotropic and its spread in cultured cells proceeds in the absence of detectable extracellular virus or syncytium formation. BDV spread has been proposed to be strictly dependent on the expression and correct processing of BDV G. Here we present evidence that cell-to-cell spread of BDV required neither the expression of cellular receptors involved in virus primary infection, nor the furin-mediated processing of BDV G. We also show that in furin-deficient cells, the release of BDV particles induced by the treatment of BDV-infected cells with hypertonic buffer was not significantly affected, while virion infectivity was dramatically impaired, correlating with the decreased incorporation of BDV G species into viral particles. These findings support the view that the propagation of BDV within the central nervous systems of infected hosts involves both a primary infection that follows a receptor-mediated endocytosis pathway and a subsequent cell-to-cell spread that is independent of the expression of the primary receptor and does not require the processing of BDV G into GP1 and GP2.     

Biochemical and functional analysis of the Borna disease virus G protein.

The Borna disease virus (BDV) antigenome is comprised of five major open reading frames (ORFs). Products have been reported only for ORFs I, II, and III, encoding N (p40), P (p24/p23), and M (gp18), respectively. ORF IV predicts a 57-kDa protein with several potential glycosylation sites. Analysis of radiolabeled extracts from BDV-infected C6 cells and BHK-21 cells transfected with a Semliki Forest virus vector that contains ORF IV demonstrated the presence of a 94-kDa protein (G protein) which was sensitive to tunicamycin, endoglycosidase F/N-glycosidase, and endoglycosidase H but not to O-glycosidase. Sera from BDV-infected rats detected the G protein and had neutralization activity that was reduced following immunoadsorption with the G protein. Preincubation of cells with the G protein interfered with BDV infectivity. This effect was enhanced by treatment of the G protein with the exoglycosidase alpha-mannosidase and reduced after subsequent treatment with N-acetyl-beta-D-glucosaminidase. In concert these findings indicate that ORF IV encodes a 94-kDa N-linked glycoprotein with extensive high mannose- and/or hybrid-type oligosaccharide modifications. The presence of neutralization epitopes on the G protein and its capacity to interfere with infectivity suggest that the G protein is important for viral entry.     

Effect of Immune Priming on Borna Disease

Borna disease virus (BDV) is a neurotropic virus with a broad host and geographic range. Lewis rats were immunized against BDV with a recombinant vaccinia virus expressing the BDV nucleoprotein and were later infected with BDV to evaluate protection against Borna disease (BD). Relative to animals that were not immunized, immunized animals had a decreased viral burden after challenge with infectious virus, more marked inflammation, and aggravated clinical disease. These data suggest that a more robust immune response in Borna disease can reduce viral load at the expense of increased morbidity.     

Neurons are MHC class I-dependent targets for CD8 T cells upon neurotropic viral infection

Following infection of the central nervous system (CNS), the immune system is faced with the challenge of eliminating the pathogen without causing significant damage to neurons, which have limited capacities of renewal. In particular, it was thought that neurons were protected from direct attack by cytotoxic T lymphocytes (CTL) because they do not express major histocompatibility class I (MHC I) molecules, at least at steady state. To date, most of our current knowledge on the specifics of neuron-CTL interaction is based on studies artificially inducing MHC I expression on neurons, loading them with exogenous peptide and applying CTL clones or lines often differentiated in culture. Thus, much remains to be uncovered regarding the modalities of the interaction between infected neurons and antiviral CD8 T cells in the course of a natural disease. Here, we used the model of neuroinflammation caused by neurotropic Borna disease virus (BDV), in which virus-specific CTL have been demonstrated as the main immune effectors triggering disease. We tested the pathogenic properties of brain-isolated CD8 T cells against pure neuronal cultures infected with BDV. We observed that BDV infection of cortical neurons triggered a significant up regulation of MHC I molecules, rendering them susceptible to recognition by antiviral CTL, freshly isolated from the brains of acutely infected rats. Using real-time imaging, we analyzed the spatio-temporal relationships between neurons and CTL. Brain-isolated CTL exhibited a reduced mobility and established stable contacts with BDV-infected neurons, in an antigen- and MHC-dependent manner. This interaction induced rapid morphological changes of the neurons, without immediate killing or impairment of electrical activity. Early signs of neuronal apoptosis were detected only hours after this initial contact. Thus, our results show that infected neurons can be recognized efficiently by brain-isolated antiviral CD8 T cells and uncover the unusual modalities of CTL-induced neuronal damage.     

Borna disease virus phosphoprotein binds a neurite outgrowth factor, amphoterin/HMG-1

The Borna disease virus (BDV) p24 phosphoprotein is an abundant protein in BDV-infected cultured cells and animal brains. Therefore, there is a possibility that binding of the p24 protein to cellular factor(s) induces functional alterations of infected neural cells in the brain. To identify a cellular protein(s) that interacts with BDV p24 protein, we performed far-Western blotting with extracts from various cell lines. Using recombinant p24 protein as a probe, we detected a 30-kDa protein in all cell lines examined. Binding between the 30-kDa and BDV p24 proteins was also demonstrated using BDV p24 affinity and ion-exchange chromatography columns. Microsequence analysis of the purified 30-kDa protein revealed that its N terminus showed complete homology with rat amphoterin protein, which is a neurite outgrowth factor abundant in the brain during development. Mammalian two-hybrid and immunoprecipitation analyses also confirmed that amphoterin is a specific target for the p24 protein in vivo. Furthermore, we showed that infection by BDV, as well as purified p24 protein in the medium, significantly decreased cell process outgrowth of cells grown on laminin, indicating the functional inhibition of amphoterin by interaction with the p24 protein. Immunohistochemical analysis revealed decreased levels of amphoterin protein at the leading edges of BDV-infected cells. Moreover, the expression of the receptor for advanced glycation end products, of which the extracellular moiety is a receptor for amphoterin, was not significantly activated in BDV-infected cells during the process of extension, suggesting that the secretion of amphoterin from the cell surface is inhibited by the binding of the p24 protein. These results suggested that BDV infection may cause direct damage in the developing brain by inhibiting the function of amphoterin due to binding by the p24 phosphoprotein.