Borna disease virus and the brain

Viruses with the ability to establish persistent infection in the central nervous system (CNS) can induce progressive neurologic disorders associated with diverse pathological manifestations. Clinical, epidemiological, and virological evidence supports the hypothesis that viruses contribute to human mental diseases whose etiology remains elusive. Therefore, the investigation of the mechanisms whereby viruses persist in the CNS and disturb normal brain function represents an area of research relevant to clinical and basic neurosciences. Borna disease virus (BDV) causes CNS disease in several vertebrate species characterized by behavioral abnormalities. Based on its unique features, BDV represents the prototype of a new virus family. BDV provides an important model for the investigation of the mechanisms and consequences of viral persistence in the CNS. The BDV paradigm is amenable to study virus-cell interactions in the CNS that can lead to neurodevelopmental abnormalities, immune-mediated damage, as well as alterations in cell differentiated functions that affect brain homeostasis. Moreover, seroepidemiological data and recent molecular studies indicate that BDV is associated with certain neuropsychiatric diseases. The potential role of BDV and of other yet to be uncovered BDV-related viruses in human mental health provides additional impetus for the investigation of this novel neurotropic infectious agent.     

Fine structure and morphogenesis of Borna disease virus

Borna disease virus (BDV), a negative nonsegmented single-stranded RNA virus, has not been fully characterized morphologically. Here we present what is to our knowledge the first data on the fine ultrastructure and morphogenesis of BDV. The supernatant of MDCK cells persistently infected with BDV treated with n-butyrate contained many virus-like particles and more BDV-specific RNA than that of untreated samples. The particles were spherical, enveloped, and approximately 130 nm in diameter; had spikes 7 nm in length; and reacted with BDV p40 antibody. A thin nucleocapsid, 4 nm in width, was present peripherally in contrast to the thick nucleocapsid of hemagglutinating virus of Japan. The BDV particles reproduced by budding on the cell surface.     

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.     

Evaluation of serological diagnosis of Borna disease virus infection using recombinant proteins in experimentally infected rats

We produced two recombinant Borna disease virus (BDV) proteins, p40 and p24, by using a baculovirus vector as a diagnostic antigen. Antigenicities of these recombinant proteins were evaluated by immune rabbit sera. Recombinant p40 was a more sensitive antigen than p24 for the detection of antibodies in infected rats. Rats inoculated with BDV within 24 hr after birth showed higher detection rates of viral RNA and viral proteins from the brain than rats inoculated at 4 weeks-old. Depending on the age of infection and the time postinfection, the detection of BDV RNA, protein, or anti-BDV antibody did not always correlate in individuals. We suggest both serological and molecular biological methods are needed in the diagnosis of BDV infection.     

Ion channels: structural basis for function and disease

Ion channels are ubiquitous proteins that mediate nervous and muscular function, rapid transmembrane signaling events, and ionic and fluid balance. The cloning of genes encoding ion channels has led to major strides in understanding the mechanistic basis for their function. These advances have shed light on the role of ion channels in normal physiology, clarified the molecular basis for an expanding number of diseases, and offered new direction to the development of rational therapeutic interventions.     

Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication initiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase alpha as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase alpha are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.     

Processing of the Borna disease virus glycoprotein gp94 by the subtilisin-like endoprotease furin

Open reading frame IV (ORF-IV) of Borna disease virus (BDV) encodes a protein with a calculated molecular mass of ca. 57 kDa (p57), which increases after N glycosylation to 94 kDa (gp94). The unglycosylated and glycosylated proteins are proteolytically cleaved by the subtilisin-like protease furin. Furin most likely recognizes one of three potential cleavage sites, namely, an arginine at position 249 of the ORF-IV gene product. The furin inhibitor decRVKRcmk decreases the production of infectious BDV significantly, indicating that proteolytic cleavage of the gp94 precursor molecule is necessary for the full biological activity of the BDV glycoprotein.     

Isolation of Borna disease virus from the autopsy brain of a schizophrenia patient]

Mineralization of aorta is known to occur late in life and appears to be a pathological phenomenon. In vitro studies revealed that the matrix prepared from the thoracic aorta pieces after their extraction with 3% Na2HPO4 and 0.1 mM CaCl2 were mineralized under physiological conditions of temperature, pH, and ionic strength of the media to form matrix-bound mineral phase resembling hydroxyapatite in nature. However, the matrix identically prepared from the unextracted rabbits aortae failed to mineralize under identical assay conditions. The addition of the aorta extract in the assay system inhibited the above mineralization process. Standard biochemical techniques, e.g., dialysis, ion exchange, and molecular sieve chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino acid analysis by high-performance liquid chromatography were employed to isolate, purify, and characterize the potent inhibitory biomolecules from the aorta extract. The inhibitory activity of the aorta extract was found to be primarily due to the presence of three biomolecules having molecular weights of 66, 45, and 27-29 kDa. The above inhibitory biomolecules loosely associated with aorta may be involved in the control of calcification associated with arteriosclerosis.     

Molecular basis of proteolytic activation of Sendai virus infection and the defensive compounds for infection

It has been proposed that the pathogenicity of Sendai virus is primarily determined by a host cellular protease(s) that activates viral infectivity by proteolytic cleavage of envelope fusion glycoproteins. We isolated a trypsin-like serine protease, tryptase Clara, localized in and secreted from Clara cells of the bronchial epithelium of rats. The enzyme specifically cleaved the precursor of fusion glycoprotein F0 of Sendai virus at residue Arg116 in the consensus cleavage motif, Gln(Glu)-X-Arg, resulting in the presentation of the membrane fusion domain in the amino-terminus of the F1 subunit. Administration of an antibody against tryptase Clara in the airway significantly inhibited the activation of progeny virus and multiple cycles of viral replication, thus reducing the mortality rate. These findings indicate that tryptase Clara in the airway is a primary determinant of Sendai virus infection and that proteolytic activation occurs extracellularly. We identified two cellular inhibitory compounds against tryptase Clara in bronchial lavage. One was a mucus protease inhibitor, a major serine protease inhibitor of granulocyte elastase in the lining fluids of the human respiratory tract, and the other was a pulmonary surfactant which may adsorb the enzyme, resulting in its inactivation. These compounds inhibited virus activation by tryptase Clara in vitro and in vivo, but did not themselves affect the hemagglutination and the infectivity of the virus. The functional domain of the mucus protease inhibitor against the enzyme, which is organized in two homologous N- and C-terminal domains, is located in the C-terminal. Administration of these compounds in the airway may be useful for preventing infection with Sendai virus.     

Molecular basis of changes in biological properties of foot and mouth disease virus of subtype A22

Primary structure of capsid proteins and RNA polymerase of three closely related strains of foot and mouth disease virus (FMDV), subtype A22, differing by biological properties (the initial epitheliotropic strain A22 550 and its derivatives: thermoresistant myotropic A22 550/4 and thermosensitive attenuated A22 645) are compared by nucleic acid sequencing and analysis of the amino acid sequencing. The study revealed 1 substitute in VPI and 8 in RNA polymerase in the myotropic variant and 1 substitute in VP2, 2 in VP3, 13 in VP1, and 3 in RNA polymerase. Alteration of A22 550/4 tropism is probably due to a single substitution Gly 145-->Thr in the RGD site of capsid protein VP1. Analysis of the origin and biological properties of the attenuated strain A22 645 and the results of studies of the primary structure of proteins permit us to hypothesize that attenuation is polygenic, caused by adaptation to a heterologous host (continuous porcine cell culture), and can be expressed by changes in the structure of virus antireceptor providing its binding to cell receptors. Sites responsible for the reproduction of A22 FMDV at certain temperatures are presumably located in RNA polymerase.     

Molecular cloning, expression and subcellular localization of a BiP homolog from rice endosperm tissue

The ER luminal binding protein, BiP, has been linked to prolamine protein body formation in rice. To obtain further information on the possible role of this chaperone in protein body formation we have cloned and sequenced a BiP cDNA homolog from rice endosperm. The rice sequence is very similar to the maize BiP exhibiting 92% nucleotide identity and 96% deduced amino acid sequence identity in the coding region. Substantial amino acid sequence homology exists between rice BiP and BiP homologs from several other plant and animal species including long stretches of conservation through the amino-terminal ATPase domain. Considerable variation, however, is observed within the putative carboxy-terminal peptide-binding domain between the plant and nonplant BiP sequences. A single hand of approximately 2.4 kb was visible when RNA gel blots of total RNA purified from seed tissue were probed with radiolabeled rice BiP cDNA. This band increased in intensity during seed development up to 10 days after flowering, and then decreased gradually until seed maturity. Protein gel blots indicated that BiP polypeptide accumulation parallels that of the prolamine polypeptides throughout seed development. Immunocytochemical analysis demonstrated that BiP is localized in a non-stochastic fashion in the endoplasmic reticulum membrane complex of developing endosperm cells. It is abundant on the periphery of the protein inclusion body but not in the central portion of the protein body or in the cisternal ER membranes connecting the protein bodies. These data support a model which proposes that BiP associates with the newly synthesized prolamine polypeptide to facilitate its folding and assembly into a protein inclusion body, and is then recycled.     

Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel alpha 1 subunits

To identify and localize the protein products of genes encoding distinct L-type calcium channels in central neurons, anti-peptide antibodies specific for the class C and class D alpha 1 subunits were produced. Anti-CNC1 directed against class C immunoprecipitated 75% of the L-type channels solubilized from rat cerebral cortex and hippocampus. Anti-CND1 directed against class D immunoprecipitated only 20% of the L-type calcium channels. Immunoblotting revealed two size forms of the class C L-type alpha 1 subunit, LC1 and LC2, and two size forms of the class D L-type alpha 1 subunit, LD1 and LD2. The larger isoforms had apparent molecular masses of approximately 200-210 kD while the smaller isoforms were 180-190 kD, as estimated from electrophoresis in gels polymerized from 5% acrylamide. Immunocytochemical studies using CNC1 and CND1 antibodies revealed that the alpha 1 subunits of both L-type calcium channel subtypes are localized mainly in neuronal cell bodies and proximal dendrites. Relatively dense labeling was observed at the base of major dendrites in many neurons. Staining in more distal dendritic regions was faint or undetectable with CND1, while a more significant level of staining of distal dendrites was observed with CNC1, particularly in the dentate gyrus and the CA2 and CA3 areas of the hippocampus. Class C calcium channels were concentrated in clusters, while class D calcium channels were generally distributed in the cell surface membrane of cell bodies and proximal dendrites. Our results demonstrate multiple size forms and differential localization of two subtypes of L-type calcium channels in the cell bodies and proximal dendrites of central neurons. The differential localization and multiple size forms may allow these two channel subtypes to participate in distinct aspects of electrical signal integration and intracellular calcium signaling in neuronal cell bodies. The preferential localization of these calcium channels in cell bodies and proximal dendrites implies their involvement in regulation of calcium-dependent functions occurring in those cellular compartments such as protein phosphorylation, enzyme activity, and gene expression.     

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.     

The HHQK domain of beta-amyloid provides a structural basis for the immunopathology of Alzheimer's disease

The beta-amyloid peptide 1-42 (Abeta1-42), a major component of neuritic and core plaques found in Alzheimer's disease, activates microglia to kill neurons. Selective modifications of amino acids near the N terminus of Abeta showed that residues 13-16, the HHQK domain, bind to microglial cells. This same cluster of basic amino acids is also known as a domain with high binding affinity for heparan sulfate. Both Abeta binding to microglia and Abeta induction of microglial killing of neurons were sensitive to heparitinase cleavage and to competition with heparan sulfate, suggesting membrane-associated heparan sulfate mediated plaque-microglia interactions through the HHQK domain. Importantly, small peptides containing HHQK inhibited Abeta1-42 cell binding as well as plaque induction of neurotoxicity in human microglia. In vivo experiments confirmed that the HHQK peptide reduces rat brain inflammation elicited after infusion of Abeta peptides or implantation of native plaque fragments. Strategies which exploit HHQK-like agents may offer a specific therapy to block plaque-induced microgliosis and, in this way, slow the neuronal loss and dementia of Alzheimer's disease.     

Intra-renal and subcellular distribution of the human chloride channel, CLC-5, reveals a pathophysiological basis for Dent's disease

Dent's disease, which is a renal tubular disorder characterized by low molecular weight proteinuria, hypercalciuria and nephrolithiasis, is associated with inactivating mutations of the X-linked chloride channel, CLC-5. However, the manner in which a functional loss of CLC-5 leads to such diverse renal abnormalities remains to be defined. In order to elucidate this, we performed studies to determine the segmental expression of CLC-5 in the human kidney and to define its intracellular distribution. We raised and characterized antisera against human CLC-5, and identified by immunoblotting an 83 kDa band corresponding to CLC-5 in human kidney cortex and medulla. Immunohistochemistry revealed CLC-5 expression in the epithelial cells lining the proximal tubules and the thick ascending limbs of Henle's loop, and in intercalated cells of the collecting ducts. Studies of subcellular human kidney fractions established that CLC-5 distribution was associated best with that of Rab4, which is a marker of recycling early endosomes. In addition, confocal microscopy studies using the proximal tubular cell model of opossum kidney cells, which endogenously expressed CLC-5, revealed that CLC-5 co-localized with the albumin-containing endocytic vesicles that form part of the receptor-mediated endocytic pathway. Thus, CLC-5 is expressed at multiple sites in the human nephron and is likely to have a role in the receptor-mediated endocytic pathway. Furthermore, the functional loss of CLC-5 in the proximal tubules and the thick ascending limbs provides an explanation for the occurrences of low molecular weight proteinuria and hypercalciuria, respectively. These results help to elucidate further the patho-physiological basis of the renal tubular defects of Dent's disease.