Peripheral expression of ocular antigens in regulation and therapy of ocular autoimmunity.

The retina is a well-known immune-privileged tissue in the eye. Gene therapy and transgenic strategies have been taken to explore the relationship between the immune system and retinal antigens. Retroviruses were used to express retina-specific antigens or fragments systemically, leading to an antigen-specific loss of susceptibility to autoimmune disease. Transgenic strategies used a neo self-antigen, beta-galactosidase, or a known retinal antigen, interphotoreceptor retinoid-binding protein, to show that immune recognition of antigen by mice, which express solely in the retina, is not detectably different than that of mice that don't express this antigen. Together, these studies show that antigens expressed solely in the retina do not appear to be seen by the immune system, demonstrating that sequestration contributes to the lack of antigen recognition and absence of tolerance. Provision of these antigens outside of the retina provides the opportunity for development of peripheral tolerance, protection from autoimmunity, and potential therapies.

Site-directed mutagenesis and X-ray crystallography of the PQQ-containing quinoprotein methanol dehydrogenase and its electron acceptor, cytochrome c(L).

Two proteins specifically involved in methanol oxidation in the methylotrophic bacterium Methylobacterium extorquens have been modified by site-directed mutagenesis. Mutation of the proposed active site base (Asp303) to glutamate in methanol dehydrogenase (MDH) gave an active enzyme (D303E-MDH) with a greatly reduced affinity for substrate and with a lower activation energy. Results of kinetic and deuterium isotope studies showed that the essential mechanism in the mutant protein was unchanged, and that the step requiring activation by ammonia remained rate limiting. No spectrally detectable intermediates could be observed during the reaction. The X-ray structure, determined to 3 A resolution, of D303E-MDH showed that the position and coordination geometry of the Ca2+ ion in the active site was altered; the larger Glu303 side chain was coordinated to the Ca2+ ion and also hydrogen bonded to the O5 atom of pyrroloquinoline quinone (PQQ). The properties and structure of the D303E-MDH are consistent with the previous proposal that the reaction in MDH is initiated by proton abstraction involving Asp303, and that the mechanism involves a direct hydride transfer reaction. Mutation of the two adjacent cysteine residues that make up the novel disulfide ring in the active site of MDH led to an inactive enzyme, confirming the essential role of this remarkable ring structure. Mutations of cytochrome c(L), which is the electron acceptor from MDH was used to identify Met109 as the sixth ligand to the heme.

The nucleoporin Nup98 associates with the intranuclear filamentous protein network of TPR.

The Nup98 gene codes for several alternatively spliced protein precursors. Two in vitro translated and autoproteolytically cleaved precursors yielded heterodimers of Nup98-6kDa peptide and Nup98-Nup96. TPR (translocated promoter region) is a protein that forms filamentous structures extending from nuclear pore complexes (NPCs) to intranuclear sites. We found that in vitro translated TPR bound to in vitro translated Nup98 and, via Nup98, to Nup96. Double-immunofluorescence microscopy with antibodies to TPR and Nup98 showed colocalization. In confocal sections the nucleolus itself was only weakly stained but there was intensive perinucleolar staining. Striking spike-like structures emanated from this perinucleolar ring and attenuated into thinner structures as they extended to the nuclear periphery. This characteristic staining pattern of the TPR network was considerably enhanced when a myc-tagged pyruvate kinase-6kDa fusion protein was overexpressed in HeLa cells. Double-immunoelectron microscopy of these cells using anti-myc and anti-TPR antibodies and secondary gold-coupled antibodies yielded row-like arrangements of gold particles. Taken together, the immunolocalization data support previous electron microscopical data, suggesting that TPR forms filaments that extend from the NPC to the nucleolus. We discuss the possible implications of the association of Nup98 with this intranuclear TPR network for an intranuclear phase of transport.

Evidence that the hypermutated M protein of a subacute sclerosing panencephalitis measles virus actively contributes to the chronic progressive CNS disease.

Subacute sclerosing panencephalitis (SSPE) is a progressive degenerative disease of the brain uniformly leading to death. Although caused by measles virus (MV), the virus recovered from patients with SSPE differs from wild-type MV; biologically SSPE virus is defective and its genome displays a variety of mutations among which biased replacements of many uridine by cytidine resides primarily in the matrix (M) gene. To address the question of whether the SSPE MVs with M mutations are passive in that they are not infectious, cannot spread within the CNS, and basically represent an end-stage result of a progressive infection or alternatively SSPE viruses are infectious, and their mutations enable them to persist and thereby cause a prolonged neurodegenerative disease, we utilized reverse genetics to generate an infectious virus in which the M gene of MV was replaced with the M gene of Biken strain SSPE MV and inoculated the recombinant virus into transgenic mice bearing the MV receptor. Our results indicate that despite biased hypermutations in the M gene, the virus is infectious in vivo and produces a protracted progressive infection with death occurring as long as 30 to 50 days after that caused by MV. In primary neuron cultures, the mutated M protein is not essential for MV replication, prevents colocalization of the viral N with membrane glycoproteins, and is associated with accumulation of nucleocapsids in cells' cytoplasm and nucleus.

Measles virus infection in a transgenic model: virus-induced immunosuppression and central nervous system disease.

Measles virus (MV) infects 40 million persons and kills one million per year primarily by suppressing the immune system and afflicting the central nervous system (CNS). The lack of a suitable small animal model has impeded progress of understanding how MV causes disease and the development of novel therapies and improved vaccines. We tested a transgenic mouse line in which expression of the MV receptor CD46 closely mimicked the location and amount of CD46 found in humans. Virus replicated in and was recovered from these animals' immune systems and was associated with suppression of humoral and cellular immune responses. Infectious virus was recovered from the CNS, replicated primarily in neurons, and spread to distal sites presumably by fast axonal transport. Thus, a small animal model is available for analysis of MV pathogenesis.

The molecular structure of an unusual cytochrome c2 determined at 2.0 A; the cytochrome cH from Methylobacterium extorquens.

Cytochrome cH is the electron donor to the oxidase in methylotrophic bacteria. Its amino acid sequence suggests that it is a typical Class 1 cytochrome c, but some features of the sequence indicated that its structure might be of special interest. The structure of oxidized cytochrome cH has been solved to 2.0 A resolution by X-ray diffraction. It has the classical tertiary structure of the Class 1 cytochromes c but bears a closer gross resemblance to mitochondrial cytochrome c than to the bacterial cytochrome c2. The left-hand side of the haem cleft is unique; in particular, it is highly hydrophobic, the usual water is absent, and the "conserved" Tyr67 is replaced by tryptophan. A number of features of the structure demonstrate that the usual hydrogen bonding network involving water in the haem channel is not essential and that other mechanisms may exist for modulation of redox potentials in this cytochrome.

Regulation of the initiation of coronavirus JHM infection in primary oligodendrocytes and L-2 fibroblasts.

Upon maturation, primary rat oligodendrocytes become resistant to coronavirus JHM (JHMV) infection at an early stage. Involvement of cAMP-dependent protein kinase (PK) in the regulation of oligodendrocyte differentiation has been established (S. Beushausen et al. (1987). J. Virol. 61, 3795-3803). An inducer which accelerates maturation, dibutyryl cyclic AMP (dbcAMP) also upregulates the expression of the regulatory subunit, R1 of PK1. Since (i) early block preventing infection of mature oligodendrocytes can be bypassed when transfection with genomic RNA is used and (ii) inhibitors of PKs counteract the dbcAMP effect, so as to alleviate the inhibition of JHMV, enhanced expression of R1 appeared to be connected with virus restriction. This idea was confirmed following upregulation of the R1 gene in fully permissive L-2 cells. There was a connection between an effect due to R1 and dephosphorylation of the nucleocapsid protein N by an endosomal phosphoprotein phosphatase (PPPase) having the properties of types 1 or 2A enzyme which occurs during penetration of inoculum virions. An inhibition in vitro (cell free) of N dephosphorylation by R1 together with evidence that in vivo (cell culture) overexpression of R1 inhibited the endosomal PPPase as well as replication of JHMV supports the hypothesis that uncoating of the JHMV inoculum occurs after dephosphorylation, a step obligatory for dissociation of the N protein from the genome. Thus inhibition by R prevents uncoating and thereby interferes with the commencement of replication. These observations intimate the existence of a novel mechanism controlling a virus infection of specific cell target(s) undergoing a process of differentiation and maturation in the central nervous system.

Reconstitution of the quinoprotein methanol dehydrogenase from inactive Ca(2+)-free enzyme with Ca2+, Sr2+ or Ba2+.

The reconstitution of active holoenzyme containing calcium from inactive calcium-free methanol dehydrogenase, isolated from a moxA mutant of Methylobacterium extorquens, has a pH optimum of about pH 10, with a well defined pK for the process at pH 9.3. Two Ca2+ ions were irreversibly incorporated per alpha 2 beta 2 tetramer. Calcium could be replaced in the incorporation process by strontium or barium, the affinities for these ions being similar to that for Ca2+. Arrhenius plots for measurement of the activation energy of reconstitution were biphasic; the lower activation energy was typical of most biological processes, while the higher activation energy was at least three times greater, implying the involvement of a large conformational change during incorporation of the cations. The activation energy for incorporation of Ba2+ was considerably higher than that for incorporation of Ca2+. The novel disulphide bridge that is at the active site of the enzyme was not involved in the incorporation process. Studies of the time courses for incorporation of 45Ca2+, production of active enzyme and changes in absorption spectra failed to show any intermediates in the incorporation process.

The murine coronavirus as a model of trafficking and assembly of viral proteins in neural tissue.

The replication of JHM, a murine coronavirus, provides a useful model of the assembly and dissemination of viral components in neuronal cells. Involvement of microtubules in virus trafficking is an important feature which may explain dissemination of the infection from primary cell targets at olfactory, hippocampal and cerebellar sites within the central nervous system, resulting in severe neuropathies.

Involvement of spicules in the formation of vaccinia virus envelopes elucidated by a conditional lethal mutant.

The envelope of immature vaccinia virions consists of a lipoprotein bilayer upon which a precise curvature is imposed by acquisition of an external scaffold of spicules. Self-assembly of this tegument was examined employing our ts 6757 mutant, which induces accumulation of immature envelopes at the restrictive temperature. With ts 6757 the envelope bilayers were also assembled into an alternative membrane configuration in the form of flexible cylinders or tubes of uniform width, lacking the spicule coat. Such tubes became extensions of or were continuous with the spherical virion envelopes. The approximately 65 kDa spicule protein, L65, product of gene D13L on the HindIII map, generally designated as a late protein, was expressed as an early function in presence of hydroxyurea, an inhibitor which entirely blocked vaccinia DNA synthesis without stopping assembly of immature envelopes. Labeling of thin sections by immunogold for electron microscopy demonstrated that L65 is present at the surface of immature virions, consistent with the position of spicules on envelopes. Transiency of the spicule scaffold was documented by (a) absence of L65 from intracellular mature virions (IMV) and (b) rapid turnover of L65 during ts 6757 virus replication at the permissive temperature but conservation of this protein at restrictive temperature, as demonstrated in pulse-chase experiments. Time-related decrease in MW of L65 to a smaller polypeptide is interpreted as evidence suggesting that the spicules attached to the envelope are assembled from a higher MW precursor.

Organization of the vaccinia envelope and relationship to the structure of intracellular mature virions.

Protrusions covering the surface of intracellular mature virions (IMV), termed by us surface tubular elements (STE), are released in a quasi-intact form during stripping of the envelope. The concentrated, reproducibly isolable STE were shown to contain the 58-kDa 4c polypeptide and prominent protein antigens residing at the surface of IMV. The major core protein 4b, identified as a minor contaminant of STE, presumably became detached along with STE during the shearing off process. Antibodies against protein 4b became specifically bound to the surface of isolated cores, where a palisade layer of rodlets occurs. The same antibodies absorbed onto isolated STE where similar rodlets were evident. Based on the new observations we constructed a model of the organization of the IMV envelope and its relationship to the core.

The interaction of methanol dehydrogenase and its cytochrome electron acceptor.

A fluorescence method is described for direct measurement of the interaction between methanol dehydrogenase (MDH) and its electron acceptor cytochrome cL. This has permitted a distinction to be made between factors affecting electron transfer and those affecting the initial binding or docking process. It was confirmed that the initial interaction is electrostatic, but previous conclusions with respect to the mechanism of EDTA inhibition have been modified. It is proposed that the initial 'docking' of MDH and cytochrome cL is by way of ionic interactions between lysyl residues on its surface and carboxylate groups on the surface of cytochrome cL. This interaction is not inhibited by EDTA, which we suggest acts by binding to nearby lysyl residues, thus preventing movement of the 'docked' cytochrome to its optimal position for electron transfer, which probably involves interaction with the hydrophobic funnel in the surface of MDH.

Factors controlling coronavirus infections and disease of the central nervous system. A review.

There is a correlation between specificity of tropism of JHMV for O-2A lineage cells from the rat and demyelination of white matter, associated with chronic disease. Susceptibility to infection, which can occur in O-2A cells before terminal differentiation may be influenced by cytokines. During the normal, age-related or rapidly induced maturation/differentiation of rat oligodendrocytes, suppression of JHMV replication is correlated with upregulation of the subunit R1 of the cAMP-dependent protein kinase. Virus inhibition occurs at a stage between penetration and initiation of genome expression. Regulation over coronavirus infection of oligodendroglia is strictly controlled by the host cell. There is evidence that induction of R1 subunit of protein kinase A influences uncoating, illustrated in Figure 9, by suppression of dephosphorylation during penetration. Our former working hypothesis, now borne out by recent data predicts that the infection in mature oligodendrocytes is blocked because specific dephosphorylation of the capsid protein N, required for uncoating, etc., is suppressed.

Mapping of functional domains for HIV-2 gag assembly into virus-like particles.

The human immunodeficiency virus type 2 gag precursor protein, pr41, self assembles as virus-like particles (VLP) when the gag gene is expressed in insect cells. To map the functional domains for HIV-2 gag VLP formation, a series of deletion mutants was constructed by removing sequentially the C-terminal region of HIV-2 gag precursor protein and expressing the truncated gag genes in SF9 insect cells by means of recombinant baculoviruses. We found that deletion of up to 143 amino acids at the C-terminus of HIV-2 gag, leaving 376 amino acids at the N-terminus of the protein, did not prevent VLP formation. However, an additional four amino acids deletion from the C-terminus, which represents 372 amino acids at the N-terminus, made gag protein fail to form VLP. There is a proline-rich region at amino acid positions 372 and 377 of HIV-2 gag. To analyze the role of these proline residues, we generated five mutants in which proline was changed sequentially into leucine. Our results showed that replacement of one or two prolines did not stop gag VLP formation, whereas replacement of all three prolines by leucine residues completely abolished VLP assembly. Our data demonstrate that the C-terminal p12 region of HIV-2 gag precursor protein and the zinc finger domain are dispensable for gag VLP assembly, but the presence of at least one of the three proline residues located between amino acid positions 372 and 377 of HIV-2NIH-Z is required.

Lewis rat T cells can reutilize, process, and present myelin basic protein to antigen-specific T cell lines.

MHC class-II-negative astrocytes prevented from intracellular antigen (Ag) processing induce myelin basic protein (MBP)-specific short-term T cell lines to proliferate. This process results from the ability of the T cells themselves to take up, process, and present Ag to each other. The Ag-presenting function of the T cells occurred in the absence of any conventional antigen-presenting cell (APC), was independent of their T cell receptor specificity, was sensitive to chloroquine, and was prevented by anti-class-II MHC antibody. Both native and HPLC-purified MBP were effective in stimulating T cell lines, and there was no obvious benefit in using either enzymatically digested or synthetic peptide preparations of the Ag. Furthermore, the Ag-presenting T cells could take up, reutilize, and re-present Ag adsorbed to the surface of histoincompatible astrocytes. Responding T cells activated by Ag-presenting T cells in the absence of other conventional APC were fully encephalitogenic upon transfer to syngeneic recipients. These results have relevance for understanding pathogenetic mechanisms in T cell-mediated autoimmunity in the central nervous system.

Distribution and trafficking of JHM coronavirus structural proteins and virions in primary neurons and the OBL-21 neuronal cell line.

The neurotropic murine coronavirus JHM is capable of inducing various forms of neurologic diseases, including demyelination. Neurons have been shown to act as a repository site at the early stages of the disease process (O. Sorensen and S. Dales, J. Virol. 56:434-438, 1985). JHM virus (JHMV) replication and trafficking of viral proteins and virions in cultured rat hippocampal neurons and a neuronal cell line, OBL-21, were examined, with an emphasis placed on the role of the microtubular network. We show here that JHMV spread within the central nervous system occurs transneuronally and that virus protein trafficking was dependent upon microtubules. Viral trafficking occurred asymmetrically, involving both the somatodendritic and the axonal domains. Thus coronavirus can be disseminated from neurons at either the basolateral or the apical domains. A specific interaction between antibodies derived against the microtubule-associated protein tau and JHMV nucleocapsid protein (N) was observed, which can presumably be explained by an overall amino acid similarity of 44% and an identity of 20% between proteins N and tau, with optimal alignment at the microtubule binding domain of tau. Collectively, our data suggest an important role of the microtubule network in viral protein trafficking and distribution. They also draw attention to protein sequence mimicry of a cell component by this coronavirus as one strategy for making use of the host's functions on behalf of the virus.

Vaccinia virion surface polypeptide Ag35 expressed from a baculovirus vector is targeted to analogous poxvirus and insect virus components.

Polypeptide Ag35, a major early component of the vaccinia surface, is integrated into the formative viral lipoprotein tegument. To ascertain whether positioning of Ag35 is due to its general affinity for newly assembled viral membranes we created a recombinant A12 vector to express the vaccinia protein. The baculovirus system was chosen because intranuclear virions of this agent are likewise enclosed inside newly formed envelopes. Comparable infections of two insect cell lines established that more abundant synthesis occurred in High Five (H5) than in SF9 cells. We, therefore, used H5 cells for most experiments reported here. Combined analyses by PAGE, Western blotting, and immunocytology, using light and electron microscopy, revealed a dissemination of Ag35 throughout the cell. Higher concentrations were evident at the cell surface, nuclear perimeter, and within intranuclear virogenic stroma. The association with the virogenic stroma was of specific interest with respect to vaccinia development because it showed a similarity in the targeting of Ag35 toward intranuclear DNA-protein foci of baculovirus which are analogous to the vaccinia-specified cytoplasmic "factories." A further remarkable analogy concerns association of Ag35 with intranuclear baculovirus envelopes, revealing a propensity of Ag35 for nascent viral lipoprotein membranes.