Localization at high resolution of antibody-induced mobilization of vaccinia virus hemagglutinin and the major histocompatibility antigens on the plasma membrane of infected cells.

We examined the consequence of simultaneous or independent binding of monospecific antibody to the hemagglutinin (HA) of vaccinia virus and the A-, B- and -determinants of HLA on HeLa or Raji cells or KkDk determinants of H-2 on L929 cells. The bound antibodies were marked by goat-anti-mouse (GAM) or goat-anti-rabbit (GAR) fluorochrome conjugates suitable for light microscopy and GAM or GAR gold conjugates, used in electron microscopy. Specificity and amount of antibody adsorbed was ascertained by complement-mediated lysis of 51Cr-labeled cells and by fluorescence-activated cell sorter analysis. Regardless of the order of either antibody to major histocompatibility complex (MHC) or antibody to HA addition after warming to 37 degrees C, there was evidence by light microscopy for co-patching and co-capping of the viral and host antigens. Electron microscopic examination revealed that goat-anti-rabbit 20 nM gold conjugate and goat-anti-mouse 5 nM gold conjugate, marking respectively the HA and MHC molecules, became concentrated in patched or caps in which the two antigens frequently overlapped or were closely associated. The contiguous MHC and HA antigens were also engulfed, as evidenced from the of two sizes of gold particles inside endocytic vacuoles. The significance of these observations is discussed in relation to the cytotoxic T lymphocyte-mediated killing virus-infected targets.

The penetration of reovirus RNA and initiation of its genetic function in L-strain fibroblasts.

Reovirus type 3 is phagocytized by L cells and rapidly sequestered inside lysosomes. Hydrolases within these organelles are capable of stripping the viral coat proteins, but they fail to degrade the double-stranded RNA genome. These observations support the view that sojourn of reovirus in lysosomes, when the lytic enzymes uncoat its genome, is an obligatory step in the sequence of infection. Although the mechanism for transferring the uncoated RNA out of lysosomes remains to be elucidated, evidence is presented suggesting that progeny genomes are bound to site(s) possessing the fine structure of viral inclusions or factories. It appears that both the synthesis of single- and double-stranded viral RNA and the morphogenesis of progeny virus particles occur in such factories.

Membrane synthesis in Bacillus subtilis. 3. The morphological localization of the sites of membrane synthesis.

The results of several lines of investigation indicate that membrane growth in Bacillus subtilis does not occur at one or a small number of discrete zones. No indications of large regions of membrane conservation were observed. Kinetic labeling experiments of mesosomal and plasma membrane lipids indicate that the mesosomal lipids are not precursors of the plasma membrane lipids. Density shift experiments, in which the changes in buoyant density of membranes were studied after growth in deuterated media, showed no indication of large zones of conservation during membrane growth. Radioautography of thin sections of cells pulse labeled with tritiated glycerol showed no indication of specific zones of lipid synthesis. The consequences of these results for models of cell growth and division are discussed.

Evidence for RNA linked to nascent DNA in HeLa cells.

Rapidly labeled, i.e., nascent, DNA from HeLa cells was separated from the bulk DNA by ultracentrifugation. Further characterization of the rapidly labeled component revealed that its sedimentation coefficient is in the range of 4S and that it exists in a single- and double-stranded conformation. Moreover, analysis by nitrocellulose chromatography and CsSO4 density sedimentation of the nascent DNA labeled with 3H-uridine revealed that it is covalently linked to short chains of RNA, indicating that in HeLa cells RNA primer is involved in DNA replication.

The fine structure and immunological labeling of the achromatic mitotic apparatus after disruption of cell membranes.

After treatment of HeLa and L cells with vinblastine sulfate the material of microtubules (tubulin) was reorganized into (a) large paracrystals (PC) of tightly packed tubules; (b) smaller aggregates of tubules with greater diameter whose walls are constituted from well defined, helically arranged morphological subunits; and (c) microtubules associated with helices of polyribosomes of uniform size. All of these structures survived disruption of cellular membranes by means of a nonionic detergent. Following a thorough stripping of membranes there remained a subcellular fraction sedimenting at 1,500 g for 15 min, in which were contained nuclei, centrioles, and the above mentioned microtubular elements, maintained as a complex of organelles by an interconnecting network of 80 A microfibrils. As a result of membrane disruption it was possible to localize precisely in the electron microscope the binding of ferritin antibody conjugates. Specific labeling at the surface of PC and microtubule aggregates could be demonstrated. This result was substantiated by means of the immunoperoxidase method of labeling the PC. A concentrated deposit of ferritin was also found in the vicinity of centrioles and related structures, the annuli of the nuclear pore complex and the annulate lamellae. However, the specificity of the label on these organelles remains questionable because ferritin, albeit in lower concentration, was also present on them in control preparations reacted with preimmune sera.

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.

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.

Endosomal association of a protein phosphatase with high dephosphorylating activity against a coronavirus nucleocapsid protein.

On the assumption that dephosphorylation of the neurotropic coronavirus JHM (JHMV) nucleocapsid protein (N) may be connected with initiation of the infectious cycle we searched for a relevant host enzyme activity. Analysis of subcellular fractions from L-2 murine fibroblasts, separated by dual Percoll density gradients, revealed the presence of a phosphoprotein phosphatase (PPPase), co-sedimenting with the endososomal/prelysosomal material, which possesses high activity against N. With purified [32P]N as substrate it was demonstrated that this PPPase, distinguishable from acid and alkaline phosphatases, acts optimally at neutral pH in the presence of Mn2+ following treatment with a detergent. Complete inhibition with okadaic acid at 0.9-4.5 microM but not at 1-10 nM relegates this PPPase to a type 1 protein phosphatase. Similar PPPase activity for N was present in the endosome fraction of a rat Roc-1 astrocytoma-oligodendrocyte cell line and in homogenates of brain and cultured oligodendrocytes. Our data suggest that the phosphorylated N of the inoculum may be modified by the endosomal PPPase in host cells, including those from the CNS so as to facilitate the JHMV infectious process.

In vivo and in vitro models of demyelinating diseases. III. JHM virus infection of rats.

Suckling rats of three inbred and three outbred strains were inoculated intraperitoneally (P) or intracerebrally (IC) with the JHM strain of mouse hepatitis virus (JHMV) and were monitored for evidence of neurologic diseases. Consequences of varying age at inoculation, route of injection, and virus dose were ascertained. No disease was evident after IP injection but IC inoculation with at least 10(4) plaque-forming units at 2 days of age resulted in either a rapidly fatal encephalitis or a chronic, progressive, fatal neurologic disease in most rats, regardless of strain. Inoculation at 5 or 10 days of age predominantly caused the chronic neurologic disease, characterized by demyelinating lesions in the brain, spinal cord, or optic nerve, which sometimes were evident as late as several months postinoculation. Demyelination in the optic nerve proved to be concurrent with demyelinating lesions elsewhere in the CNS. Occasionally, clinical remissions were observed in rats in which posterior paralysis developed, suggesting that remyelination in the rat can occur. Demonstration of virus replication, by infectivity, in rats exhibiting neurologic disease and in rats without clinical symptoms was substantiated by electron microscopic observations of virus development and assembly in oligodendroglia of the optic nerve and spinal cord. In view of the protracted course of the disease in some rats, presence of demyelinating lesions confirmed by light and electron microscopy, and remissions of clinical symptoms, the JHMV-infected rat seems to be an appropriate animal model to study virus-mediated progressive demyelinating disease.

Probing for the human coronavirus OC43 in multiple sclerosis.

Tissues acquired at autopsy from four patients with MS and biopsies from one patient with a probable diagnosis of MS were probed for the presence of OC43 RNA. This human coronavirus was not detected.

Influence of RNA polymerase II upon vaccinia virus-related translation examined by means of alpha-amanitin.

Our previous studies employing alpha-amanitin-sensitive H-9 and resistant Ama 102 mutant host cells demonstrated that polymerase II (Pol II), or a drug-sensitive component of the enzyme, is required for replication of vaccinia virus. Evidence was also obtained indicating that transcription from the host genome does not appear to be involved (Silver et al., 1979; Silver and Dales, 1982), suggesting a possible role for Pol II in transcription from the viral genome. This idea is consistent with the present findings, based on immunofluorescence analysis, which revealed that upon infection Pol II antigen is mobilized out of the nucleus into discrete cytoplasmic foci. Effects of treating H-9 rat myoblasts with alpha-amanitin upon vaccinia-specific protein synthesis were also examined. Under the experimental conditions employed, the toxin drastically curtailed in vivo translation into early, late and late-late proteins without altering the spectrum of polypeptides produced. By contrast, treatment with the drug affected, only minimally, the rate of transcription into viral RNA, whether in vivo or from isolated vaccinia factories. The mRNA isolated from infected and treated or untreated cells was translated in a reticulocyte lysate with equal efficiency and general fidelity. This finding suggests that Pol II may be involved in transcription into RNAs related to factors controlling the in vivo translation process. The possible mechanisms for exercising such controls are discussed in relation to factors regulating transcription by host RNA polymerases from a viral DNA genome.

In vivo and in vitro models of demyelinating diseases. XII. Persistence and expression of corona JHM virus functions in RN2-2 Schwannoma cells during latency.

The coronavirus JHMV persistently infects rat Schwannoma cells RN2-2 at 32.5 degrees C and enters a host-imposed reversible, latent state at 39.5 degrees C. JHMV can remain up to 20 days in the latent state and about 14 days before the cultures lose the capacity to resume virus production upon return to 32.5 degrees C. Although persistently and latently infected RN2-2 cells display resistance to superinfection by a heterologous agent VSV, these cells do not release detectable soluble mediators (e.g., interferon) of the antiviral state. Nevertheless, RN2-2 cells are competent to synthesize and release interferon when treated with the appropriate inducers. These observations suggest that interferon does not play any role or may not be the major factor in the control of latency in the Schwannoma cell. Hybridization with virus-specific cDNAs shows that all viral mRNAs are present during latency and that viral mRNAs are present in the polysomes of infected cells at 39.5 degrees C. Western immunoblotting with hybridoma antibodies demonstrates that viral specific proteins are produced at the restrictive temperature. These results suggest that despite the absence of production of infectious virus at 39.5 degrees C, there is active transcription and translation into virus-specified products.

In vivo and in vitro models of demyelinating disease X. A Schwannoma-L-2 somatic cell hybrid persistently yielding high titres of mouse hepatitis virus strain JHM.

Following infection of RN2 rat Schwannoma cells with unfiltered JHMV inocula, a cell line with an altered phenotype evolved, which was shown to be a somatic cell hybrid of RN2 and mouse L-2 cells. This cell line, EJ, persistently yields JHMV at titres greater than 10(6) pfu/ml and does not show the suppression of virus production at 39.5 degrees C that is characteristic of a persistently infected RN2 line. Intracellular viral nucleocapsids are demonstrated. Cloning of EJ hybrids yields cell lines that show a variety of responses to infection by JHMV or MHV3.

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.

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.

In vivo and in vitro models of demyelinating disease: a phosphoprotein phosphatase in host cell endosomes dephosphorylating the nucleocapsid protein of coronavirus JHM.

We have identified a phosphoprotein phosphatase which dephosphorylates efficiently the NC protein of coronavirus JHM. The activity was found in L-2 murine fibroblasts, Wistar Furth rat neonatal brain extracts, Wistar Furth rat oligodendrocyte primary cells and in Roc-1 cells, an oligodendrocytic hybrid cell line. In both L-2 cells and Roc-1 cells the enzyme was found to be localized predominantly in the endosomal fraction. The enzyme is optimally active at pH 7.0 and has a requirement for Mn++ ions. This PPPase activity is distinguishable from acidic and alkaline phosphatases. In view of the specificity of the endosomal PPPase for the phosphory-lated NC protein it is hypothesized that this enzyme may have a function during early stages of coronavirus infection.

In vivo and in vitro models of demyelinating diseases, XX: Replication of coronaviruses in primary neural cultures from genetically resistant and susceptible mice.

Resistance of SJL mice to JHMV could be associated with explanted oligodendrocytes and astrocytes in primary neural cultures from newborn mice. The restriction demonstrated is not at the stage of adsorption, uptake by the host or RNA synthesis and does not occur with the serorelated MHV3 strain. Experiments with neural cells from F1 hybrid mice bred from resistant and sensitive parents show that resistance is a recessive trait. It is hypothesized that the defect may involve proteolysis at the cell surface, but to date no clear cut experimental data have been obtained to substantiate this idea. In more general terms our in-vitro observations demonstrate that resistance of SJL mice pertains to both embryonic and newborn animals and involves neural and non neural cell types. Thus, the SJL restriction of JHMV, evident at the cellular level, does not involve an age-related maturation process associated with rat and mouse oligodendrocytes studied previously (1, 14).

In vivo and in vitro models of demyelinating disease, XXIII: Infection by JHM virus of athymic rats.

Wistar Lewis (WL), Long Evans (LE) and other rat strains develop complete resistance to CNS disease when inoculated intracerebrally with 5 x 10(4) PFU/ml of murine hepatitis JHM virus (JHMV) after the 10th day of age (1). Immunosuppression of WL rats following onset of the age-related resistance demonstrated that cyclosporin A (CsA) was partially able to abrogate resistance. Studies on nude (rnu/rnu) rats, their heterozygous (rnu/+) litter mates and genetically related LE rats showed that rnu/+ and LE animals became completely resistant to JHMV before the age of weaning, whereas some rnu/rnu rats, challenged as late as 70 days of age, developed disease symptoms, albeit after a long latent period. These observations indicated that the cellular immune system plays an important role in suppressing the viral disease process in the CNS. When the infection of nude rats was initiated on or after the 15th day of life, the histological lesions were generally small and present in both grey and white matter but were seldom seen in the spinal cord. By contrast in rnu/+, LE and WL rats with late-onset disease symptoms, only the demyelinating-type white matter lesions were present. Mononuclear infiltrates, evident throughout the CNS, of nude rats were sometimes massive near the meningies and within ventricular spaces. JHMV RNA was detectable by dot-blotting analysis in the CNS of both paralysed and asymptomatic rnu/rnu and rnu/+ rats. In-situ hybridization with cDNA probes for JHMV RNA showed that neurons in the hippocampus and cerebellum, as well as cells in the white matter, were frequently infected. The present data indicate that in the rat T cells have an important function in maintaining resistance to the JHMV-related CNS disease. However, even without a functional T cell compartment after 15 days of age nude rats did not develop an acute encephalitis, suggesting that an age-dependent, non-immunological mechanism is also involved in restricting the spread of infection.