Synthesis, tautomerism, and antimicrobial, anti-HCV, anti-SSPE, antioxidant, and antitumor activities of arylazobenzosuberones.

2-Dimethylaminomethylene-1-benzosuberone 1 was coupled with diazotized aniline derivatives to afford a series of the hitherto unreported 2-arylazo-1-benzosuberones 3a-i. The tautomeric structure and the effect of substituents on the tautomeric form (s) of the products 3a-i were discussed. Similar coupling of the enaminone 1 with diazonium salts of heterocyclic amines gave the respective fused azolotriazino-benzosuberones. Some of the newly synthesized compounds showed potent antimicrobial, anti-HCV, antioxidant, antitumor (as topoisomerase I inhibitors), and antimicrobial activities.

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.

Growth of defective subacute sclerosing panencephalitis viruses in human neural cells and their neurovirulence in mice.

A defective subacute sclerosing panencephalitis (SSPE) virus which had been passaged in human embryonic lung cells was transferred to cultures of three neural cell types: neuroblastoma, oligodendroglioma and glioblastoma. The growth characteristics of the virus in these cells were essentially similar to those in non-neural cells. On the other hand, a marked difference in neurovirulence was noticed for the virus grown in neural cells when examined by intracerebral inoculation into mice. The virus passaged in neuroblastoma and oligodendroglioma cells showed high neurovirulence, inducing an acute encephalitis, whereas the virus passaged in human embryonic lung cells and that in glioblastoma cells did not show neurovirulence. These results suggest that the virus recovered its neurovirulence after passage in certain human neural cells.

Growth of measles and subacute sclerosing panencephalitis viruses in human neural cell lines.

Growth of cell-free subacute sclerosing panencephalitis (SSPE) virus was compared with that of measles virus in three human neural cell lines; neuroblastoma, oligodendroglioma, and glioblastoma. The Edmonston strain of measles virus replicated in these neural cells as efficiently as in Vero cells. In contrast, the growth of the Mantooth strain of SSPE virus was suppressed moderately in neuroblastoma cells and markedly in oligodendroglioma and glioblastoma cells in spite of the induction of apparent cytopathic effects in these cells. Virus adsorption, defective interfering particles, interferon, and temperature sensitivity were not responsible for this low yield of SSPE virus in neural cell lines. Synthesis of viral proteins of SSPE virus was slower than that of measles virus in oligodendroglioma and glioblastoma cells. These results suggest that the slow rate of synthesis of viral proteins may be relevant to the low yield of SSPE virus in neural cells.

Persistent and lytic infections with SSPE virus: a comparison of the synthesis of virus-specific polypeptides.

The synthesis of virus-specific polypeptides and messenger RNA in cell cultures persistently infected with an isolate of measles virus from a patient with subacute sclerosing panencephalitis (SSPE) has been compared to that found in a lytic infection with the homologous virus. The persistent infection described here was chosen as its biological characteristics reflect those of virus-infected brain cells from SSPE patients. The synthesis of H, N and possibly F protein was seen in both lytic and persistent infections, but the synthesis of M protein was only detected in the lytic infection. However, messenger RNA isolated from either the lytic or persistent infection directed the synthesis in a cell-free translation system of all structural polypeptides, including M, and also three non-structural polypeptides, with mol. wt. of 34 000, 30 000 and 18 000. Messenger RNAs coding for the virus-specific polypeptides were also shown to be polyadenylated. In addition, those polypeptides made in vitro which were antigenically related to the haemagglutinin, demonstrated structural changes after passage through a persistent infection.

An interference phenomenon associated with a measles virus SSPE isolate (Halle).

A measles virus (Halle SSPE isolate) induced ring plaque phenomenon (concentric rings of living and dead cells) has been shown to be associated with the temperature-dependent production of interfering particles. The interfering particles have been purified by potassium tartrate linear gradient centrifugation and have buoyant densities of 1.15 g/ml (M particle) and 1.06 g/ml (L particle) respectively. Both interfering particle population have been shown to decrease the yield of wild-type measles virus from infected cells by 50% when co-infection experiments were performed. Neither the M or L particle population interfered with the growth of VSV in the same host-cell system.

Mode of subacute sclerosing panencephalitis (SSPE) virus infection in tissue culture cells. II. Cell-free viruses in cell cultures infected with Kitaken-1 and Biken strains of SSPE virus.

Cell-free infectious viruses were successfully recovered by the aid of freezing and thawing from cultures infected with the Kitaken-1 and Biken strains of subacute sclerosing panencephalitis (SSPE) virus. Our results including those in a previous report which dealt with the Niigata-1 strain of SSPE virus show that cell-free viruses can be detected from all of the SSPE virus-carrying cultures established in Japan. It was also found that cell-free infectious viruses can be recovered efficiently by dispersing the virus-carrying cultures with EDTA. The inclusion of trypsin in the EDTA solution, however, caused a poor recovery of the infectious viruses. Infection of cells with the cell-free viruses readily established the virus-carrying cultures that have characteristics comparable to those of their original cultures. The culture infected with the Kitaken-1 strain produced infectious viruses in about ten times the amount of the other two infected cultures. The buoyant densities of the cell-free infectious viruses were almost the same among the three strains, the values being 1.120 to 1.132, but significantly less than that of 1.164 of measles virus. The low density can be ascribed to one of the characteristics of these SSPE viruses.

Cyclic expression of antigen and infectious virus in a BHK cell line (0-853) persistently infected with an SSPE strain of measles virus.

Establishment and characteristics of a baby hamster kidney cell line (BHK 0-853) persistently infected with a subacute sclerosing panencephalitis (SSPE) strain of measles virus (Lec strain) is described. The persistent infection was easily and repeatedly established and no special conditions were required. There was a predictable fluctuation in expression of virus intracellular and membrane antigens which varied from greater than 90% to less than 1% of the cells demonstrating these antigens during the first 6 or 7 passages. Thereafter, fluctuation of antigen and infectious virus expression continued in an unpredictable fashion.

Impairment of hormone dependent signal transfer by chronic SSPE virus infection.

In a CNS-derived cell line, the cellular response to hormonal stimulation, represented by the rise of intracellular cAMP levels, is impaired under the influence of a persisting neurotropic virus infection. This dysfunction is caused by the decrease in adenylate cyclase activity, most probably due to the virus-induced loss of active catalytic units.

Mode of subacute sclerosing panencephalitis (SSPE) virus infection in tissue culture cells. I. Detection of infectious virions from cells infected with Niigata-1 strain of SSPE virus.

By the aid of freezing and thawing, cell-free infectious virions were detected from an apparently nonproductive Vero cell line infected with Niigata-1 strain of subacute sclerosing panencephalitis virus. The production of infectious virions was limited in amount and such virions were detectable only during a limited period after cell subculture. The infectious virions were filtrable through a 0.65 mu membrane filter and neutralized completely by an antiserum against measles virus. The virions were banded at the density of 1.132, while Edmonston strain of measles virus banded at 1.164 in potassium tartrate density gradients. Infectious virions were also released from infected Vero cells by treatment of the cells in a hypotonic solution to an amount comparable to that obtained by freezing and thawing. Infection of normal culture of Vero cells with the infectious virions readily established a virus-cell interaction identical to that in the original infected culture from which the virions were recovered.

In vitro inhibition of subacute sclerosing panencephalitis virus by the antiviral agent ribavirin.

The multiplication in tissue cultures of three isolates of subacute sclerosing panencephalitis virus as well as the Edmonston strain of measles virus was observed to be inhibited by various concentrations of the antiviral agent ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide). The blockage of viral replication by ribavirin could be reversed by the simultaneous addition of equimolar concentrations of either guanosine or adenosine. Expression of virus-specific antigens was greatly reduced in the presence of the antiviral drug.

Establishment and characterization of a subacute sclerosing panencephalitis (measles) virus persistent infection in BGM cells.

Infection of BGM cells with the Halle isolate of subacute sclerosing panencephalitis (SSPE) gave rise to a persistent infection (BGM/Halle), whereas infection of another African green monkey kidney cell line (Vero) under identical conditions led to a lytic infection. The BGM/Halle cells multiplied more slowly than the non-infected cells (even when the medium was changed daily). Under such conditions 10(7) to 10(8) p.f.u./ml/24 h of measles virus was released into the medium. It was established that the persistent infection was not due to the accumulation of thermosensitive mutants and that the virus was not modified as measured by several biological parameters. The virus released from BGM/Halle cells had, however, acquired an ability to give rise to a persistent infection in Vero cells. The quantity of virus released from persistently infected Vero cells was very low (10(2) to 10(3) p.f.u./ml). It was concluded that a host-cell factor plays a role in the restriction of virus replication.

Biological and biochemical characterization of a latent subacute sclerosing panencephalitis (SSPE) virus infection in tissue culture.

The present investigation describes the biological and biochemical properties of a persistent SSPE virus infection. Persistently infected cells were derived by cocultivation of infected brain cells and uninfected Vero cells, and cultures were maintained by normal subculturing methods. No infectious virus was ever released from these cultures, and all attempts to induce infectious virus release were unsuccessful. Biological assays showed that infected cells contained nucleocapsid and salt-dependent hemagglutinin antigens, whereas the normal hemagglutinin appeared not to be present. Electron microscopic examination demonstrated the presence of both intranuclear and cytoplasmic nucleocapsids together with the release of virus particles (defective?) from the cell membrane. Biochemical analysis demonstrated that approximately 90% of the intracellular genomic RNA was defective or subgenomic although a small quantity of infectious genomes was present. It is proposed that the large quantities of defective genomes in the infected cells are the major factor in the maintenance of this persistent infection.