Meihan Nonoyama

Inhibition of herpes simplex virus infection by tannins and related compounds

Several chemically defined plant extracts were investigated for their antiviral action on herpes simplex virus (HSV-1, HSV-2)-infected African green monkey kidney cells and human adenocarcinoma cells, using a plaque formation assay. Among them, the monomeric hydrolyzable tannins, oligomeric ellagitannins and condensed tannins, having galloyl groups or hexahydroxydiphenoyl groups, had the most potent anti-HSV activity. Their 50% effective doses (0.03-0.1 microgram/ml) were by two-three orders of magnitude lower than their 50% cytotoxic doses (greater than 10 micrograms/ml). On the other hand, gallic acid, neutral polysaccharides, chemically modified (N,N-dimethylaminoethyl-, carboxymethyl-, and sulfated-) glucans, sialic acid-rich glycoproteins, and uronic acid-rich pine cone polysaccharide showed little or no activity. Using radiolabeled virus particles, we demonstrated that the anti-HSV effect of the tannins is due to inhibition of virus adsorption to the cells.

Restriction enzyme map of herpesvirus of Turkey DNA and its collinear relationship with Marek's disease virus DNA

The genome of herpesvirus of turkey (HVT) was shown to consist of long and short unique regions flanked by inverted repeats (J. Cebrian, Kaschka-Dietrich, C., Berthelot, N., and Sheldrick, P., 1982, Proc. Natl. Acad. Sci. USA 79, 555-558). In this paper we report the construction of the linkage map of HVT DNA for BamHI, HindIII, and PstI restriction endonucleases. The maps were constructed by hybridization of 19 cloned BamHI fragments of HVT DNA to electrophoretically separated digests of genomic DNA. Our results indicate that the terminal and internal inverted repeats (TRL and IRL) flanking the long unique sequences (UL) are spanned by BamHI-F fragment and a -F-related terminal fragment, respectively, whereas the terminal and internal inverted repeats (TRS and IRS) flanking the short unique sequences (US) are mostly contained in BamHI-A fragment. Both BamHI-A and -F showed a heterogeneity in size, suggesting the presence of amplification of certain sequences in the inverted repeats. We also report that the HVT genome is collinear with the genetically related Mareks disease virus (MDV) genome, as determined by hybridization of labeled cloned HVT DNA fragments with electrophoretically separated MDV DNA fragments.

Biochemical evidence of the nonintegrated status of Marek's disease virus DNA in virus-transformed lymphoblastoid cells of chicken.

Abstract The status of latent virus genomes in MKT-1 cells, a lymphoblastoid cell line derived from a kidney tumor in a Mareks disease virus (MDV)-infected chicken, has been studied. The cells are virus nonproductive and contain 15 virus genomes per cell, the majority of which can be separated from high molecular weight cell DNA. The fast-sedimenting virus DNA from MKT-1 cells showed an S value of 100 S in a neutral gradient and 200 S in an alkaline gradient by centrifugation and banded at the position of SV40 component 1 DNA in ethidium bromide-cesium chloride equilibrium centrifugation. The data indicate that the 100 S molecule has all the characteristics of closed circular DNA. Thus, the latent MDV DNA in MKT-1 cells is another example of the circular plasmid form of latent herpesvirus DNA. The possibility of integration of a small portion of the virus genomes has not been determined.

Inhibition of influenza virus infection by pine cone antitumor substances

The anti-influenza virus activity of polysaccharides and other high molecular weight fractions from pine cone extract (PCE) of Pinus parviflora Sieb. et Zucc. was investigated. None of the fractions affected the growth of MDCK cells. The acidic PCE substances markedly suppressed the growth of the influenza virus in MDCK cells. Significant inhibition of both the viral protein synthesis in infected cells and virion-associated RNA-dependent RNA polymerase activity was observed with these acidic fractions. Although amantadine inhibited virus plaque formation as effectively as PCE fractions, it was less effective in inhibiting the RNA polymerase activity. These results suggest that PCE, which has been shown to contain antitumor substance(s), also contains anti-influenza virus substance(s).

The inhibitory effects of oligonucleotides, complementary to Marek's disease virus mRNA transcribed from the BamHI-H region, on the proliferation of transformed lymphoblastoid cells, MDCC-MSB1.

An oligonucleotide complementary to the splice donor sequence of the 1.8 kb gene family produced from the BamHI-H region of Mareks disease virus (MDV) DNA inhibited the proliferation of the MDV-derived lymphoblastoid cell line, MDCC-MSB1 (MSB-1), but not that of the avian lymphoid leukosis-derived lymphoblastoid cell line, LSCC-BK3. Colony formation in soft agar was also inhibited by treatment of MSB-1 cells with the antisense oligonucleotide. It is hypothesized that expression of the 1.8 kb gene family produced from the BamHI-H region is directly associated with the maintenance of the tumorigenic state of transformed Mareks disease-derived lymphoblastoid cells.

Minor DNA homology between herpesvirus of Turkey and Marek's disease virus?

Abstract Herpesvirus of turkey (HVT), which prevents Mareks disease in chickens, shares only 1 to 4% of DNA homology with MDV. The experiments indicate that the effective vaccination against virus-induced tumors does not necessarily require a close DNA homology between virus strain for vaccination and virus strain for tumor induction.

Latent DNA of Epstein-Barr Virus: Separation from High-Molecular-Weight Cell DNA in a Neutral Glycerol Gradient

Epstein-Barr virus DNA in virus non-productive cells was separated from high-molecular-weight cell DNA by sedimentation through a neutral glycerol gradient after gentle lysis of cells by Pronase and Sarkosyl. The isolated Epstein-Barr virus DNA had a density of 1.716-1.717 g/cm(3) in CsCl equilibrium centrifugation, which is very close to the virus DNA density of 1.718 g/cm(3). The results indicated that the majority of Epstein-Barr virus DNA in virus nonproductive cells is not covalently integrated into high-molecular-weight cell DNA.

Transcription of the Marek's disease virus genome in a nonproductive chicken lymphoblastoid cell line

Abstract Transcription of the MDV genome in virus-nonproducing MKT-1 cells, a lymphoblastoid cell line derived from a Mareks disease tumor, was studied by analyzing the hybridization kinetics of 3 H-labeled MDV DNA with unlabeled RNA extracted from whole cells or from polyribosomes. Twelve to 14% of the viral DNA template was transcribed and only a portion (60–70%) of the viral-specific RNA sequences found in whole cells could be detected in the polyribosomal fraction. When MKT-1 cells were treated with 5′-iododeoxyuridine (IUdR), transcription of the viral genome was induced so that 42% of the MDV DNA template was transcribed. This represents nearly the same degree of transcription as in chicken embryo fibroblasts productively infected with MDV. Viral antigens also were produced following IUdR induction, but viral DNA replication remained restricted. Only 60–70% of the viral-specific RNA sequences found in whole cells of IUdR-treated MKT-1 cells were found in the polyribosomal fraction. The data suggest that there exists within the cell a post-transcriptional control mechanism which selectively excludes certain RNA transcripts from stable association with the polyribosomes and may therefore be responsible, in part, for the repressed expression of viral genetic information in MKT-1 cells.

Identification of Epstein-Barr virus genes expressed during the early phase of virus replication and during lymphocyte immortalization

Transcription of the Epstein-Barr virus (EBV) genome in Raji cells superinfected with P3HR-1 EBV in the presence of cycloheximide was compared to transcription in human lymphocytes infected with transforming EBV (B95-8). This was done to identify regions of the EBV genome which contain genes that may mediate initiation of virus replication. Hybridization of 32P-labeled cDNA to cloned fragments of EBV DNA (dot blot hybridization) was employed to identify transcriptionally active regions of the viral genome in these cells. DNA in the BamHI A, F, H, and M restriction fragments was found to encode poly(A) RNA during the early phase of EBV replication. In the absence of cycloheximide the earliest detectable transcripts were transcribed from the BamHI M region. The most transcriptionally active region of the EBV genome in lymphocytes following infection with EBV (B95-8) was the BamHI W-Y-H-F region and, to a lesser extent, the K region. Transcription of the BamHI M region was not detected in these cells. The data suggest that expression of a gene or genes located in the BamHI M region of the EBV genome is an important event in the initiation of EBV replication, whereas expression of the genes in the BamHI W-Y-H-F and K regions may be important in the establishment of latency and cellular immortalization.

The Structure of Marek's Disease Virus DNA: Amplification of Repeat Sequence in IRs and TRs

The structure of Mareks disease virus (MDV) DNA was investigated by using Southern blot hybridization analysis. A heterogeneous region was observed in the inverted repeats region, IRs and TRs, as well as in the TRL and IRL. The results of DNA sequencing of the heterogeneous region showed that the heterogeneity of IRs and TRs was due to amplification of a 178‐bp repeat sequence. Amplification of IRs and TRs was found in viral DNA from both pathogenic and nonpathogenic strains. The structure of DNA from the latent MDV genome present in established lymphoblastic cells was also determined. Amplification of the 132‐bp repeat sequence in IRL and TRL was not found in latent MDV DNA of established lymphoblastic cells, whereas amplification of the 178‐bp repeat sequence in IRs and TRs was found in the same DNA.