Amphibian and piscine iridoviruses proposal for nomenclature and taxonomy based on molecular and biological properties.

We have compared a number of properties of the well-characterized iridovirus, frog virus 3, with two other iridoviruses from amphibia, bullfrog edema virus and Lucké triturus virus, and with a piscine iridovirus, goldfish virus (GFV), to provide information for developing taxonomic classification of these viruses and establishing their ecological niche. Purified virions had similar size and shape (icosahedral) for each virus, and the genomic DNAs of each virus were methylated by a virus-induced DNA methyltransferase. The three amphibian viruses replicated equally well in fish (FHM), hamster (BHK), and human (WI-38) cell monolayer with identical cytopathology, while GFV failed to replicate in these cell lines.

Methylation of the promoter for an immediate-early frog virus 3 gene does not inhibit transcription.

Methylation of critical sites within the promoter region of eucaryotic genes has been shown to inhibit transcription by RNA polymerase II. However, although the large DNA virus frog virus 3 (FV3) has a highly methylated genome, it uses host RNA polymerase II for at least the immediate-early stage of transcription. We have previously shown that an FV3-induced trans-acting protein allows transcription from adenovirus promoters inactivated by methylation.

Mutation in a DNA-binding protein reveals an association between DNA-methyltransferase activity and a 26,000-Da polypeptide in frog virus 3-infected cells.

The DNA of frog virus 3 (FV3), an iridovirus, is highly methylated; more than 20% of the cytosine bases are methylated at the 5-carbon position by an FV3-induced DNA methyltransferase (DNA-mt). To determine the role of this enzyme in virus replication and regulation of gene expression, we have analyzed an FV3 mutant that lacks DNA-mt activity and is resistant to 5-azacytidine (an inhibitor of DNA-mt). Comparative polypeptide analysis, using cytoplasmic extracts from the wild-type FV3 and mutant-infected cells, revealed that a single protein of 26,000 (26K) molecular weight was altered in the mutant-infected cells.

Infection with frog virus 3 allows transcription of DNA methylated at cytosine but not adenine residues.

The genome of the iridovirus, frog virus 3, is highly methylated at cytosine residues by a virus-encoded DNA methyltransferase. We have shown previously that an FV3-induced trans-acting protein alters either host RNA polymerase II or methylated template to allow transcription from promoters inactivated by methylation. We now present evidence that the ability of FV3-infected cells to transcribe methylated DNA is specific for DNA methylated at cytosine residues.

Trans-activation of a methylated adenovirus promoter by a frog virus 3 protein.

The high degree of methylation of the frog virus 3 (FV3) genome suggests that FV3-infected cells are capable of transcribing highly methylated DNA. We tested this hypothesis by assaying the transcriptional activity of adenovirus promoters known to be inhibited by methylation. Plasmid constructs containing the E1a and E2aE promoters of adenovirus type 12 linked to the gene for chloramphenicol acetyltransferase [(CAT) EC 2.

Adrenal steroid secretion in girls with pseudoprecocious puberty due to autonomous ovarian cysts.

To evaluate the role of adrenal steroids in pseudoprecocious puberty due to large ovarian follicular cysts, we studied the serum 17-hydroxyprogesterone response to a combination of dexamethasone suppression followed by iv ACTH administration in two girls and compared the results to those in girls with premature thelarche, normal prepubertal girls, and a girl with true precocious puberty. Although basal serum 17-hydroxyprogesterone levels were normal in all subjects, there was incomplete suppression of 17-hydroxyprogesterone with dexamethasone in the two girls with pseudoprecocious puberty and large ovarian cysts. The 17-hydroxyprogesterone response to ACTH was much greater in these girls (360 and 540 ng/dl) than in the girls with other types of precocious puberty (mean +/- SD, 71 +/- 15 ng/dl) or in normal prepubertal girls (80 +/- 20 ng/dl).

Temperature-sensitive mutants of frog virus 3: biochemical and genetic characterization.

Nineteen frog virus 3 temperature-sensitive mutants were isolated after mutagenesis with nitrosoguanidine and assayed for viral DNA, RNA, and protein synthesis, as well as assembly site formation at permissive (25 degrees C) and nonpermissive (30 degrees C) temperatures. In addition, mutants were characterized for complementation by both quantitative and qualitative assays. Based on the genetic and biochemical data, the 19 mutants, along with 9 mutants isolated earlier, were ordered into four phenotypic classes which define defects in virion morphogenesis (class I), late mRNA synthesis (class II), viral assembly site formation (class III), and viral DNA synthesis (class IV).

trans activation of an immediate-early frog virus 3 promoter by a virion protein.

We investigated the protein and DNA sequence requirements for the expression of an immediate-early frog virus 3 (FV3) gene, infected-cell RNA (ICR) 169. We used a plasmid containing the 78 nucleotides 5' to the transcription start site of ICR-169 placed upstream from the coding sequence for the bacterial enzyme chloramphenicol acetyltransferase (CAT). This construction, when introduced by CaPO4-mediated transfection into various eucaryotic cell lines, promoted CAT synthesis only if the transfected cells were subsequently infected with FV3.

17-Hydroxyprogesterone responses to adrenocorticotropin in children with premature adrenarche.

The adrenal secretory response to an iv bolus dose of ACTH was measured in 10 girls (4-8 yr of age), 5 boys (4-9 yr) with premature adrenarche (PA), and 20 normal children. The evening before the ACTH test, each subject took dexamethasone (1 mg at bedtime) to suppress the early morning surge of ACTH. The next morning, 2 serum samples were obtained before the administration of ACTH (Cortrosyn; 0.

Nucleotide sequence of an immediate-early frog virus 3 gene.

We have used "gene walking" with synthetic oligonucleotides and M13 dideoxynucleotide sequencing techniques to obtain the complete coding and flanking sequences of the gene encoding a major immediate-early RNA (molecular weight, 169,000) of frog virus 3. R-loop mapping of the cloned XbaI K fragment of frog virus 3 DNA with immediate-early RNA from infected cells showed that an RNA of approximately 500 to 600 nucleotides (the right size to code for the immediate-early viral 18-kilodalton protein of unknown function) hybridized to a region within 100 base pairs of one end of the XbaI K fragment; no evidence for splicing was observed in the electron microscope or by single-strand nuclease analysis. Further restriction mapping narrowed the location of the gene to the XbaI end of a 2-kilobase-pair XbaI-Bg/II fragment, which was bidirectionally subcloned into the bacteriophage pair mp10 and mp11 for sequencing.

Isolation and characterization of a frog virus 3 variant resistant to phosphonoacetate: genetic evidence for a virus-specific DNA polymerase.

A variant of frog virus 3 (FV3) resistant to 200 micrograms/ml phosphonoacetate was isolated, and used to establish that the DNA polymerase induced in FV3-infected cells was virus coded. In addition, inhibitor studies showed that the FV3 polymerase is similar to eukaryotic polymerase alpha in its sensitivity to aphidicolin, and that resistance to phosphonoacetate does not confer cross-resistance to thymidine arabinoside or acycloguanosine.

The role of DNA methylation in virus replication: inhibition of frog virus 3 replication by 5-azacytidine.

Frog virus 3 (FV3) DNA is the most highly methylated DNA of any known DNA virus; about 20% of the cytosine residues in FV3 DNA are methylated (D. Willis and A. Granoff, 1980, Virology 107, 250-257).

Localization of frog virus 3 proteins using monoclonal antibodies.

Thirty-seven monoclonal antibodies to seven frog virus 3 (FV3) structural proteins were isolated and used to examine the distribution of viral proteins within virions and infected cells. Three monoclonal antibodies, one to the major capsid protein, VP55, and two to VP38 had detectable neutralizing activity suggesting that these proteins are located on the surface of virions. Immunofluorescent studies showed that VP108, VP57, VP55, and VP16 were localized mainly within virus assembly sites, while VP17 was detected in both assembly sites and the surrounding cytoplasm.

Early proteins are required for the formation of frog virus 3 assembly sites.

The formation of frog virus 3 virions takes place within morphologically distinct regions of the cytoplasm termed assembly sites. These sites are formed within infected BHK cells by 6-7 hr after infection, a time when viral DNA and both early and late proteins are present. To identify macromolecules involved in assembly site formation, a temperature-sensitive mutant ( ts9467 ) was used which is not only defective in the synthesis of late RNA and proteins (D.

DNA methyltransferase induced by frog virus 3.

Over 20% of the cytosine bases in frog virus 3 DNA are methylated at the 5-carbon position. To determine whether this high degree of methylation is the result of a virus-specific enzyme, we examined the kinetics of induction and the substrate specificity of a DNA methyltransferase from frog virus 3-infected fathead minnow cells. A novel DNA methyltransferase activity appeared in the cytoplasm of infected cells at 3 h postinfection.

Cell-free translation of frog virus 3 messenger RNAs. Initiation factors from infected cells discriminate between early and late viral mRNAs.

Cell-free protein-synthesizing extracts prepared from rabbit reticulocytes, wheat germ, or cultured baby hamster kidney cells efficiently translated frog virus 3 early mRNAs; in contrast, late mRNAs were translated poorly under similar conditions. However, the translational efficiency of the late viral mRNAs was markedly enhanced in cell-free extracts prepared from frog virus 3 (FV 3)-infected baby hamster kidney cells and in nuclease-treated rabbit reticulocyte extracts by the addition of a 0.5 M KCl wash from FV 3-infected cell ribosomes; the 0.

Treatment of menstrual irregularities with dexamethasone in congenital adrenal hyperplasia.

Abnormal menstrual patterns occur frequently in adolescent girls with congenital adrenal hyperplasia (CAH). In this report, two sisters with CAH secondary to the 21-hydroxylase defect are described in whom the administration of dexamethasone, a long-acting glucocorticoid, initiated or regulated their menstrual cycles. Plasma levels of 17-hydroxyprogesterone and androstenedione were elevated while on therapy with 80 mg/day of hydrocortisone, and became normal after treatment with daily dexamethasone, 1.

Psychiatric presentation of an arachnoid cyst.

A 23 year old white male, successful until 18 months prior to presentation, began noticing a decrease in mental functions which began to speed up with time. This progressed to a point where he had changes in his personality which appeared schizophreniform in nature. The patient was admitted to a general hospital for evaluation and a large arachnoid cyst was found in his left temporal lobe.