Dawn B. Willis

Frog virus 3 DNA is heavily methylated at CpG sequences

Abstract DNA extracted from frog virus 3 (FV 3) virions was resistant to the action of restriction endonucleases that cannot cleave DNA methylated at the sequence me CpG. However, viral DNA was susceptible to digestion with at least one enzyme that recognizes an identical sequence, independent of methylation. When we analyzed the 5-methylcytosine content of FV 3 DNA by thin-layer chromatography, we found that over 20% of the cytosine residues were methylated, in contrast to 6 to 8% 5-methylcytosine found in the host fathead minnow cells. This degree of cytosine methylation exceeds that reported for any animal virus.

Macromolecular Synthesis in Cells Infected by Frog Virus 3

The best studied member of the Iridoviridae is frog virus 3 (FV3), isolated by Granoff et al. (1966) from a renal adenocarcinoma of the leopard frog, Rana pipiens. Similar isolates were made from normal frog kidney and liver (FV1, FY 2, FV5–FY24), but FV3 was chosen for further investigation because of its presence in the tumor. As it turned out, this unusual cytoplasmic DNA virus bore no causal or even helper relationship to the tumor, which was the result of infection with the Lucke herpesvirus (Naegele et al. 1974), but it did have a number of interesting characteristics that made it a worthy object of study in its own right. FV3 is probably similar or identical to other amphibian virus isolates obtained from bullfrogs, newts, and toads (Wolf et al. 1968; Clark et al. 1969).

Lipid composition of frog virus 3

Abstract The lipid composition of purified frog virus 3 was determined and compared with that of host cells of avian and piscine origin. Unenveloped, infectious icosahedral virions contained approximately 9% lipid, at least 90% of which was phospholipid. The ratios of the various phospholipid classes of the virion to those of the host cells, as well as the low amount of cholesterol present in the virions, led to the conclusion that the viral membrane was not derived from preexisting host membranes. The incorporation of 32 P into phosphatidylserine and phosphatidylinositol, the phospholipid classes that were present in greater amounts in virions than in host cells, was increased in infected cells. Infectivity was abolished by treatment with Nonidet P-40 or phospholipase A but not by treatment with phospholipase C. These results indicate that although an intact lipid membrane was required for infectivity, the loss of certain phospholipid polar groups did not affect the biological function of this membrane.

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). To understand the role of DNA methylation in virus replication, we have examined the effect of 5-azacytidine, a drug that inhibits DNA methylation. 5-Azacytidine (10 microM) reduced the production of infectious FV3 by 100-fold or more and inhibited methylation of viral DNA by about 80%. Inhibition of DNA methylation did not affect viral gene expression since there was no detectable inhibition of virus-specific RNA or protein synthesis in 5-azacytidine-treated cells. In contrast, the size of the replicating DNA measured under completely denaturing conditions, was much smaller than that found during infection in the absence of drug. These results suggest that the undermethylated DNA was susceptible to endodeoxyribonuclease(s). Additionally, electron microscopic examination of FV3-infected, 5-azacytidine-treated cells revealed that preformed capsids remained empty or were only partially filled with viral DNA. Based on these data, it is suggested that methylation of DNA protects it from endonucleolytic cleavage and that the integrity of genomic DNA is required for its proper packaging into virions.

Macromolecular synthesis in cells infected by frog virus 3 IX. Two temporal classes of early viral RNA

Abstract By selectively inhibiting functional viral protein synthesis with cycloheximide or fluorophenylalanine (FPA) and analyzing the resultant viral RNAs by polyacrylamide gel electrophoresis and hybridization, we have shown that early frog virus 3 (FV3) RNA can be subdivided into two sequentially synthesized classes. The polyacrylamide gel pattern of viral RNA synthesized 6 hr postinfection (p.i.) in cells treated with FPA was identical to that of the RNA synthesized at 1 hr p.i. in the absence of drug. RNA synthesized at 6 hr p.i. in the presence of FPA hybridized to 36% of the viral genome, whereas RNA synthesized 6 hr p.i. in its absence contained many additional species and hybridized to 50% of the genome. Therefore, the FPA RNAs appeared to represent the same sequences as the early viral RNA. In contrast, viral RNAs synthesized when protein synthesis was inhibited by cycloheximide did not contain all of the species present at 1 hr p.i. or in FPA RNA. The cycloheximide RNAs, which hybridized to only 16% of the genome, comprised a subset of the FPA RNAs. One of the viral RNA species was synthesized at a very high rate in cells exposed to cycloheximide, but at a much lower rate during infection in the presence or absence of FPA. When we delayed the addition of cycloheximide until 1 or 2 hr p.i., all of the remaining early viral RNA species were synthesized and the rate of synthesis of the overproduced RNA decreased. We could, therefore, divide FV3 early RNA synthesis into two temporal classes: “immediate early,” which took place in the absence of protein synthesis (cycloheximide), and “delayed early,” which occurred under conditions of restricted protein synthesis (FPA). In addition, our data suggest that at least one species of immediate early viral RNA was subject to negative transcriptional control.

Restriction endonuclease mapping of the frog virus 3 genome

A physical map for the frog virus 3 (FV 3) genome was constructed after digestion with the following restriction endonucleases: EcoRI, HindIII, KpnI, and XbaI. Mapping of the DNA was accomplished by partial digestion and recutting, double-digestion, and Southern blot hybridization with deduction of overlaps. Although the virion DNA is physically linear, the restriction map was circular, supporting the data that the FV 3 genome is circularly permuted (Goorha and Murti, Proc. Nat. Acad. Sci USA 79, 248-252, 1982), a unique feature among eukaryotic viruses.

Taxonomy of Iridoviruses

Originally grouped together on the basis of morphology, the iridoviruses are now beginning to reveal biochemical and genetic similarities—e.g. circularly permuted and terminally redundant genomic DNA—that set them apart from other eukaryotic virus groups and justify their inclusion in the same family.

Nongenetic reactivation of frog virus 3 DNA

Abstract Fathead minnow cells co-infected with purified frog virus 3 DNA and either ultraviolet light-irradiated virus or a temperature-sensitive mutant at a nonpermissive temperature produced infectious progeny with the genotype of the purified DNA. This is the first reported example of nongenetic reactivation of viral DNA by proteins associated with nonreplicating virions.

Characterization of a temperature-sensitive mutant of frog virus 3 defective in DNA replication

We have characterized the defect of a temperature-sensitive (ts) DNA− mutant (ts 6642) of frog virus 3 (FV 3). At the nonpermissive temperature (30°) ts 6642 synthesized <3% of the viral DNA that was synthesized at the permissive temperature (23°). When ts 6642-infected cells were incubated at 30° for 4.0 hr and then shifted to permissive temperature, viral DNA synthesis started immediately even when protein synthesis was inhibited at the time of shiftdown. This result implies that at 30°, ts 6642 synthesized all the proteins required for viral DNA replication but that one of these was nonfunctional at the nonpermissive temperature. Further characterization revealed that ts 6642 was probably defective in the initiation of DNA replication. This conclusion was based on the following data: When ts 6642-infected cells incubated at 23° for 4.0 hr were shifted to 30°, there was a gradual decrease in viral DNA synthesis. By 1 to 1.5 hr after the shiftup, viral DNA synthesis was completely inhibited. Analysis of the density of the DNA synthesized after a shiftup in the presence of BUdR and FUdR suggested that residual viral DNA synthesis represented chain elongation, and not initiation of new rounds of DNA replication. The defective protein was therefore involved in the initiation process. Both wild-type FV 3 (FV 3+) and ts 6642 induced the synthesis of thymidine kinase and DNA polymerase at 30°. Therefore, neither of these enzymes was involved in the DNA replication defect of ts 6642. At the nonpermissive temperature, ts 6642 synthesized all the viral proteins that were detectable at the permissive temperature. However, synthesis of late proteins was delayed, and never reached wild-type levels. Furthermore, the rate of synthesis of late proteins at 30° became dependent upon the multiplicity of infection. These results reinforce our previous conclusion (R. Goorha and A. Granoff, 1974, Virology60, 237–250) that in FV3+-infected cells late proteins (and by implication late mRNAs) were synthesized in the absence of viral DNA replication.

Macromolecular synthesis in cells infected with frog virus 3. V. The absence of polyadenylic acid in the majority of frog virus 3-specific mRNA species.

Abstract Most of the virus-specific RNA molecules isolated from the cytoplasm of BHK-21 cells infected with frog virus 3 (FV 3) did not contain poly(A) tracts of sufficient length to permit the RNA to bind to oligo(dT) cellulose. The 14% of [ 3 H]adenosine-labeled RNA molecules that did bind were 40–55% RNAse-resistant and sedimented at about 6 S. In contrast, 40–70% of the pulse-labeled RNA isolated from vaccinia virus-infected BHK cells was bound to oligo(dT) cellulose, and the bound RNA was representative of the whole spectrum of vaccinia virus RNA size classes.