Allan Granoff

Viruses and renal carcinoma of Rana pipiens: I. The isolation and properties of virus from normal and tumor tissue☆

Abstract Properties of a virus, frog virus 1 (FV 1), isolated from plaques appearing in uninoculated frog kidney cell monolayers were studied. The virus multiplied at 24 ° in frog, fish, chick embryo, and hamster cell monolayers with cytopathology resulting in cell death. Although each cell system could be used for plaque assay the efficiency of plating was higher (10- to 100-fold) in chick embryo and hamster cells. Growth curves in cultured cells from both poikilothermic and homothermic animals were similar with a latent period between 6 and 10 hours. Most of the virus (50–99%) remained cell-associated. Virus multiplication was temperature dependent. At 33 ° plaques did not develop and little or no infectious virus was produced although adsorption and penetration occurred. Virus multiplication ceased when infected cultures were removed from 24 ° to 33 ° at any time during the growth cycle, a phenomenon suggesting that virus maturation was thermosensitive. The site of virus synthesis appeared to be the cell cytoplasm where DNA, identified by cytochemical stains and isotope incorporation, accumulated during infection. Infectivity was lost by exposure of virus to lipid solvents or to pH 3.0. It was stable to storage at low temperature (−20 ° to −60 °) but was rapidly inactivated at 50 °. Additional virus isolations were made from spontaneously degenerating frog kidney cell cultures and from homogenates of frog livers and frog renal tumors (Lucke adenocarcinoma). There was no correlation between the presence of intranuclear inclusions or virus particles (as observed by light and electron microscopy) and the ability to isolate virus from tumor homogenates. Comparative tests showed no distinguishing differences between properties of an isolate from a Lucke tumor, FV 3, and FV 1. The results are discussed as they relate to a viral etiology of the Lucke tumor.

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: I. Virus-specific protein synthesis and its regulation☆

Abstract Heat-inactivated frog virus 3 inhibited protein synthesis in fathead minnow and baby hamster kidney cells but did not affect the replication of superinfecting infectious frog virus 3 in these cells. This method of controlling host-cell protein synthesis enabled us to identify 20 proteins induced by frog virus 3 infection. All detectable virus-specific proteins were synthesized within 2 hr after infection. Three viral structural proteins (VSP)—7, 9, and 13—were synthesized at maximum rates early in infection, and two others, VSP 5 and 11, reached their peak rate of synthesis late in infection. All structural and nonstructural proteins were made in the presence of cytosine arabinoside, an inhibitor of DNA synthesis. In the absence of viral DNA replication, the kinetics of synthesis of VSP 5 and 11 were similar to those during normal infection, but there was no reduction in the synthesis of VSP 7, 9, and 13 late in the infection. These results suggest that synthesis of all viral structural proteins is an early event in the FV 3 replication cycle, with progeny viral DNA required to regulate the synthesis of certain viral proteins.

Macromolecular synthesis in cells infected by frog virus — VIII. The nucleus is a site of frog virus 3 DNA and RNA synthesis

Abstract The replication of frog virus 3 (FV 3) had been thought to occur exclusively in the cytoplasm. However, we recently reported that FV 3 did not replicate in enucleated or ultraviolet light (uv)-irradiated cells nor was viral DNA or RNA synthesized [Goorha, R., Willis, D. B., and Granoff, A. (1977).]. To elucidate the role of the nucleus in FV 3 replication further, we studied viral DNA and RNA synthesis in infected cells by electron microscopic autoradiography and by biochemical techniques. The results show that a fraction of FV 3 DNA is synthesized in the nucleus and then transported into the cytoplasm. The same is true for a fraction of FV 3-specific RNA. We have characterized the DNA molecules in these cell compartments. Both nuclear and cytoplasmic DNA appeared between 1 and 2 hr after infection. The molecular weight of newly synthesized nuclear viral DNA, as measured by sedimentation in alkaline sucrose gradients, did not exceed 8 × 106 regardless of the labeling period. In contrast, the cytoplasmic fraction contained only DNA sedimenting at a rate expected for a single strand of the viral genome (50 × 106 daltons). Pulse-chase experiments provided evidence for a precursor-product relationship between the nuclear and cytoplasmic DNA: About 35% of the nuclear DNA was chased into the cytoplasm and was genome size at the end of a 4-hr chase period. Nuclear viral DNA synthesis was more sensitive to cycloheximide, which inhibits initiation of DNA synthesis by inhibiting viral protein synthesis, than was cytoplasmic viral DNA synthesis. These data provide strong evidence for the nucleus as a site of FV 3 DNA and RNA synthesis and support the conclusion that FV 3 DNA synthesis is initiated in the nucleus and completed in the cytoplasm.

Viruses and renal carcinoma of Rana pipiens: II. Ultrastructural studies and sequential development of virus isolated from normal and tumor tissue☆

Abstract The sequential development of a virus (FV 1) isolated from cultured frog kidney cells has been studied by electron microscopy in fathead minnow (FHM) and baby hamster kidney (BHK 21/13) cells. Regions of virus synthesis were first seen in the cell cytoplasm at 7 and 10 hours after infection of FHM and BHK 21/13 cells, respectively. These sites contained virus particles in all stages of development from partial capsids to complete capsids with nucleoids. The virus was hexagonal and measured approximately 120 by 130 mμ. At 12 hours after infection, virus was seen budding from the cytoplasmic membrane, thus acquiring an envelope. This process continued throughout the course of the infection. Late in the infection (24–48 hours) one or more virus crystals were regularly seen in the cytoplasm of FHM cells and occasionally in BHK 21/13 cells. FV 1 replication in adult and embryonic frog cell monolayers, cultured Lucke tumor cells, and chick embryo monolayers was identical to that seen in FHM and BHK 21/13 cells. Eleven additional isolates from both normal and tumor (Lucke) frog tissue were examined in FHM cells. The morphology and site of synthesis of these isolates were identical to FV 1 and contrasted with the nuclear site of synthesis of virus seen in the Lucke tumor.

Viruses and renal carcinoma of Rana pipiens: IV. Nucleic acid synthesis in frog virus 3-infected BHK 2113 cells☆

Abstract Infection of BHK 21 13 cells with FV 3 inhibited host cell RNA synthesis within 1 hour and DNA synthesis between 2 and 4 hours after infection. Between 3 and 4 hours a new species of DNA was synthesized in the cell cytoplasm and by 5 hours reached a maximal rate equal to 6 times that of DNA synthesis in uninfected cells. This new DNA had a G + C content of 53% (ϱ = 1.712 g/ml). Virus-induced RNA synthesis began within 2 hours after infection and reached a maximal rate at 5–6 hours. This new RNA was found in the cytoplasm at the periphery of areas of DNA synthesis. Infectious virus was detected 6 to 7 hours after infection.

Icosahedral Cytoplasmic Deoxyriboviruses

Deoxyriboviruses having an apparent icosahedral symmetry and replicating in the cytoplasm have been titled “icosahedral cytoplasmic deoxyriboviruses” (ICDVs) (Kelly and Robertson, 1973; McAuslan and Armentrout, 1974). Members of this group are widely distributed in nature in a variety of hosts and include iridescent viruses from insects, lymphocystis virus from fish, amphibian viruses, African swine fever virus, and cauliflower mosaic and related viruses from plants (Granoff, 1969; Stoltz, 1971; Plowright, 1972; Kelly and Robertson, 1973). In this chapter we have chosen to retain the term “icosahedral cytoplasmic deoxyribovirus,” but because of the diversity of properties of these viruses this term is inadequate for classification and nomenclature pur poses. We might compare this term to grouping papovaviruses, adenoviruses, and herpesviruses under the heading of “icosahedral nuclear deoxyriboviruses.” Our usage of this term merely defines those viruses that have or may have icosahedral symmetry, appear to replicate in the cytoplasm, and contain DNA. It does not imply any taxonomic relationships and includes viruses from plants, invertebrates, and vertebrates. The designation “ICDV” is useful in distinguishing these viruses from the morphologically distinct and structurally complex poxviruses, which also replicate in the cytoplasm (Moss, 1974).

Viruses and renal carcinoma of Rana pipiens: XI. Isolation of frog virus 3 temperature-sensitive mutants; Complementation and genetic recombination

Abstract Eleven temperature-sensitive (ts) mutants were obtained from frog virus 3 (FV 3) grown in the presence of bromodeoxyuridine. Six mutants were selected for further study; each gave plaque counts at the nonpermissive temperature (30°) which ranged from 1.4 × 10 −4 to 3.5 × 10 −5 of the plaque count at the permissive temperature (25°). The yields of mutant viruses at the nonpermissive temperature as determined by single-step growth cycles were from 0.4% to 3.6% of the yield obtained at permissive temperature. Heat inactivation kinetics indicated that none of the mutants was more temperature-sensitive than the wild-type FV 3. Mixed infection experiments showed that five of the six mutants complemented each other with complementation levels ranging from 9 to 126. High recombination frequencies (9 to 63%) were obtained with all mutants. Evidence is presented which rules out factors other than genetic exchange to account for the recombination frequencies obtained.

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.