Frog Virus Research Articles

Probable role of endogenous endotoxins in hepatocytolysis during murine hepatitis caused by frog virus 3.

Three new observations bear out the role of endogenous endotoxins in the pathogenesis of murine hepatitis caused by frog virus 3. First, the LD50 of endotoxin is 20 times lower in mice pretreated for 2.5 hr with a sublethal dose of frog virus 3 than in untreated mice. Animals inoculated with one sublethal dose of lipopolysaccharide 2.5 hr after injection of one sublethal dose of virus die, all having developed extensive hepatocellular necrosis. This hypersensitivity varies according to the intensity of virus-induced destruction of Kupffer cells, which are the intrahepatic target of the virus. Second, mortality is significantly lower and the interval between infection and death longer in axenic mice, which are largely protected from portal endotoxemia. Third, the impairment of some biologic activities of endotoxin (through treatment with polymyxin B or indomethacin, for example) protects mice against hepatic damage and death. Likewise, mice rendered tolerant to endotoxins, and C3H/HeJ mice, which are genetically resistant to endotoxins, survive challenge with frog virus 3 and are refractory with regard to hepatocytolysis . These findings suggest that, in hepatitis caused by frog virus 3, endogenous endotoxins are responsible for extensive hepatocytolysis since virus-induced damage to the hepatic reticuloendothelial system prevents their detoxification.

Fate of frog virus 3 DNA replicated in the nucleus of arginine-deprived CHO cells.

In a productive infection, frog virus 3 (FV 3) DNA was synthesized in both the cell nucleus and cytoplasm. The infection was aborted in arginine-starved Chinese hamster ovary cells and viral DNA replication was then restricted to the nuclear compartment. It has been demonstrated that the newly synthesized FV 3 DNA present in the nucleus is of genomic size. After the addition of arginine, this DNA is transferred into the cytoplasm and can be encapsidated. The formation of infectious particles occurred even if an inhibitor of DNA synthesis was added simultaneously with arginine. Although the synthesis of early FV 3 polypeptides and their intracellular distribution were comparable in the presence or in the absence of arginine, the production of late species was greatly reduced by arginine deprivation; one of these late proteins must be involved in the passage from the nuclear to the cytoplasmic phase of FV 3 DNA replication. This system made it possible to carry out an independent analysis of the nuclear events that comprise the first stage of FV 3 multiplication.

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. This activity was induced in the absence of viral DNA replication and was therefore probably an early viral enzyme. In contrast to the methyltransferase activity extracted from uninfected cell nuclei, the cytoplasmic enzyme showed a strong template preference for double-stranded over single-stranded and for unmethylated over hemimethylated DNA. The dinucleotide sequence dCpdG was a necessary and sufficient exogenous substrate for methylation in vitro. A mutant of frog virus 3, isolated as resistant to 5-azacytidine and having unmethylated virion DNA, did not induce cytoplasmic DNA methyltransferase, leading to the conclusion that this activity is coded for by the virus.

Ultrasonic absorption evidence for structural fluctuations in frog virus 3 and its subparticles.

The structural fluctuations specific to self-assemblies of biological molecules have been investigated further with ultrasonic techniques by using frog virus 3 (FV3). We compared the ultrasonic properties of complete FV3 virions and of several subparticles that may be obtained from this DNA virus: (i) the central nucleoprotein core versus its component DNA and proteins in a dissociated state; (ii) the core versus the capsidless subparticle, consisting of the core surrounded by the lipid membrane; and (iii) the complete virus versus the capsidless subparticle. The ultrasonic absorption by the core particle was quite large compared with the absorption by other nucleoprotein assemblies, suggesting that the core contains some organized structure. Both the core and the complete virus absorbed ultrasound more than did the capsidless subparticle. The difference spectrum for the virion relative to the capsidless subparticle may represent a single relaxation and is analyzed, by using a recent model, in terms of volume fluctuations due to radial movements in the virion. These fluctuations are much smaller than can be detected in virus crystals with present-day x-ray techniques.

Interaction of frog virus-3 with the cytoskeleton. I. Altered organization of microtubules, intermediate filaments, and microfilaments.

The progressive cytoskeletal alterations of frog virus 3-infected baby hamster kidney (BHK) and fathead minnow (FHM) cells were studied by immunofluorescence and electron microscopy. The virus assembly sites, which contain viral genomes and viral proteins, were detected in the cytoplasm at 4 h (FHM) or 6 h (BHK) and mature virions appeared 2 h later. When infected cells were treated with Triton X-100, the assembly sites were found in association with the cytoskeleton. In infected cells, the number of microtubules progressively decreased but a few microtubules traversing in the vicinity of the assembly sites remained intact. Early in infection, the intermediate filaments retracted from the cell periphery, delimited the forming assembly sites, and remained there throughout infection. We suggest that intermediate filaments are involved in the formation of assembly sites. In addition, the filaments either by themselves or in conjunction with microtubules may anchor the assembly sites near the nucleus. The microfilament bundles (stress fibers) disappeared with the formation of assembly sites, and late in infection many projections containing microfilaments and virus particles appeared at the cell surface. The observation suggests a role for microfilaments in virus release. Taken together, these results provide the first example of a virus-infected cell in which all three cytoskeletal filaments show profound organizational changes and suggest an active participation of the host cytoskeleton in viral functions.

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.

Analysis of the genome of fish lymphocystis disease virus isolated directly from epidermal tumours of pleuronectes

Virions of fish lymphocystis disease virus (FLDV), a member of the iridovirus family, were isolated directly from lymphocystis disease lesions of individual flatfishes and purified by sucrose and subsequent cesium chloride gradient centrifugation to homogeneity as judged by electron microscopy. The isolated FLDV DNAs appear to be heterogeneous in size. Contour length measurements of 43 DNA molecules gave an average length of 49 +/- 23 microns, corresponding to 93 +/- 44 X 10(6) D. Molecular weight estimations of FLDV DNA by restriction enzyme analysis resulted in only 64.8 X 10(6) D indicating an excess length of the DNA of about 50%. FLDV DNA was sensitive to lambda 5'-exonuclease and to E. coli 3'-exonuclease III without preference of any one terminal DNA restriction fragment. Denaturation and reannealing experiments of FLDV DNA resulted in the formation of circular DNA molecules of 34.25 microns contour length (= 65.22 X 10(6) D). This result suggests that FLDV DNA contains directly repeated sequences at both ends and that it is terminally redundant. FLDV DNA is methylated in cytosine. FLDV DNA did not hybridize with frog virus DNA indicating that the two iridoviruses are not closely related to each other. Restriction enzyme analysis and Southern blot hybridizations revealed that FLDV isolates can be classified into two different strains: FLDV strain 1 occurs in flounders and plaice, whereas strain 2 is usually found in lesions of dabs.

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 0.5 M KCl wash (initiation factors) from uninfected cells had no such effect. Total cytoplasmic RNA from infected cells was fractionated according to size on sucrose gradients and fractions containing different concentrations, and relative proportions of early and late mRNAs were translated in either native or initiation factor-supplemented extracts. Under these conditions, the translation efficiency of early mRNAs was unchanged, while the translation of late mRNAs increased 2-7-fold. Thus, the in vitro discriminatory activity of the 0.5 M wash was not dependent on the complexity of the mRNAs present in the translation mixture. We show also that in native extracts, under conditions of blocked polypeptide chain elongation, early mRNAs are initiated preferentially. However, late as well as early mRNAs are initiated equally well in reticulocyte extracts under similar experimental conditions when supplemented with crude initiation factors from infected cells. These data support the conclusion that the translational enhancement of FV 3 mRNAs in vitro is mediated by a virus-specified or virus-modified initiation factor(s) and likely represents a regulatory mechanism of protein synthesis operative in vivo (Willis, D. B., Goorha, R., Miles, M., and Granoff, A. (1977) J. Virol. 24, 326-342).

Murine hepatitis induced by frog virus 3: a model for studying the effect of sinusoidal cell damage on the liver.

Murine hepatitis induced by frog virus 3: a model for studying the effect of sinusoidal cell damage on the liver.

Protective effect of colectomy in frog virus 3 hepatitis of rats: possible role of endotoxin.

Four to six days after colectomy, rats resisted a challenge of frog virus 3 that in sham-operated animals led to lethal hepatitis. Furthermore, the beneficial effect of colectomy was lost after intravenous administration of a dose of bacterial endotoxin as small as 0.01 100% lethal dose. The protection was related to neither a different distribution of the virus in body organs nor a stimulation of the reticuloendothelial system. The virus-induced early events--destruction of liver sinusoidal cells with leakage of cathepsin D into serum and inhibition of liver macromolecular synthesis--evolved similarly in both groups of rats. After an identical consumption of complement at the beginning of infection, a renewal in complement activity in the protected rats contrasted with an increasing deficiency in the control animals. The protective role of colectomy seems to be related to the suppression of the main source of bacterial endotoxin.

Expression of frog virus 3 early genes after ultraviolet irradiation

The synthesis of early gene products was studied in Chinese hamster ovary (CHO) cells infected with FV 3 virions irradiated with various doses of uv light. The transcription of the immediate early units, occurring in the presence of cycloheximide, was analyzed by quantitation of the polypeptides synthesized after drug removal, pulse labeled with [ 35S]methionine, and separated by gel electrophoresis. A distinct inactivation rate of protein synthesis was observed for four immediate early species (54K, 42K, 20K, 16K) and the uv target sizes were found to be proportional to the molecular weight of the encoded polypeptides. Delayed early species were analyzed in the absence of genome replication. Under these conditions the delayed early genes (coding for polypeptides 90K and 31K) and the gene for one immediate early species 16K showed target sizes greater than that of polypeptide 42K. The results suggest that the immediate early mRNA species whose translation was detectable in vivo are transcribed from individual promoter regions; they also reflect the fact that the expression of the gene coding for polypeptide 42K is controlled by at least one of the early products and/or the dependence of early genes upon the immediate early protein 42K.

Frog virus 3 DNA replication occurs in two stages.

Viral DNA synthesis in frog virus 3 (FV3)-infected cells occurs both in the nucleus and in the cytoplasm (Goorha et al., Virology 84:32-51, 1978). Relationships between viral DNA molecules synthesized in these two compartments and their role in the virus replication were examined. The data presented here suggest that (i) FV3 DNA replicated in two stages and (ii) nucleus and cytoplasm were the sites of stages 1 and 2 of DNA replication, respectively. Stages 1 and 2 were further distinguished by their temporal appearance during infection and by the sizes of the replicating DNA as determined by sedimentation in neutral sucrose gradients. In stage 1, replicating molecules, between the size of unit and twice the unit length, were produced early in infection (2 h postinfection). In contrast, stage 2 of DNA replication occurred only after 3 h postinfection, and replicating molecules were large concatemers. Results of pulse-chase experiments showed that the concatemeric DNA served as the precursor for the production of mature FV3 DNA. Denaturation of concatemeric DNA with alkali or digestion with S1 nuclease reduced it to less than genome size molecules, indicating the presence of extensive single-stranded regions. Analysis of replicating DNA by equilibrium centrifugation in CsCl gradients after a pulse-chase suggested that these single-stranded regions were subsequently repaired. Based on these and previous data, a scheme of FV3 replication is presented. According to this scheme, FV3 utilizes the nucleus for early transcription and stage 1 of DNA replication. The viral DNA is then transported to the cytoplasm, where it participates in stage 2 DNA replication to form a concatemeric replication complex. The processing of concatemers to produce mature viral DNA and virus assembly also occurs in the cytoplasm. This mode of replication is strikingly different from any other known DNA virus.

Endocytic capacities of Kupffer cells isolated from the human adult liver.

Kupffer cells isolated from human adult livers and maintained in culture display the main structural features of macrophages. They are able to phagocytose latex particles and opsonized sheep erythrocytes in vitro. Vaccinia virus as well as Frog Virus 3 are taken up and uncoated in cultured Kupffer cells. Such a model may be used advantageously for studying different problems in liver physiopathology.

Localization of some frog virus 3 structural polypeptides

The localization of several frog virus 3 structural polypeptides has been established by stripping the different layers of the virion with controlled degradations using Brij, NP 40, and Pronase. The degradation products were further separated on sucrose gradients and characterized by electron microscopy and SDS-gel electrophoresis. This study allowed us to determine the radial distribution of the following viral polypeptides (VPs): VP 58 in the viral envelope, VP 48 in the capsid subunits, and VPs 44 and 63, beneath the icosahedral lattice, probably associated with the lipids. Additional information is also provided on FV 3 ultrastructure from the study of the morphology of different types of well-characterized subviral particles.

The genome of frog virus 3, an animal DNA virus, is circularly permuted and terminally redundant.

We examined the structure of the frog virus 3 (FV 3) genome by using electron microscopic and biochemical techniques. The linear FV 3 DNA molecules (Mr approximately 100 x 10(6) formed circles when partially degraded with bacteriophage lambda 5'-exonuclease and annealed, but not when the annealing was done without prior exonuclease digestion. The results suggest that the DNA molecules contain direct terminal repeats. The repeated region composed about 4% of the genome. Complete denaturation of native FV 3 DNA molecules followed by renaturation produced duplex circles each bearing two single-stranded tails at different points along the circumference. The tails presumably represent the terminal repeats. The formation of duplex circles suggests that the FV 3 genome is circularly permuted. This is further borne out by (i) failure to identify a specific restriction endonuclease fragment containing the label when the molecular ends were radiolabeled by using the polynucleotide kinase procedure, and (ii) similarity in the restriction patterns of virion DNA and large concatemeric replicating viral DNA as revealed by endonucleolytic cleavage of both DNAs with HindIII. From the above data, we conclude that the FV3 genome is both circularly permuted and terminally redundant--unique features for an animal virus.

Structure of frog virus 3 genome: size and arrangement of nucleotide sequences as determined by electron microscopy

The structural features of frog virus 3 (FV 3) DNA have been analyzed by electron microscopy. The FV 3 genome is a linear duplex molecule of 52.00 ± 5.60 μm in length which corresponds to 98.28 ± 10.58 X 106 daltons. To compare the structure of the genomes of FV 3, vaccinia virus, and herpes simplex virus, the three viral DNAs were denatured, quickly renatured, and examined in the electron microscope. The FV 3 DNA consisted of linear single strands while the vaccinia virus DNA contained reannealed duplexes and the herpesvirus DNA contained single-stranded circles with bushes or molecules in which two single-stranded circles were joined by a duplex region. These data suggest that the FV 3 genome contains neither crosslinked terminii nor inverted repeats. To construct a partial denaturation map of the FV 3 genome, the DNA was denatured with 85% formamide and examined in the electron microscope. The partially denatured molecules failed to match by their denatured regions when lined up end to end, suggesting that the molecules were dissimilar with respect to the arrangement of nucleotide sequences. However, a distinct denaturation map was obtained when the molecules were matched by their denatured regions rather than by the physical ends. The map revealed (1) a continuous stretch of about 40 μm (or 75.60 × 10 6 daltons) of uniquely ordered sequences common to all molecules, and (2) a distribution of the ends within 20 to 28% of the map length. These observations suggest that FV 3 DNA molecules contain various circular permutations of a common overall nucleotide sequence and that the extent of circular permutation is restricted as in bacteriophage P22 ( B. K. Tye, J. A. Huberman, and D. Botstein, 1974, J. Mot Biol. 85, 501–532).

DNA-binding proteins in frog virus 3-infected cells.

At least 12 virus-induced DNA-binding proteins with mol. wt. ranging from 14 X 10(3) have been isolated from frog virus (FV 3)-infected fathead minnow cells by DNA affinity chromatography. Two enzymic activities, DNA-dependent DNA polymerase and endodeoxyribonuclease, were present in the DNA-binding proteins; these enzymic activities were similar to those induced by FV 3 in infected cells. A single species of DNA-binding proteins with a mol. wt of 36 000 had very high affinity for single-stranded DNA, but a low one for double-stranded DNA. Proteins with such characteristic affinity for single-stranded DNA destabilize the DNA helix and are essential for viral DNA replication. Thus, the 36 000 mol. wt. DNA-binding protein is a candidate for such a role in FV 3 DNA replication.

Penetration and uncoating of frog virus 3 (FV 3) in cultured rat Kupffer cells

The in vitro interactions between unenveloped frog virus 3 and Kupffer cells have been studied at 37°, a nonpermissive temperature for viral multiplication. Kupffer cells were isolated by collagenase perfusion of rat livers followed by purification of the cells by centrifugal elutriation. Electron microscopic examinations revealed peculiar modes of interaction with the cellular membranes. Two hours after infection most virus particles were present in phagocytic vacuoles or dense and only few in pinocytic vesicles. About 25% of the engulfed virions displayed fusion between the trilaminar internal core membrane and the membrane of the vacuoles resulting in an opening of the shell that allowed dense core material to be released into the cytoplasm. The empty capsids were integrated into the membrane of the vacuole and were often invaded by the endoplasmic reticulum. Some virions fused directly with the plasma membrane allowing their electrondense content to be shed into the cytoplasm from the outside. Finally, some particles came into contact with the plasma membrane through a stalk-like structure which permitted the passage of the dense core material into the cell.

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, Virology 60, 237–250) that in FV3 +-infected cells late proteins (and by implication late mRNAs) were synthesized in the absence of viral DNA replication.

Intracellular distribution and phosphorylation of virus-induced polypeptides in frog virus 3-infected cells

Analysis of the intracellular localization of frog virus 3 (FV 3)-infected cell polypeptides (ICPs) showed that although the largest amount of newly synthesized proteins were recovered, at any time postinfection, in the cytoplasmic fraction, most of the early viral-induced ICPs were present in the nuclei and the relative molar ratio of ICPs 90, 42, 31 was higher in the nuclei than in the cytoplasm. After 5–8 hr late polypeptides species were found concentrated in the nucleus (ICPs 70, 63, 12). The absence of viral DNA replication did not prevent the appearance of ICPs in the nucleus. Under abortive conditions produced by the replacement of arginine by canavanine, only a restricted set of ICPs was induced (early polypeptides) and the nuclear concentrations of ICPs 31 and 42 was modified. Newly phosphorylated proteins in infected cells were predominantly found in the nucleus, most species being viral induced. Moreover, following the induction of protein kinase, the specific activity of this enzyme was about 50 times higher in the nuclei than in the cytoplasmic fraction. These results extend the evidence for specific steps of FV 3 replication in the host nucleus, emphasizing the importance of the nucleus as the predominant site of viral polypeptide phosphorylation.