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Structure of Varicella-Zoster Virus DNA

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Structure of Varicella-Zoster Virus DNA JOURNAL OF VIROLOGY, Nov. 1981, p. 516-525 Vol. 40, No. 2 0022-538X/81/110516-10$02.00/0 Structure of Varicella-Zoster Virus DNA STEPHEN E. STRAUS,I* HARI S. AULAKH,' WILLIAM T. RUYECHAN,2 JOHN HAY,3 THOMAS A. CASEY,3 GEORGE F. VANDE WOUDE,4 JOHN OWENS,' AND HOLLY A. SMITH' Laboratory of Clinical Investigation, National Institute ofAllergy and Infectious Diseases,' Bethesda, Maryland 20205, and Laboratory ofMolecular Virology, National Cancer Institute,4 Department of Biochemistry2 and Department ofMicrobiology,3 Uniformed Services University of the Health Sciences, Bethesda, Maryland 20014 Received 30 March 1981/Accepted 13 June 1981 Varicella-zoster virus (VZV) DNA was prepared from nucleocapsids and from enveloped virions of a laboratory strain (Ellen) and directly from the vesicle fluids of patients with zoster infections. VZV Ellen nucleocapsid DNA was cleaved with 11 different restriction endonucleases and electrophoresed in agarose gels. The restriction profiles of the nucleocapsid DNA were identical to those of the DNA recovered from purified virions, but differed from those of another VZV strain (KM). In vitro-labeled VZV K.M. DNA purified directly from vesicle fluid yielded a distinct restriction pattern which appeared to be unchanged after several tissue culture passages of the isolate from that fluid. Restriction endonuclease analysis (EcoRI or BglII) of VZV DNA revealed the presence of four cleavage fragments with a molar ratio of -0.5. No individual fragments with molar ratios of 0.25 were noted. This observation suggests that the VZV genome may contain one invertible segment. Comparison of the electrophoretic migrations of VZV DNA fragments relative to those of DNAs of known size permitted calculation of the VZV genome size to be 72 x 106 to 80 x 106 daltons. These results were confirmed by electron microscopy which demonstrated a genome size of about 76 x 106 daltons for passaged and unpassaged VZV DNA. Electron microscopy also re- vealed that some of the DNA molecules recovered from nucleocapsids or directly from vesicle fluids were superhelical circles. Varicella-zoster virus (VZV) is a human her- nique, by which sufficient DNA was obtained to pesvirus which causes the primary infection var- permit a number of original observations (13). icella (chicken pox) and, after a highly variable The molecular mass of VZV DNA was calcu- latency period, may reactivate to produce zoster lated to range between 92 and 110 x 106 daltons (shingles) (19). Since its initial isolation and in from measurements of its rate of sedimentation vitro cultivation by Weller in 1953 (21), this in neutral sucrose (5, 13). In addition, electro- virus has been found to have a number of dis- phoretic profiles of DNA cleaved with restriction tinctive properties which have impeded detailed endonucleases suggested the presence of sub- characterization of its genome. In all tissue cul- molar fragments and revealed small but distinct ture systems described thus far, VZV remains variations among laboratory-passaged clinical highly cell associated, and only small numbers isolates (15). By electron microscopic measure- of virions are released from cells during produc- ments of molecules extracted from purified viri- tive infection. They can be more efficiently re- ons, Dumas et al. (3) have recently estimated covered from disrupted, infected cells by a vari- that the molecular mass of the VZV genome is ety of methods, but still not in quantities con- 80 x 106 daltons. ducive to extensive biochemical investigations. In this report we have further characterized Some investigators have therefore turned to the structure of VZV DNA. Biochemical, elec- direct purification of viral DNA from infected trophoretic, and electron microscopic methods cells, but they have been frustrated by yet an- were used to study both DNA recovered directly other unfortunate feature of VZV. The viral from vesicular lesions of patients with zoster and DNA possesses a buoyant density in cesium DNA of the laboratory-passaged VZV strain El- chloride of about 1.705 g/cm3 which is suffi- len. ciently similar to that of human cell DNA (about 1.697 g/cm3) to prevent ready separation by MATERIALS AND METHODS isopycnic centrifugation (7, 13). One of the more Cells and viruses. A frozen suspension of WI-38 successfully exploited means of VZV DNA pu- cells (human diploid fibroblasts) infected with VZV rification utilized a modification ofthe Hirt tech- strain Ellen (isolated originally from an adult patient 516 VOL. 40, 1981 VARICELLA-ZOSTER VIRUS DNA 517 with varicella) was obtained from the American Type Preparation ofvaricella-zoster virions. Virions Culture Collection (ATCC VR-586). The specimen were purified by the method of Dumas et al. (3). was thawed and passaged twice in several roller culture Briefly, virions released from infected cells during tubes of WI-38 cells to develop a working stock. Other treatment with trypsin and EDTA were pelleted at isolates employed were obtained from vesicular skin high speeds and then banded in 5 to 55% sucrose lesions of immunocompromised patients. VSV strain gradients. KM was from a 3-year-old girl with rhabdomyosar- Purification of viral DNA. Nucleocapsids or vir- coma and disseminated zoster. VSV strain CG was ions in 2x STE buffer were digested by the addition from a 62-year-old man with Wegener's granulomato- of proteinase K (1 mg/ml) and incubated at 50°C for sis and dermatomal zoster. VSV strain HO was from 30 min. The DNA was gently extracted one time with a 65-year-old woman with lymphoma and dermatomal redistilled phenol saturated with 0.2 M Tris-hydro- zoster. Vesicle fluid was aseptically aspirated from the chloride (pH 7.9), again with equal volumes of phenol freshest lesions, delivered into 1 cc of veal infusion and chloroform containing 2% (vol/vol) isoamyl alco- broth, and stored at 0 to 4°C for up to 18 h until a hol, and once finally with chloroform alone. The upper portion of the sample was inoculated into WI-38 and aqueous phase containing VZV DNA was very care- human embryonic kidney cell roller culture tubes. The fully removed with a wide-bore pipette and precipi- remainder of the specimens was stored at -70°C until tated in 3 volumes of absolute ethanol at -20°C. The needed for DNA purification. All isolates used in this DNA was pelleted by centrifugation at 10,000 rpm for study demonstrated characteristic cytopathic changes 30 min at 4°C. In smaller preparations precipitation of VZV in the cell lines employed. was aided by the addition of yeast RNA (10 jLg/ml). Large-scale preparations of VZV-infected cell pools DNA precipitates were gently resuspended in 10 mM were performed in 175-cm2 flasks and 850-cm2 roller Tris-hydrochloride (pH 7.5)-0.1 mM EDTA. bottles of primary human embryonic lung fibroblasts VZV DNA was recovered from human vesicle fluid (HEL, passages 10 to 14), purchased from Flow Lab- and from purified viruses by dilution into 2x STE oratories, Rockville, Md. (Flow 5000 cell line). Cell buffer, treatment with proteinase K, extraction, and sheets were infected by the addition of freshly har- precipitation, as described above. vested infected cells at low multiplicity (less than 1 Adenovirus type 2 and its DNA were prepared as infected cell per 30 uninfected cells) or at high multi- previously described (20). Phage lambda DNA was plicity (1 infected cell per 4 uninfected cells). Infected purchased from Bethesda Research Laboratories, cultures were grown at 37°C in media consisting of Rockville, Md. Simian virus 40 (SV40) form I DNA equal parts of minimal essential medium and medium was purchased from Bethesda Research Laboratories 199 supplemented with 10% heated fetal calf serum, and converted to form II molecules by repeated freez- penicillin, gentamicin, and glutamine until typical cy- ing and thawing. topathic effects were observed in most cells (5 to 7 In vitro labeling of viral DNA. Purified VZV days). The medium was decanted, and infected cells Ellen DNA was labeled in vitro to specific activities of were scraped, suspended, and dispersed by gentle mix- 1 X 107 to 2 x 107 cpm/,tg with all four tritiated ing in a Vortex miixer in a small volume of fresh media. nucleoside triphosphates by the nick translation meth- Preparation of viral nucleocapsids. VZV nu- ods of Maniatis et al. (9) and Kelly et al. (6). In cleocapsids were isolated by a method used to isolate experiments with DNA isolated from vesicle fluid, or herpes simplex virus nucleocapsids (Deniston et al., for blot hybridizations, 10 to 100 ng of DNA was Gene, in press). Freshly harvested VZV-infected HEL labeled by a similar protocol with one 32P-labeled and cells were centrifuged at 1,500 rpm at 4°C for 10 min. three unlabeled deoxynucleoside triphosphates. Spe- The cell pellet was cycled three times through freezing cific activities of about 5 x 107 cpm/4ug of DNA were in a slurry of dry ice and acetone and thawing at 37°C. routinely obtained. Normally, DNase treatment was The pellet was then suspended in 0.5 ml of lysis buffer unnecessary. (0.5% Nonidet P-40, 3.6 mM calcium chloride, 5 mM Thermal elution profiles. Thermal elution mid- magnesium acetate, 125 mM potassium chloride, 0.5 points [Tm(e)] of 3H-labeled VZV DNA was deter- mM EDTA [pH 7.5], 6 mM /?-mercaptoethanol, and mined by the methods described previously (10). The 0.5% deoxycholate) for each 75 to 150 cm2 of cell Tm(e) was calculated as the temperature at which monolayer originally harvested. To each 1 ml of this 50% of total counts were eluted as single-stranded solution was added 25 Ag (approximately 50 U) of DNA molecules.
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