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 revealed 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. The guanine-plus-cytosine mole per- DNase I and 25 itg of RNase A (both from Worthing- cent content (G+C) was calculated by the formula of ton Diagnostics, Freehold, N.J.). The cell lysate was Mandel and Marmur corrected for the 0.187 M sodium incubated at 30°C for 30 min and extracted once with ion concentration of the eluting buffer (8), namely, 0.5 volume of genosolv-D (trichloro-trifluoroethane; G+C = 2.44 [Tm(e) - 69.4]. Allied Chemicals, Morristown, N.J.). The mixture was Restriction endonuclease analysis. All restric- vigorously shaken for 1 min and centrifuged at 1,000 tion endonucleases were purchased from Bethesda rpm for 10 min at 4°C. The aqueous phase was gently Research Laboratories or New England Biolabs, Bev- removed and layered over a discontinuous gradient of erly, Mass. Viral DNA was digested by the endonucle- 5 and 40% glycerol in lysis buffer. The gradient was ases under the reaction conditions recommended by centrifuged at 40,000 rpm for 40 min at 4°C. The the manufacturer for each enzyme. Reactions were supernatant was discarded, and the pellet containing terminated by the addition of 0.1 volume of a solution viral nucleocapsids was suspended in 2% sodium do- containing 7.5% sucrose, 0.1% bromphenol blue, and decyl sulfate-0.1 M Tris-hydrochloride (pH 7.5)-20 100 mM EDTA. Samples (15 tl) were placed in pre- mM EDTA (2x STE buffer). formed wells in 20-cm by 25-cm by 6-mm horizontal 518 STRAUS ET AL. J. VIROL. slab gels of 0.5, 1.0, or 1.2% agarose in E buffer (40 pared from each 850-cm2 roller bottle of infected mM Tris [pH 7.4], 20 mM sodium acetate, 1 mM HEL cells. Only about one-fourth to one-half EDTA). Gels were run in E buffer containing 0.5 jig of that amount of DNA was obtained from virions ethidium bromide per ml at 60 mA constant current purified from the same number of cells. Nucleo- for 16 to 24 h. After electrophoresis DNA bands were capsid DNA was observed to have a ratio of visualized with a UV transilluminator and photographed. Negative photographic transparencies were optical density at 260 nm to optical density at analyzed with an Auto Scanner densitometer (Helena 280 nn of 1.90 to 1.95, indicating high purity. Laboratories, Beaumont, Tex.). Areas under the 3H-labeled VZV DNA labeled in vitro was curves were determined by weighing cutouts of the hybridized in the presence of 20 mg of DNA chart paper bearing relevant peaks. purified from uninfected HEL cultures per ml to Electron microscopy of vesicle fluid speci- a Cot of over 3,000 mol. s/liter. Different prepa- mens. Vesicle fluid specimens in veal infusion broth rations of DNA recovered from nucleocapsids were examined electron microscopically before DNA demonstrated from 5 to 20% hybridization to the extraction. Samples of the specimens were subjected to either negative staining with 2% phosphotungstic cellular DNA. The contamination of nucleocap- acid or positive staining for nucleic acids with uranyl sid DNA with cellular DNA was verified in acetate. Parlodian-coated copper grids were carbon experiments in which gels containing endonucle- coated and air glow treated before their use in negative ase-cleaved nucleocapsid DNA or HEL DNA stains. For negative staining a treated grid was touched were blotted onto nitrocellulose membranes and to a drop of vesicle fluid. The grid was blotted after a hybridized with 32P-labeled VZV nucleocapsid 10-s adsorption period and then rinsed by touching DNA (17) labeled in vitro. Autoradiographs the grid to a drop of distilled water. The water was demonstrated that the nucleocapsid DNA probe blotted, and the grid was stained for 1 min by touching hybridized to the individual VZV DNA bands as it to a drop of 2% phosphotungstic acid (pH 7.4). After well as to the lanes containing HEL cell DNA final blotting, the grid was air dried. Staining for nucleic acids utilized 80,ul of vesicle fluid to which 10 (data not shown). DNA recovered from purified ,ul of 4 M ammonium acetate and 10 pl of a 1-mg/ml virions appeared to be free of cellular DNA solution of cytochrome c were added. The mixture was contamination by these same criteria. spread onto a hypophase of 0.4 M ammonium acetate. To estimate the G+C content of VZV DNA, The resulting surface film was picked up with 200 3H-labeled VZV DNA labeled in vitro was ad- mesh Parlodian-coated copper grids which were then sorbed to a column of hydroxylapatite, and the stained for 25 to 30 s with 0.5 mM uranyl acetate, step-wise elution of counts in single-stranded rinsed with isopentane, and air dried. All grids were molecules was monitored after sequential 40C examined in a Zeiss EM10A high-resolution transmis- to sion electron microscope. temperature increments from 60 1000C. As a Electron microscopy of DNA. The method used control the Tm(e) of 3H-labeled adenovirus type for mounting the VZV DNA was the aqueous ammo- 2 DNA was measured. The Tm(e) of 91.80C for nium acetate technique of Davis et al. (2). One micro- that DNA and the calculated G+C content liter of a solution containing about 50 ,ug of purified (55%) agreed with published values (12). The VZV Ellen DNA per ml was mixed with 78 to 79 IlI of data from six individual determinations with a solution containing 10 mM Tris-hydrochloride (pH VZV DNA revealed an average Tm(e) of 89.00C 7.6), 1 mM EDTA, and 10 Ml each of 4 M ammonium and a calculated G+C content of 47.8 mol%, acetate and 1-mg/ml cytochrome c. Owing to the low which agrees well with the values of 46 and 47 concentration of DNA in extracts in vesicle fluid, they mol% previously reported in studies of the sedi- were mounted without dilution. Thus, ammonium ace- mentation of VZV DNA in cesium chloride (5, tate and cytochrome c solutions were added to 80 p1 of the given DNA solution. The DNA was then spread, 7). mounted, and stained as described above. Restriction endonuclease analyses. Unla- SV40 form II DNA (50 Lg/ml) was used as the beled DNA purified from VZV nucleocapsids or molecular weight standard. The SV40 DNA was either virions was cleaved with restriction endonucle- mixed with the VZV DNA (0.1 ,ug/80 Il of sample) or ases and subjected to electrophoresis on 0.5, 1, spread separately under identical conditions. No dif- or 1.2% agarose gels. Figure 1 displays the elec- ference in contour length was seen between SV40 trophoretic patterns ofDNA in 0.5% agarose gels DNA molecules mounted in these two ways. Negative after cleavage by one of 11 different enzymes. photographs of discrete molecules were projected onto Purified virion DNA with a blackboard with a latern slide projector, and the digested EcoRI, DNA contour lengths were determined with a Keuffel BamHI, or BglII yielded identical cleavage pat- and Esser map-measuring device. terns to DNA from nucleocapsids (data not shown). The only difference in the electrophoretic profiles observed in repeated experiments RESULTS was in the diffuse background in lanes of cleaved Yield, purity, and physical properties of nucleocapsid DNA which presumably repre- VZV DNA. Approximately 4 ug of DNA was sents unresolved small fragments of degraded routinely recovered from nucleocapsids pre- viral DNA or cleaved cellular DNA contami- VOL. 40, 1981 VARICELLA-ZOSTER VIRUS DNA 519 Ad2 VZV(Ellen)

S.-,cr 7 -4 -c4 0 E -' 0 a Q cn c -Fo E c - uLL m CDn CO LLi I ) U) O

FIG. 1. Restriction endonuclease analysis of VZV (Ellen) DNA by electrophoresis in 0.5% agarose gels. VZV DNA purified from nucleocapsids was cleaved with 11 different enzymes and run in parallel with an EcoRI-cleaved adenovirus type 2 (Ad2) DNA standard. nants or both. The cleavage patterns remained molar fragments in the VZV genome. Negative unchanged after 22 serial high-multiplicity pas- photographs of gels displaying VZV Ellen DNA sages or 14 low-multiplicity passages (data not cleaved by these enzymes were scanned with a shown). densitometer (Fig. 3), and estimates ofthe molar DNAs derived from zoster lesions were also ratios of the bands were derived (Table 1). Al- subjected to restriction endonuclease analysis. though these techniques are somewhat crude Figure 2 depicts the gel profiles of VZV Ellen and the results varied among different gels, the and KM DNAs. The electrophoretic pattern of areas under each of the individual peaks sug- VZV KM DNA differs from that of VZV Ellen gested that the submolar bands were all approx- DNA. To date, the DNAs of VZV isolates from imately 0.5 M (EcoRI-A, -E, -F, and -J; BglII-C, eight epidemiologically unrelated individuals -D, -G, and -J). No bands containing only 0.25 have been examined by restriction endonuclease M fragments were apparent in these digests. The analysis (work in progress). Each isolate pos- restriction patterns of VZV KM DNA (Fig. 2) sesses a unique set of cleavage profiles. This also revealed the presence of submolar frag- confirms an earlier observation that the DNAs ments. Quantitative densitometric studies of of different VZV isolates possess unique restric- KM DNA (data not shown) suggested that the tion patterns (15). Importantly, the restriction bands with molar ratio of 0.5 are identical in pattern of the DNA derived from passaged virus both the vesicle fluid and laboratory-passaged is identical to that of the DNA extracted directly material and correspond directly to the bands from the patient's vesicle fluid, indicating that occupying similar positions in the VZV Ellen early cell passages do not introduce a substantial cleavage patterns. proportion of defective genomes into the virus Electrophoresis of endonuclease-digested pool. VZV DNA in gels containing higher concentra- Molar ratio of VZV DNA fragments. The tions of agarose permitted identification of the electrophoretic profiles of EcoRI- or BglII- smaller molecular weight fragments (Fig. 3C cleaved DNA (Fig. 1) demonstrated unequal through F). In these analyses 0.5 M fragments intensity of some bands relative to their neigh- of VZV DNA were detected in digests with SmaI boring bands, suggesting the presence of sub- (fragments T, V, and Y), BgII (F), BamHI (I), 520 STRAUS ET AL. J. VIROL.

A ... bands is 79.4 x 10' daltons for EcoRI, BamHI .. L. 3 (Table 2), and BglII digests (not shown). BglI Ba fragments total 72.9 x 106 daltons, the Sall C fragments total about 71.5 x 106 daltons, and the sum of the masses of the SmaI fragments is DE about 73.9 x 106 daltons. Electron microscopic studies. Examina- F 1~ tion of negatively stained grids of vesicle fluid specimens revealed both enveloped and partially H disrupted virions. Nearly all of the virions purified from VZV Ellen-infected HEL cells proved to be enveloped. Very little cell debris was observed, and none of these preparations was K found to contain free DNA. Hence, essentially L all of the DNA molecules observed after extrac- tions originated from virions or nucleocapsids. M Sixteen well-extended linear molecules of VZV strain Ellen DNA extracted from purified N- virions were photographed at a magnification of 2,520x (for example, Fig. 4A). The contour o lengths of the molecules were measured (Fig. 5) and compared to those of 13 SV40 form II DNA molecules. The molecular weight of the VZV DNA was calculated from the ratio of the VZV to SV40 DNA lengths based on a molecular mass of 3.4 x 106 daltons for SV40 DNA (1, 4, FIG. 2. Autoradiographs of 0.5% agarose gels con- 14). The mean molecular mass of the VZV Ellen taining EcoRI-cleaved, 32P-labeled VZV DNAs la- DNA was 75.7 x 106 ± 3.4 x 106 daltons, which beled in vitro. Lanes: 1, 32P-labeled VZV Ellen nu- is in good agreement with the molecular masses cleocapsid DNA; 2, 2P-labeled VZVKM vesicle fluid determined from the restriction enzyme analyses DNA; 3, profile of 32P-labeled VZV KM DNA after presented above. Measurements using the Esch- tissue culture passage four times in WI-38 and two times in HEL cells. The two smallest fragments (P erichia coli plasmid pSC179 (19.6 x 106 daltons) and Q) are present in all isolates, but they have run gave approximately the same VZV DNA length too far ahead of the larger fragments to be shown measurements (data not shown). DNA from a here. second extensively passaged VZV strain (Scott) gave a compact size distribution very similar to and possibly also SalI (L and P) (Fig. 3C through that of strain Ellen, with a mean molecular mass F). Again, no individual 0.25 M fragments were of 76.5 x 106 ± 4.7 x 106 daltons (data not observed. shown). Molecular weights of VZV DNA frag- DNA extracted from the vesicle fluid samples ments. Estimates of the molecular weights of of three patients were also studied, and the one the VZV DNA restriction fragments were ob- extract examined (strain CG) was shown to be tained by measuring their electrophoretic mo- infectious (J. Hay, unpublished data), support- bilities relative to that of adenovirus type 2 DNA ing the observation of Dumas et al. (3) (Fig. 4B). EcoRI fragment F which was included as an The sizes of 29 of the CG DNA molecules re- internal marker in all gels. The molecular covered from vesicle fluid are displayed in Fig. weights were estimated with double-exponential 5. The molecules obtained from the clinical spec- regression formulas empirically derived from the imens were more heterogeneous in size than relative mobilities in 0.5 or 1.0% agarose gels of those recovered from VZV Ellen-infected cul- over 100 adenovirus type 2 and phage lambda tures. This heterogeneity may be the result of DNA fragments of known size (data not shown). the presence of defective viral genomes or of The actual VZV DNA fragment sizes may differ damage inflicted upon the DNA in the vesicle slightly from the estimated sizes because the by enzymes released during the inflammatory relative migration of DNA fragments is depend- response to the infection. Sixteen of the VZV ent upon G+C content and the combined G+C CG DNA molecules were in the molecular mass contents of the VZV and standard DNAs are range of 67 x 106 to 86 x 106 daltons and the different. Assuming that certain fragments are mean molecular mass of this subset of molecules present in molar ratios of 0.5 (Table 1), the sum was 76.1 x 106 ± 4.2 x 106 daltons. Similar of the molecular masses of the visible VZV DNA analyses yielded mean molecular masses of 72.6 VOL. 40, 1981 VARICELLA-ZOSTER VIRUS DNA 521

A EcoR B Bg: II ....I !i ....

%-'-"-I C D .. U *.1K\..\, m I wi-i-Ct / Ai- 4. I

A 1 \-i ' A;- v w E F RMIUIL"

~~~~~~~~'~

:j I

------

(K'- I

FIG. 3. Densitometric analyses of endonuclease-generated cleavage patterns of VZV DNA. Negative photographs (not shown here) of UV-transilluminated gels were scanned with a densitometer. VZV Ellen DNA purified from nucleocapsids was cleaved with six different enzymes and electrophoresed in 0.5% (panels A and B) or 1.0%o (panels C through F) agarose gels. x 106 + 3.3 x 106 daltons for HO DNA and 78.6 4C and E appear to be full length (70 x 106 to 80 x 106 + 4.4 x 106 daltons for the vesicle fluid x 106 daltons). Supercoiled molecules have been KM DNA (data not shown). Thus, these DNA observed in every preparation examined includ- molecules are comparable in size to those puri- ing both the VZV Ellen DNA and the various fied from infected cultures. vesicle fluid DNA specimens. The usual fre- Many of the DNA molecules seen in this study quency of occurrence of such molecules appears were tangled and were thus unsuitable for con- to be about 5 to 10%, although much higher tour length measurements. In addition to linear frequencies have also been observed (W. T. Roy- and tangled linear molecules, some partially re- echan, unpublished observations). laxed supercoiled molecules were observed (Fig. 4C through E). Because the supercoiled DNA is DISCUSSION more compacted than the partially relaxed re- In this communication an extensive structural gions of these molecules, their sizes could only and biochemical analysis of the DNA of the be estimated. The molecules displayed in Fig. laboratory-passaged strain VZV Ellen is pre- 522 STRAUS ET AL. J. VIROL. TABLE 1. Molar ratios of VZV Ellen DNA cleavage The results of the restriction endonuclease fragmentsa analyses reported here have extended the earlier BamHI Bglll EcoRI observations of the structural features of VZV Frag- genome in several regards. By using sufficient ment Ob- Sug- Ob- Sug- Ob- Sug- DNA to be visualized directly by ethidium bro- served gested served gested served gested mide fluorescence, we achieved a resolution of A 0.83 1.0 1166 1.0 0.48 0.5 the individual endonuclease-generated frag- B 0.79 1.0 1 * 1.0 0.78 1.0 ments that was a great improvement over that C 0.95 1.0 0.66 0.5 1.00 1.0 obtained previously by autoradiography (5, 11, 0.5 0.86 1.0 D 0.88 1.0 1 J4 15). Sharp and reproducible electrophoretic pro- E 0.92 1.0° 1 1.0 0.47 0.5 F 0.95 1.0 1.00 1.0 0.56 0.5 files were consistently observed (Fig. 1 and 3). G 1.08 1.0 0.72 0.5 0.90 1.0 Moreover, these studies strengthen the sugges- H 1.05 1.0 1.76 1.0 1.05 1.0 tion that each VZV isolate possesses a distinct M }.62 1.0 1.35 1.0 1.04 1.0 restriction pattern (15) which does not alter 2.6212.0 0.71 0.5 0.49 0.5 upon in vitro passage from vesicle fluid. K 1.03 1.0 N2D 1.0 0.95 1.0 Endonuclease analysis indicated the existence L 1.03 1.0 N 1.0 1.05 1.0 of four 0.5 M fragments in the EcoRI and BglII M 1.11 1.0 1.35 1.0 1.00 1.0 gel profiles. No individual 0.25 M fragments were N 1.09 1.0 NDb 1.0 1.05 1.0 observed in the gel profiles of DNA cleaved by 0 0.98 1.0 ND 1.0 0.92 1.0 P 1.04 1.0 ND 1.0 ND 1.0 11 different enzymes, suggesting that the VZV Q 2.24 2.0 ND 1.0 ND 1.0 R 1.13 1.0 ND 1.0 TABLE 2. Estimations of the molecular mass of S 1.15 1.0 ND 1.0 VZV Ellen DNA from the electrophoretic patterns of T 137 0.5 - - - - restriction endonuclease-generated cleavage u }13{ 1.0 - - - - fragments' V 0.92 1.0 - - - - BamHI EcoRI W 0.78 1.0 - - - - Fragment fragment mass fragment mass X ND 1.0 - - - - (megadaltons) (megadaltons) Y ND 1.0 - - - - Z ND 1.0 - - - - A 10.59 10.78 AA ND 1.0 - B 7.11 9.77 C 6.32 8.99 'Data are derived from two to five individual de- D 5.70 8.30 terminations. E 5.45 7.97 b ND, Not determined; densitometry peaks too F 5.20 7.70 small for accurate determinations. G 4.47 5.83 -, No corresponding fragments observed. H 4.13 5.67 I 3.06 5.28 sented. These results have been compared, J 3.02 5.08 where feasible, to those obtained with DNA K 2.53 4.83 L 2.36 4.37 recovered directly from unpassaged VZV-con- M 2.14 3.32 taining clinical specimens. Most of the present N 1.81 2.79 studies have utilized DNA from viral nucleocap- 0 1.71 2.68 sids rather than from virions since substantially P 1.61 1.10 more DNA could be recovered from nucleocap- Q 1.40 0.71 sid preparations. However, the nucleocapsid R 1.28 __b preparations were shown to be contaminated S 1.15 - with cellular DNA and were feared to contain T 0.99 - populations of aberrant VZV DNA molecules U 0.96 - which would never be encapsidated and V 0.89 - properly W 0.79 - enveloped. Therefore, the general structural fea- X 0.70 - tures of the VZV nucleocapsid DNA were veri- Y 0.63 - fied using the more precious but uncontami- Z 0.56 - nated virion DNA. AA 0.40 - Previous reports of the G+C content of VZV Total 79.4 ± 4.3 79.4 ± 3.3 DNA were independently affirmed by thermal aData are calculated from three to five individual elution midpoint experiments. The present re- determinations. Molecular mass estimations take ac- sult of47.8% agrees well with the values reported count of the suggested molar ratios of each fragment in earlier studies of VZV DNA sedimentation in as indicated in Table 1. cesium chloride (5, 7). b, No corresponding fragments observed. VOL. 40, 1981 VARICELLA-ZOSTER VIRUS DNA 523

I.Opm .4 lOin t0e

0.5pm %.

1. a0 AI aoa .. E ..~~~~~~~~~~~~~~~~~~~~~~~~~~

f , b ;

A1.I -e.I ^ .A *'~~~~~~ -, I

FIG. 4. Electron micrographs of VZV DNA. Examples of (A) full-length linear DNA molecules recovered from VZV Ellen virions and (B) full-length linear VZV CG DNA recovered from vesicle fluid. Also shown are partially relaxed supercoiled molecules: (C) VZV Ellen virions DNA, (D) VZV CG DNA from vesicle fluid, and (E) VZV KM DNA from vesicle fluid.

genome does not contain two invertible regions molecular would result in two isomeric forms of as does herpes simplex virus DNA (16), but is the genome. Specific nucleotide sequences which analogous to that of pseudorabies virus which reside near the DNA terminus in one confor- possesses only a single invertible segment (18). mation are near the junction with the nonin- Recognizing the inherent limitations of quanti- verting segment in the second confirmation, and tative restriction enzyme analyses, it is possible vice versa. Endonucleolytic cleavage of a genome that some bands represent superimposition of of this type would generate molar fragments 0.25 M fragments upon other bands. Examina- from sequences lying entirely within unique re- tion of all of the possible cases of this in Fig. 3, gions of the genome. The four fragments con- however, does not give rise to a consistent or taining the invertible terminus and straddling plausible model for VZV DNA structure. De- the junction site would be 0.5 M. A requirement spite the probable absence, then, of 0.25 M frag- of this model is that the sum of the molecular ments, VZV DNA may still possess a herpes weights of two of the 0.5 M fragments produced simplex DNA-like structure, but it must be pos- by cleaving one of the genome forms must equal tulated that all 11 restriction enzymes studied so the sum of the weights of the 0.5 M fragments of far (Fig. 1) fortuitously cut inside a second set of the other isomer. The present data appear to repeated sequences. fulfill this requirement. The molecular masses Inversion of but one segment of the VZV DNA calculated for the 0.5 M BglII fragments of VZV 524 STRAUS ET AL. J. VIROL. 0 10 20 30 40 50 60 70 80 90 100 lower-molecular-weight fragments observed or . from both. 5 The genome size was independently assessed Ellen electron microscopically. VZV Ellen DNA mol- 4 ecules averaged 75.7 x 106 daltons when compared with the SV40 and plasmid standards. 3 VZV CG DNA recovered directly from vesicle U) 2 fluid showed size heterogeneity, as did the DNA U1) from the other patient material studied. None- -1 theless, within the subset of larger and probably 0 II I II more complete molecules the mean molecular .0 mass was also about 76 x 106 daltons. 0 5 - C.G. These estimates are significantly smaller than E those of Rapp and associates, who calculated z 4 genome masses of 92 x 106 to 110 X 106 daltons 3 from the results of sedimentation studies of various strains of VZV DNA (13). There is no 2 definite understanding of the basis for this dis- crepancy, except to suggest that centrifugation methods are relatively more inaccurate and that I I I J II the method of DNA purification (Hirt tech- 0 10 20 30 40 50 60 70 80 90 100 nique) employed in those studies may select for Molecular Weight x 106 replicating as well as unit length molecules. If FIG. 5. Molecular weights of VZV DNA molecules concatemeric forms participate in VZV DNA calculated from electron microscopic measurements. replication, as they appear to in herpes simplex Sixteen fully extended VZV Ellen DNA molecules DNA replication (16), their presence in a Hirt purified from virions and 29 fully extended VZV CG extract may result in a high estimate for the size DNA molecules purified from vesicle fluid were of the genome. The present results agree more spread and measured along with SV40 form II molecules as size standards. closely with other electron microscopic measurements recently reported by Dumas et al. (3) wherein a genome mass of about 80 x 106 daltons Ellen (C, D, G, and J) are 7.80 x 106 7.25 x 10', was obtained. 6.30 x 106, and 5.60 x 106 daltons, respectively. One final feature of the structural characteri- The sum of the molecular masses of fragments zation of the VZV genome that bears mention is C and J is 13.40 x 106 daltons, which is very the possibility that the genome may be circular close to 13.55 x 106 daltons, the sum of the (Fig. 4). All nucleocapsid- and virion-derived masses of fragments D and G. Similarly, the sum DNA preparations examined thus far have been of the masses of EcoRI fragments A and J, 15.86 found to contain partially relaxed superhelices. X 106 daltons, is nearly equal to the sum of the The Epstein-Barr virus genome is known to fragments of fragments E and F, 15.67 x 10k' assume a superhelical conformation in the cell, daltons. Despite this evidence in support of a but is believed to be linear in virions (1). VZV, model of the VZV genome in which only one on the other hand, may be able to package segment is invertible, we must await the results superhelices. of ongoing mapping studies for a definitive ap- praisal of the genomic organization. ACKNOWLEDGMENTS Because of the high resolution of the ethidium We are indebted to David Alling for performing the regression analyses and to Peter Howley for advice and assistance bromide-stained gels it was possible to identify in the early phases of this study. We also thank Anthony many cleavage fragments and to assign approx- Fauci and Philip Pizzo for patient referrals. imate molecular weights to them by comparison with known standards. After correcting for mo- ADDENDUM IN PROOF lar ratios, summation of the weights of the in- Mapping studies recently reported by A. M. Dumas, dividual fragments yielded genome size esti- J. L. M. C. Geelen, M. W. Weststrate, P. Wertheim, mates ranging between 71.5 x 106 and 79.4 x 106 and J. van der Noorda (J. Virol. 39:390-400, 1981) also daltons (SalI and EcoRI, BamHI or BglII di- indicate that VZV possesses only one short invertible gests, respectively). The lower genome weights segment. calculated with the data from BglI, Sall, and LITERATURE CITED SmaI digests may have resulted from the lack of 1. Colby, B. M., J. E. Shaw, G. B. Elion, and J. S. detection of small fragments, from uncertainties Pagano. 1980. Effect of acyclovir [9-(2-hydroxy- in estimating the molar ratios of some of the methyl)guanine] on Epstein-Barr virus DNA replica- VOL. 40, 1981 VARICELLA-ZOSTER VIRUS DNA 525 tion. J. Virol. 34:560-568. apatite. Biochim. Biophys. Acta 474:445-455. 2. Davis, R. W., M. Simon, and N. Davidson. 1971. Elec- 11. Oakes, J. E., J. P. Iltis, R. W. Hyman, and F. Rapp. tron microscope heteroduplex methods for mapping 1977. Analysis by restriction enzyme cleavage of human regions of base sequence homology in nucleic acids. varicella-zoster virus DNAs. Virology 82:353-361. Methods Enzymol. 21:413-428. 12. Piiia, M., and M. Green. 1965. Biochemical studies on 3. Dumas, A. M., J. L. M. C. Geelen, W. Maris, and J. adenovirus multiplication. IX. Chemical and base com- van der Noordaa. 1980. Infectivity and molecular position analysis of 28 human adenoviruses. Proc. Natl. weight of varicella-zoster virus DNA. J. Gen. Virol. 47: Acad. Sci. U.S.A. 54:547-554. 233-235. 13. Rapp, F., J. P. Iltis, J. E. Oakes, and R. W. Hyman. 4. Fiers, W., R. Contreras, G. Haegeman, R. Rogiers, A. 1977. A novel approach to study the DNA of herpes Van de Voorde, H. Han Heuverswyn, J. Van Her- zoster virus. Intervirology 8:272-280. eweghe, G. Volckaert, and M. Ysebaert. 1978. Com- 14. Reddy, V. G., B. Thimmappaya, R. Dhar, K. N. Sub- plete nucleotide sequence of SV40 DNA. Nature (Lon- ramanian, B. S. Zain, J. Pan, P. K. Ghosh, M. L. don) 273:113-120. Celma, and S. M. Weissman. 1978. The genome of 5. Iltis, J. P., J. E. Oakes, R. W. Hyman, and F. Rapp. simian virus 40. Science 200:494-502. 1977. Comparison of the DNAs of varicella-zoster vi- 15. Richards, J. C., R. W. Human, and F. Rapp. 1979. ruses isolated from clinical cases of varicella and herpes Analysis of the DNAs from seven varicella-zoster virus zoster. Virology 82:345-352. isolates. J. Virol. 32:812-821. 6. Kelly, R. B., N. R. Cozzrelli, M. P. Deutscher, I. R. 16. Roizman, B. 1979. The structure and isomerization of Leluman, and A. Kornberg. 1970. Enzymatic synthe- herpes simplex virus genomes. Cell 16:481-494. sis of deoxyribonucleic acids. J. Biol. Chem. 245:39-45. 17. Southern, E. M. 1975. Detection of specific sequences 7. Ludwig, H., H. G. Haines, N. Biswal, and M. Benyesh- among DNA fragments separated by gel electrophore- Melnick. 1972. The characterization of varicella-zoster sis. J. Mol. Biol. 98:503-517. virus DNA. J. Gen. Virol. 14:111-114. 18. Stevely, W. S. 1977. Inverted repetition in the chromo- 8. Mandel, M., and J. Marmur. 1968. Use of ultraviolet some of pseudorabies virus. J. Virol. 22:232-234. absorbance-temperature profile for determining the 19. Taylor-Robinson, D., and A. E. Caunt. 1972. The var- guanine plus cytosine content of DNA. Methods En- icella virus. Virol. Monogr. 12:4-88. zymol. 12b:195. 20. Tibbetts, C., K. Johansson, and L. Philipson. 1973. 9. Maniatis, T., A. Jeffrey, and D. G. Kleid. 1975. Nu- Hydroxvapatite chromatography and formamide dena- cleotide sequence of the rightward operator of phage. turation of adenovirus DNA. J. Virol. 12:218-225. Proc. Natl. Acad. Sci. U.S.A. 72:1184-1188. 21. Weller, T. H. 1953. Serial propagation in vitro of agents 10. Martinson, H. G., and E. B. Wagenaar. 1977. Thermal producing inclusion bodies derived from varicella and elution chromatography of nucleic acids on hydroxyl- herpes zoster. Proc. Soc. Exp. Biol. Med. 83:340-346.