Herpes YECHIEL Department
of Virology, Hebrew University-Hadassah Medical Accepted
June II, 1968
Herpes simplex virus DNA was isolated in an intact form by treating the virus particles with detergents. An average molecular weight of 100 X lo6 daltons was determined for herpes simDlex virus DNA. both by electron microscopy and by zone centrifugation in sucrose gradients. INTRODUCTION
The cells were infected with 10 plaque-formHerpes simplex virus (HSV), a member of ing units (PFU) per cell, incubated at 37” the Herpes virus group, was found to in the presence of tritiated thymidine or contain a DNA genome (Ben-Porat and thymidine-2-i4C (specific activity 15,200 and 60.5 mC/mmole, respectively) with or Kaplan, 1962; Russell, 1962) of 68 million daltons (Russell and Crawford, 1964) coated without arginineJ4C (specific activity 150 The isotopes were obtained by a protein capsid (Wildy et al., 1960). mC/mmole). A number of studies have dealt with the size from the Radiochemical Centre, Amersham, England. The cells were harvested after 18 of the DNA genome present in the virions hours, and the labeled virions were isolated of the different strains of the Herpesvirus from the nuclear or cytoplasmic fractions in group. It was found, using ultracentrifugasucrose gradients as previously described tion techniques, that the molecular weight (Levitt and Becker, 1967; Olshevsky et al., of the DNA molecules obtained from bovine 1967; Becker et al., 1967). rhinotracheitis virus, pseudorabies virus, The WR strain of vaccinia virus was and equine herpes virus were 54 X 106, 68 X 106, and 84 X lo6 daltons, respec- propagated in monolayers of HeLa cells, tively (Russell and Crawford, 1963, 1964). labeled with thymidine-2-14C and isolated in sucrose gradients as previously described However, Soehner el al. (1965) demonstrated that the molecular weight of the DNA ob- (Sarov and Becker, 1967). tained from equine abortion virus was 94 X Isolation of Vaccinia DNA lo6 daltons, using both analytical ultracentrifugation and electron microscopic The vaccinia virus particles were detechniques. The development of a technique graded and the DNA was obtained as to isolate intact DNA molecules from vac- previously described (method 2, Sarov and cinia virus particles (Sarov and Becker, 1967) Becker, 1967; Becker and Sarov, 1968). enabled the molecular weight of herpes Vaccinia DNA was used at concentrations simplex virus DNA to be determined by zone of 0.050.2 pg per gradient (Sarov and centrifugation in sucrose gradients and by Becker, 1967). electron microscopy (Kleinschmidt et al., Isolation uf Herpes Simplex Virus DNA 1961). Herpes virions from the cytoplasm of inMATERIALS AND METHODS fected cells were isolated in sucrose gradients. Viruses and Cells The viral DNA was then isolated by the following procedures : The HF strain of herpes simplex virus Procedure 1. The purified herpes virions was propagated in monolayer cultures of BSC, cells grown in Eagle’s medium (1959). were treated with sodium deoxycholate 184
Proceche 2. Purified herpes simplex virions, prepared in a solution of 0.001 M phosphate cont’aining 0.2 M ?;aCl and 0.001 111EDTA, pH 7.0 were treat’ed with sodium dodecyl sulfate with or without
(Difco Co.) 1% (w/v) final concentration (Becker and Chen, 1966). The suspension ~-as carefully layered with a plastic spoon onto sucrose gradients (15-30 % w/w) made in 0.001 M phosphate, pH 7.4.
I ci c-i 250-
FRACTION FIG. 1. Sedimentation of native herpes simplex virus DNA. Purifiedvirions, labeled in t,he DNA with t’ritiated thymidine and in the proteins with arginine-14C were treated with sodium deoxycholate (15; w/v) and layered on a sucrose gradient (&30’% w/w) made in 0.001 M phosphate. The gradients were centrifuged for 13 hours at. 24,000 rpm. Radioactive proteins (5971 cpm) were found at. the bottom of the centrifuge tube. Ivo radioactive DNA was demonstrable in the pellet of the gradient. l -0, IIerpes simplex virus DNA; 0- - -0, viral proteins. TABLE
CUNFOKMATION AND LENGTH OF HERPES %IMPI,FX VIRCS DNA4 RIOLIXULES Partially Number of --~--
34.88 41.16 41.80 47.24 55.70 -. -
1 0 0 0 2
ends 1 2 3 4 5 (i
7 8 9 10 11 12 13 1-l 15 1G
x.24 37.86 39.15 40.71 JO.76 41.25 44.31 48.89 50.92
1 0 0 0 0 2 1 2 0
38.13 40.01 4G.52 49.00 50.00 53.70 53.96 X. 59 57.29
0 2 0 2 0 1 1 0 0
-. . .._ -
4x.90 48.98 50.77 52.59 54.45 -
2 2 1 0 1
44.50 41.91 45 i?J 46.52 4ti.m 49 .89 53.21 53.77 54 26 54.20 55.34 55.80 50.27 56.61 57. fil 50.59
2 2 1 1 1 2 I 1 U 0 1 0 1 0 1 1
FIG. 2. Herpes simplex
sodium deoxycholate, each at a final concentration of 1% (w/v). The preparations were dialyzed against SSC buffer (0.15 M sodium chloride-O.015 M sodium citrate) at 4”. Procedure 3. The suspension of virions in 0.001 M phosphate was brought to a final concentration of 27% (w/w) sucrose, 0.001 M EDTA and 0.2 M NaCl. To the virus suspension pronase grade B (Calbiochem, USA), 500 Kg/ml and sodium dodecyl
sulfate (SDS), 0.2 % final concentration, were also added. The preparations were incubated overnight at 37” in order to release the viral DNA. Part of each DNA preparation was treated with phenol (Sarov and Becker, 1967). The DNA preparations were dialyzed against SSC buffer at 4”. Procedure 4. The purified herpes simplex virions were treated with an equal volume of a solution of 7.4 M sodium perchlorate.
FIG. 3. A flllly spread indicated by arrows.
of herpes simplex
Aftcrwwds, the DNA preparations were carefully diluted 15fold with a solution of 0.03 111XaClM.015 31 n’a2HP04, pH 6.7. Herpes simplex virus DNA was used at concernrations of 0.02-0.2 fig per sucrose gradient. Sedimentntion of Native Viral DNL4 Two different, types of gradients were used for the centrifugation of the DNA preparations: (1) 15-30 % (w/w) sucrose gradients prepared in 0.001 !l/ phosphate were centrifuged at 24,000 rpm for 13 hours at 7” in t’he Beckman model L-2 preparative ultracentrifuge; (2) 5-20 % (w/v) sucrose gradients were made in 0.1 Al NaCl, 0.05 M phosphate, pH 6.7 (Burgi and Hershey, 1963) centrifuged in the SW9 rotor for 4 hours at 25,000 rpm. The gradient’s were fractionated, treated n-it,h 20 % trichloroacctic acid, and
The two ends of the molecldea
the precipitates Jvere collected on nlillipore filters. The radioactivity n-as determined in a Packard liquid scintillation counter.
DKA molecules, isolated from purified herpes virions, were carefully added to a 0.06% solution of cytochrome c (Sigma Chemical Co., USA) in 4 .lI NaCl. The preparation was spread over a clean \\-atei molecules were surface, and the DSA picked up on carbon-coated grids, according to the met,hod described by Kleinschmidt, et al. (1961) and modified by XIacHattie and Thomas (1964). The DNA molecules were examined in the RCA EMU-3 electron magnitica microscope. The instrumemal tion was determined several times daily with a carbon grating replica (E. 1’. l;ullam Inc., US.4). The cont’our of each individual DNA
that the viral DNA molecules which were released from the protein coat sedimented in an almost homogeneous band in sucrose gradients and were only slightly contaminated with radioactive proteins. These DNA molecules were studied by electron microscopy.
molecule was traced, a thin thread was attached to the tracing, and its length was measured. The molecular weight of the DNA molecules was calculated assuming that the mass per Angstrom length of the DNA in its B configuration is 192 daltons (Langridge et al., 1960; MacHattie and Thomas, 1964).
Conformation of Herpes Simplex Viws DNA Molecules
of Native Herpes Simplex Virus DNA in Sucrose Gradients
Purified virus particles, labeled in the DNA and the protein coat with radioactive isotopes, were treated with sodium deoxycholate (procedure 1) and centrifuged in sucrose gradients. It was found (Fig. 1)
FIG. 4. A herpes simplex one end of the molecule.
The DNA molecules that were released from virions by treatment with sodium deoxycholate (procedure 1) were diluted in a solution of cytochrome c and spread on a water hypophase. It was found that the molecules attained different conformations (Table 1). Part of the molecules had one
FIG. .j. A highly by the arrows.
of herpes simplex
center (monocenter, Table l), due either to the effect, of the wat’er hypophase (Lang et al., 1967; Inman, 1967) or to an incomplete release of the DNA from the coat components (partially released; Table 1, Fig. 2). Part of t’he molecules mere fully spread (no center; Table 1, Fig. 3) or with several centers (multicenters; Table 1, Fig. 4). Some molecules had a high degree of coiling of the DNA strands (highly looped; Table 1, Fig. 5). In this respect the herpes simplex DKA molecules had conformations similar to those of vaccinia DXA (Becker and Sarov, 1968). It is possible that the highly looped molecules retained t’he molecular conformation of the DNA genomes inside the virus particles.
The looped regions are indicated
Most’ of the DXA molecules displayed one or two distinct ends (Table 1) indicating the linear nature of the herpes simplex DNA genome. DNA molecules that were treat,ed wit’h sodium perchlorate (procedure 4) also demonstrated a linear conformation. Determination of the Length and Molecular Weight of Herpes Simplex Trims DNA by Electron RIicroscopy The length of 44 DNA molecules was measured. It Tvas found (Table 1) that the length of the DNA molecules differed according to the conformation which they attained. RIolecules which were eit,her partially released, highly looped or with a monocenter were regarded unreliable for
was calculated for herpes simplex DNA by comparison to the sedimentation of vaccinia virus DNA, which was found to have a molecular weight of 150 X lo6 daltons (Sarov and Becker, 1967; Becker and Sarov, 1968). DISCUSSION
I 40 LENGTH
FIG. 6. Molecular
length distribution DNA molecules.
of the viral
molecular weight determination. The length of the DNA molecules which were fully spread (multicenter and no center) ranged from 44.5 p to 59.6 p with an average length of 52.6 P, calculated from the modal length distribution of the DNA molecules (Fig. 6). The average molecular weight of herpes simplex DNA is therefore 100X lo6 daltons, ranging from 86 to 115 X lo6 daltons. The reason for the variation in length is not yet known. Determination of the Molecular Weight of Herpes Simplex DNA by Zone Centrifugation in Sucrose Gradients In order to obtain a molecular weight estimation for herpes simplex DNA by an independent method, the molecular weight of the viral DNA was determined by zone centrifugation (Burgi and Hershey, 1963). Herpes simplex virus DNA sedimented behind the vaccinia virus DNA, which was used as a marker (Fig. 7). Table 2 shows the molecular weight determinations of 9 herpes simplex virus DNA preparations, prepared by 3 different techniques. An average molecular weight of 110 X lo6 daltons (ranging from 100 to 115 X lo6 daltonsl~, . -I
The present study is concerned with the molecular weight determination of herpes simplex virus DNA, using electron microscopy (Kleinschmidt et al., 1961) and zone centrifugation in sucrose gradients (Burgi and Hershey, 1963). The effect of the water hypophase on the configuration of the DNA molecules was also taken into consideration (Lang et al., 1967; Inman, 1967). It was found that sodium dodecyl sulfate completely released the viral DNA from the viral coat proteins and further deproteinization with pronase or phenol did not affect its sedimentation behavior in sucrose gradients. Sodium deoxycholate also released DNA from the virus particles but, as revealed by electron microscopy, in some molecules remnants of proteins were found. The sensitivity of the virions to deoxycholate was attributed to the integration of cellular lipids into the envelopes of the mature virus particles (Asher et al., 1968). The DNA molecules were found to sediment in the sucrose gradients in a homogeneous peak. The molecular weight of herpes simplex virus DNA was determined by t’wo independent techniques: (a) electron microscopy (Kleinschmidt et al., 1961) and (b) zone centrifugation in sucrose gradients, using vaccinia virus DNA as a marker (Sarov and Becker, 1967; Becker and Sarov, 1968). Studies by electron microscopy demonstrated that the viral DNA molecules attained different molecular conformations which resembled those of vaccinia virus DNA (Becker and Sarov, 1968). DNA molecules which displayed their entire molecular length were used for the determination of the molecular weight. From the modal length distribution (Fig. 6) it was calculated that the average length of a herpes simplex virus DNA is 52.6 CL,which is equivalent to a molecular weight of 101 X lo6 daltons. The molecular weight values
FRACTION FIG. 7. Zone cent,rifugation of herpes simplex and vaccinia virus DKA molecules. Herpes simplex virus l)NA (prepared by procedure 3) was mixed with vaccinia virus DN.4, and the t,wo DNA species were relltrifaged in a sllcrose gradient. O---O, Herpes simplex virus DNA; O---O, varciuia virlls r)PiA
Procedure of DNA preparation 1 2 :i -i .i
ci 7 8 !) .4ver:rpe
2 ‘2 3 3 3 3 3 3 + phenol 1
Molecul3r weight X lo6 daltons 107.2 112.7 100.i 103.6 112.i 114.2 115.8 114.2 107.2 110.03
were found to range from 86 to 115 X 10fi daltons. This result is in agreement with t’he molecular weight’ report,ed by Soehner et al. (1965) for equine abortion virus and is higher than the molecular ITeight determinations previously reported for herpes simplex virus DKA (Russell and Crawford, 1963, 1964). The molecular weight of herpes simplex virus DNA was also determined by zone centrifugation. By comparison to vaccinia virus DNA, a molecular weight, of 110 X lo6 daltons was calculated for herpes simplex virus DSA. This value is in accordance with t,he molecular weight, d&rmined by eleckon microscopy. The techniques developed for the isolation of intact DKA genomes from purified
vaccinia and herpes virions enabled an accurate determination of the molecular conformation and weight of viral DNA genomes. These measurements could be applied also to virus classification (Becker, 1966). On the basjs of the present results we suggest that the molecular weight of herpes simplex virus DNA genome is about 100 X lo6 daltons. ACKNOWLEDGMENT The expert technical assistance of Miss Miriam Adler and Mr. G. Eynav is greatly appreciated. REFERENCES ASHER, Y., HELLER, M., and BECKER, Y. (1968). Incorporation of lipids into herpes simplex virus particles. J. Gen. Viral. in press. BECKER, Y. (1966). An approach to the organization and classification of animal viruses. Nature 210, 1019-1021. BECKER, Y., and CHEN, Y. (1966). The reaction of vaccinia virus with deoxycholate. Israel J. Med. Sci. 2, 417-423. BECKER, Y., and SAROV, I. (1968). Electron microscopy of vaccinia virus DNA. J. Mol. Biol. 34, 655-660. BECKER, Y., OLSHEVSKY, U., and LEVITT, J. (1967). The role of arginine in the replication of herpes simplex virus. J. Gem Viral. 1, 471-478. BEN-P• RAT, T., and KAPLAN, A. S. (1962). The chemical composition of herpes simplex and pseudorabies virus. Virology 16, 261-266. BURGI, E., and HERSHEY, A. D. (1963). Sedimentation rate as a measure of molecular weight of DNA. Biophys. J. 3, 309-321. EAGLE, H. (1959). Amino acid metabolism in mammalian cell cultures. Science 130, 432-437. INMAN, R. B. (1967). Some factors affecting electron
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