Chromosomal organization of the herpes simplex virus type 2 genome

VIROLOGY123,344-356 (1982)

Chromosomal

Organization of the Herpes Simplex Virus Type 2 Genome’

MARK R. HALL,2 NIKOU AGHILI, CLAIRE HALL, JESSE MARTINEZ, AND STEPHEN ST. JEOR Department

of Microbiology,

School of Medicine,

University

of Nevada, Reno, Nevada

89557

Received August 10, 1982; accepted August 11, 1982

Nucleoprotein complexes containing herpes simplex virus type 2 DNA were extracted from CV-1 cells with NP-40 and (NH&SO+ The viral DNA-protein complex sedimented in neutral sucrose as a relatively homogeneous peak at around 120 S. Pooled material from the 120 S region of sucrose gradients contained DNA which, when deproteinized, sedimented at 55 S and had a density of 1.728 g/cmS as expected for herpesvirus DNA. Buoyant density studies with glutaraldehyde-fixed complexes indicate that protein is bound to viral DNA at a ratio of approximately 1.1 to 1. Micrococcal nuclease digests of isolated viral nucleoprotein complexes indicate that these complexes do not have a nucleosomal structure. Chromatin prepared from nuclei following isolation of viral nucleoprotein complexes contained viral DNA. When infected nuclei were treated with micrococcal nuclease, nucleosome-sized fragments were obtained which contained both virus and cell DNA. INTRODUCTION

structure with 140 base pairs of DNA together with a flexible spacer region of 40The chromatin of eucaryotic cells is 60 base pairs associated with histone Hl packaged into a repeating regular array (Weintraub et al., 1976). of histone-containing structures termed Chromatin-like nucleoprotein comnucleosomes (Kornberg, 1974; Kornberg, plexes (NPC) have been isolated from 19’77;Felsenfeld, 1978). Nucleosome monomers can be isolated by controlled diges- polyoma virus (Green et al., 1971; Goldtion of chromatin or nuclei by micrococcal stein et al., 1973; Seebeck and Weil, 1974) nuclease (Noll, 1977; Lohr et al., 1977). and SV40 virus-infected cells and from Each nuclease-isolated nucleosome con- mature virions (White and Eason, 1971; tains 185 to 200 base pairs of DNA com- Hall et al., 1973; Howe and Tan, 1977; plexed with two each of histones H3, H2B, Meinke et al., 1975; Christiansen et al., H2A, and H4 and one molecule of histone 1977). The major protein components of Hl (Kornberg, 1977; Felsenfeld, 1978). Ex- these viral chromosomes have been identended times of nuclease digestion of chro- tified as host cell histones (McMillan and matin lead to the production of core par- Consigli, 1974; Sen et al., 1974; MacGregor ticles which contain 140 base pairs of DNA et al., 1978; Chen et al., 1979). Micrococcal associated with a histone octomer but de- nuclease digests of polyoma virus and void of histone Hl (Varshavsky et al., 1976; SV40 virus chromatin yield discrete nuNo11and Kornberg, 1977). Thus, it appears cleosomal size units similar to those of cell that eucaryotic DNA is arrayed into nu- chromatin (Ponder and Crawford, 1977; cleosomes consisting of a compact core Klempnauer et al., 1980). Similarly, recent investigations have shown that adenovirus DNA is also pres1Presented in part at the Annual Meeting of the American Society for Microbiology, Atlanta, Ga, ent in cell nuclei (Sergeant et al., 1979;Tate and Philipson, 1979) and mature virions March 7-12, 1982. ‘To whom reprint requests should be addressed. (Corden et al., 1976) in a chromosomal 0042-6822/82/160344-13$02.00/O Copyright All rights

Q 1982 hy Academic Press. Inc. of reproduction in any form reserved.

344

HSV-2

NUCLEOPROTEIN

form. Micrococcal nuclease digestion of infected cell nuclei produces a nucleaseresistant fraction containing viral deoxyribonucleoprotein monomers and oligomers. These nucleosome-like structures contain viral DNA fragments with a unit length of 185 base pairs (Sergeant et al., 1979). DNA fragments from virus cores have a repeat size of 165 base pairs (Tate and Philipson, 1976). Recently, it has been suggested that genomes of members of the herpesvirus group are present in the form of DNAprotein complexes in nuclei of infected cells (Kierszenbaum and Huang, 1978; Pignatti and Cassai, 1980; St. Jeor et al., 1982). The presence of proteins on HSV-1 viral DNA extracted from infected cells by detergents is supported by finding that the viral DNA is associated with an endogenous DNA polymerizing activity (Pignatti et al., 1979a), has a lowered sedimentation coefficient when treated with deproteinizing enzymes (Pignatti et al., 1979b), and has a markedly different restriction nuclease XbaI cleavage pattern than that of purified HSV-1 DNA (Hyman et al., 1979). In addition, Pignatti and Cassai (1980) have shown that at least one form of extractable HSV-1 nucleoprotein complex contains several virion polypeptides. Although there are conflicting reports regarding a nucleosomal organization of HSV-1 chromosomes (Mouttet et al., 1979; Leinbach and Summers, 1980), micrococcal nuclease digestion of nuclei from Epstein-Barr virus-transformed cell lines and of chromatin from human cytomegalovirus (HCMV)-infected cells produced viral DNA fragments in a nucleosomal pattern (Shaw et al., 1979; St. Jeor et al., 1982). Results outlined in the preceding paragraph suggest that intracellular genomes of viruses of the herpesvirus group exist in chromosomal form, possibly analogous to that of host cell chromatin. The present investigation was undertaken to determine whether the HSV-2 genome is present in infected cells in a chromosome-like DNA-protein complex. The results of these experiments show that a nucleoprotein complex containing the HSV-2 genome can

345

COMPLEXES

be extracted from infected cells by nonionic detergents. Furthermore, micrococcal nuclease digestions of chromatin-associated HSV-2 genomes in infected nuclei indicate that a fraction of the intranuclear viral chromosomes have a nucleosomal structure analogous to that of host cell chromatin. MATERIALS

AND

METHODS

Solutions. The following solutions were used: TE buffer (0.02 M Tris-HCl, 0.002 M EDTA; pH 8.3); Tris-buffered saline (TBS; 0.05 M Tris-HCl, pH 7.4, 0.15 M NaCl, 0.0015M MgClz); TCM buffer (0.05 M TrisHCl, pH 7.9, 0.01 M CaClz, 0.01 M MgClz). virus and ceZZ.s. Herpes simplex virus type 2 (Strain MS) (Dowdle et al., 1967) was propagated in and assayed on the CV1 monkey kidney cell line. Cell monolayers were grown to confluence in lo-cm plastic petri dishes containing 10.0 ml of Eagle medium supplemented with 10% newborn calf serum. Cultures were infected approximately 24 hr postconfluence with 1.0 ml of virus inoculum (10 to 20 PFU/cell) and the virus was allowed to absorb for 90 min at 37”. After absorption, 10.0 ml of Eagle medium containing 2% horse serum was added to each culture. Labeling and isolation of HSV-2 chromosomes. Following infection, (procedure described above), cultures were incubated at 37” for 2 hr in Eagle medium containing 2% horse serum. At several selected intervals postinfection, [3H]thymidine was added to the culture medium to a final concentration of 10 &i/ml and cultures were incubated at 37” for selected time intervals. Mock-infected cultures were similarly treated. For some experiments, subconfluent monolayers were prelabeled with 5.0 ml of Eagle medium containing 10% newborn calf serum and 2.0 &i/ml [14C]thymidine. When labeled cultures had been confluent for 24 hr, labeling medium was removed and cells were infected as described above. Viral chromosomes were extracted from labeled cultures by a modification of the method of Wilhelm et al. (1976). Labeled cells were harvested by rinsing twice with

346

HALL ET AL.

ice-cold TBS and then scraping them into 1.0 ml of TBS per culture dish. Each dish was then rinsed with 1.0 ml of TBS and the rinse combined with initial scraped cell fractions. Cell suspensions were centrifuged for 10 min at SOOg(4”), the supernatant was discarded, and the cell pellet resuspended in 0.5 ml of TBS. An equal volume (0.5 ml) of 0.2% NP-40 was added to cells in TBS and the suspension was incubated for 10 min at 4”. At the end of 10 min over 95% of cells had lysed (monitored by observation with a phase-contrast microscope), however, nuclei remained intact. The NP-40-treated lysate was centrifuged at 800~ for 10 min at 4’, the supernatant was discarded, and the pellet (nuclei) was resuspended in 0.5 ml of TE. Nuclear suspensions were treated with 50 ~1 of 1.5 M (NH&SO4 and incubated for 15 min at 4” with occasional gentle mixing. Nuclei were pelleted at 8OOgfor 10 min at 4”. The supernatant containing HSV-2 chromosomes was removed carefully and stored at 4”. Isolation of chrmatin, Chromatin from HSV-2-infected and normal CV-1 cells was extracted as described by Axe1 (1978). Nuclei, prepared as described in the preceding section, were suspended in TBS containing 0.3 M sucrose and centrifuged at 1OOOg for 10 min. Nuclei were then treated in TBS containing 0.025M EDTA and 0.25 M NaCl and pelleted at 1000~. The nuclear pellet was then successively suspended and pelleted in 0.05,0.01,0.005, and 0.001 M Tris-HCl buffer, pH 7.9.

fection, carrier-free [32P]phosphate was added to each culture to a final concentration of 10 &i/ml. Labeled cultures were incubated for selected time intervals until nuclei were extracted and digested with micrococcal nuclease. In some experiments (described in the text) [14C]thymidine (2 &i/ml) was substituted for [3H]thymidine as a prelabel and [3H]thymidine (10 &i/ml) was substituted for [32P]phosphate as the “postchase” label. Nuclei from labeled HSV-2-infected and mock-infected cultures, prepared as described above, were suspended in TBS containing 0.3 M sucrose (TBSS). Generally, we utilized 150 ~1 of TBSS to suspend nuclei from each loo-mm culture dish. Digestion was carried out at 37” for either 15, 30, or 60 min with micrococcal nuclease (Worthington Biochemicals Corp., 50 units/ml). Reactions were terminated by addition of an equal volume of TE to each digestion mixture. The digestion mixture was pipetted into a cold (4”) Dounce homogenizer and sheared by two strokes with a tight-fitting (A) pestle. Digested nuclear suspensions were transfered to corex centrifuge tubes, heated to 37” for 2 min, cooled to 4”, and centrifuged for 10 min at 10,000 rpm (Beckman J21B, JA20 rotor). The supernatant was collected and used as a source of HSV-2 and CV-1 nucleosomes. When 32P-labeled nucleosomes were to be analyzed by velocity sedimentation in sucrose gradients, 150 ~1 of the nucleosomal digest was treated with 150 ~1 of RNase (Sigma Chemical Co., 200 units/ml). Micrococcal nuclease digestion of nuclei. When nucleosomal DNA was to be anSubconfluent monolayers in loo-mm culture dishes were labeled by addition of 5.0 alyzed by equilibrium centrifugation, 150 ml of Eagle medium containing 10% calf ~1 of digestion supernatant was treated serum and 10 &i/ml rH]thymidine. Ap- with 150 ~1 RNase (Sigma Chemical Co., proximately 20 hr postconfluence, labeling 200 units/ml), 50 ~1 Pronase (1 mg/ml), medium was removed and replaced with and 50 ~1 of 0.2 M EDTA for 120 min at “chase” medium containing unlabeled thy- 37”, followed by phenol extraction. Following extraction of nucleoprotein midine at 100 pg/ml, and cultures were incubated for 4 hr at 37°C. After 4 hr, complexes, HSV-2 DNA was extracted chase medium was removed and cultures from infected cell nuclei by the method of were infected with HSV-2 as described Hirt (1967) as modified for HSV-2 DNA above. Following infection, cultures were isolation by Pater et al. (1976). DNA-DNA hybridization using jlter incubated for 4 hr in phosphate-free Eagle medium containing 2% horse serum and disks. Samples of DNA to be immobilized 100 pg/ml thymidine. Four hours postin- onto filters were prepared by pooling con-

HSV-2

NUCLEOPROTEIN

secutive pairs of fractions across sucrose gradients of micrococcal nuclease digests of nuclei from uninfected and HSV-2-infected CV-1 cells. Each sample (0.2 ml) was then diluted to 2.0 ml with 0.01X SSC (SSC is 0.15M NaCl and 0.015M Nas-citrate) and denatured by boiling for 15 min followed by quick-cooling in an ice-water bath (Aloni et al., 1969). Denatured DNA was immobilized on cellulose nitrate filter disks (Schleicher and Schuell Type B-6) as described by Denhardt (1966). Filters were preincubated for 12 hr at 37” in a solution containing 10.0 ml of 100% formamide, 1.0 ml of 20% glycine, 5.0 ml of 20X SSC, 1.0 ml of 1 M Na2HP04 (pH 7.0), 2.6 ml of 20X Denhardt solution (Denhardt, 1966), 2.0 ml of 0.5% salmon sperm DNA. Hybridization was carried out for 48 hr at 37” in a solution containing 10.0 ml 100% formamide, 5.0 ml of 20X SSC, 0.4 ml of 1.0 M NaPO, (pH ‘7.0), 0.5 ml of 20X Denhardt solution (Denhardt, 1966), 0.4 ml of 0.1% salmon sperm DNA, and 3.0 ml of denatured probe DNA in distilled water. Labeled probe DNA was prepared from purified HSV-2 viral DNA (Kieff et al., 1971) by the methods of Rigby et al. (1977). Generally, a 32P-labeled probe DNA with a specific activity of lo7 cpm/pg was utilized. As a rule, 1 X lo6 cpm of DNA probe was utilized for each 10 filters being hybridized. Following hybridization, the filters were washed, dried, and counted as described by Denhardt (1966). Centrifugal analyses of chromosomes and DNA. Sedimentation characteristics of samples of HSV-2 chromosomes as well as viral and cellular nucleosomes were determined on 4.8 ml 5% to 20% (w/w) linear sucrose gradients. Sucrose for velocity gradients was prepared in TE plus 0.15 M NaCl. Centrifugal analyses of chromosomes were performed at 4” for 90 min at 35,000 rpm (Spine0 SW 50.1 rotor). Samples were collected dropwise, processed, and counted in a liquid scintillation counter as previously described (Hall et al., 1973). The distribution of viral and cellular DNA in chromosomal and nuclear extracts were determined by equilibrium centrif-

347

COMPLEXES

ugation in CsCl gradients. Aliquots of selected DNA preparations were diluted to a total volume of 3.0 ml with 0.01 M TrisHCl; 0.001 M EDTA, pH 8.0. The density of these samples was adjusted to 1.716 g/ cm3 by addition of CsCl and the gradients were centrifuged to equilibrium at 21” for 42 hr at 32,000 rpm (Spinco SW 50.1 rotor). Samples were collected dropwise, processed, and counted as described previously (Hall et al., 1973). The buoyant density of HSV-2 chromosomes was determined by centrifugation of glutaraldehyde-fixed samples (Baltimore and Huang, 1968) in preformed linear CsCl gradients as previously described (Hall et al., 1973). Analyses of the density distributions of chromatin preparations were determined in preformed lo-40% metrizamide gradients (Nyegaard and Co. Hs., Oslo, Norway). Gradients were centrifuged to equilibrium at 32,000 rpm for 48 hr at 18” in a Spine0 SW 50.1 rotor. RESULTS

Extraction of HSV-2 Nucleoprotein Complexes HSV-2-infected CV-1 cells or mockinfected cultures were labeled with [3H]thymidine for 120 min beginning at 10 hr postinfection. Cells were scraped from culture dishes and lysed with NP-40, and viral nucleoprotein was extracted from nuclei as described above. Aliquots of nuclear extracts from HSV-2-infected and mock-infected cells were mixed with [‘4C]thymidine-labeled phage T4 DNA marker and analyzed by centrifugation in 5 to 20% neutral sucrose gradients. The radioactive profiles from separate sucrose gradient analyses of extracts from infected and mock-infected cells have been combined (Fig. 1) for comparative purposes. A relatively homogenous peak of 3H-labeled material sedimenting at about 120 S was observed in samples from HSV2-infected cells (closed circles). A low background of 3H-labeled material, lacking distinct peaks, was present in extracts of mock-infected cells (open circles). Sim-

848

HALL

ET AL.

ilar experiments, performed with [‘“Clthymidine-prelabeled cells, produced results equivalent to those described above. Sucrose gradient profiles of extracts from HSV-2-infected cells had peaks of 3H-labeled material at 120 S with a heterogenous low 14C-labeled background and extracts of mock-infected cells had low heterogenous backgrounds of both 3H- and 14C-labeled material (data not shown). The 120 S material observed in nuclear extracts of HSV-2-infected cells does not appear to be formed by random binding of cellular macromolecules to viral DNA since labeled deproteinized viral DNA added to nuclear preparations during extraction sediments in sucrose gradients to the position characteristic of free viral DNA. Characterization of DNA in HSV-2 Nucleoprotein Complexes To determine the origin of the DNA extracted from nuclei by 0.15 M (NH4)zS04, peak fractions of the gradient shown in Fig. 1 were pooled and deproteinized by addition of Pronase and Sarcosyl. Deproteinized samples were analyzed by velocity 605

5

n

FRACTION

1

1

1

NUMBER

FIG. 1. Velocity sedimentation of HSV-2 nucleoprotein complexes in 5 to 20% neutral sucrose gradient. Extracts were prepared from HSV-2 infected (0) and mock-infected (0) CV-1 cells which had been labeled from 10 to 12 hr postinfection. Aliquots (0.19 ml) of each extract were mixed with 10 ~1 of “Clabeled T4 DNA and centrifuged as described under Materials and Methods. An arrow indicates the position of the T4 marker. Sedimentation is from right to left.

ICI 20 FRACTION

30 NUMBER

40

FIG. 2. Velocity sedimentation, filter disk hybridization, and CsCl equilibrium centrifugation of DNA released from HSV-2 nucleoprotein complexes. Pooled peak fractions from the gradient shown in Fig. 1 were deproteinized with SDS and Sarcosyl. One aliquot of the deproteinized material was mixed with 14C-labeled T4 marker DNA and analyzed by velocity sedimentation in a 5 to 20% sucrose gradient (A, closed circles). Two-sample pools of the fractions collected from the sucrose gradient were fixed to nitrocellulose filter disks and hybridized to a “P-labeled HSV-2 viral DNA probe (A, open circles). An arrow marks the position of the T4 DNA marker. A second aliquot was mixed with r4C-labeled K. pneumonia marker DNA and centrifuged to equilibrium in a CsCl density gradient (B). Sedimentation is from right to left in A.

centrifugation in linear sucrose gradients, by DNA-DNA hybridization and by equilibrium centrifugation in CsCl gradients. In velocity gradients (Fig. 2A, closed circles), DNA from the pooled fractions sedimented slightly more slowly than the 60 S T4 marker (Dubin et al., 1970). The single peak of 3H-labeled DNA has a sedimentation coefficient of about 55 S as expected for HSV-2 DNA (Kieff et al., 1971). In addition, pooled samples from the sucrose velocity gradient hybridized to a 32P-labeled HSV-2 viral DNA probe (Fig. 2A, open circles).

HSV-2

NUCLEOPROTEIN

-1.6

-1.5 % \u 2 z -1.4 g x

r

-1.3

FRACTION

NUMBER

FIG. 3. Equilibrium centrifugation of glutaraldehyde-fixed HSV-2 nucleoprotein complexes in preformed CsCl gradients. Peak fractions from gradients similar to those in Fig. 1 were pooled and treated with glutaraldehyde (Baltimore and Huang, 1963). Aliquots (0.2 ml) of the glutaraldehyde-fixed material were layered on preformed CsCl gradients and centrifuged to equilibrium (Hall et al., 1973).

Analyses of DNA from the pooled fractions in CsCl equilibrium density gradients (Fig. 2B) clearly show a peak of 3Hlabeled DNA at a density of approximately 1.728 g/cm3, the density expected for HSV2 DNA (Kieff et al., 1971). The [14C]-labeled material present in the gradient is Kleb siella pneumonia DNA (density, 1.716 g/ cm3), which was included as a density marker. Buoyant Densities of Glutaraldehgde-Fixed HSV-2 Nucleoprotein Complexes Estimates of the amount of protein bound to HSV-2 in complexes were obtained by glutaraldehyde fixation of pooled peak fractions from sucrose gradients and analysis of fixed samples by equilibrium centrifugation in preformed CsCl density gradients. As shown in Fig. 3, HSV-2 nucleoprotein complexes banded in CsCl at a density of 1.475 g/cm’. Assuming that the density contributions of the separate components are additive, an estimate of the molecular weight of HSV-2 nucleoproteins can be made from their buoyant density. Experimentally determined values for the density of HSV-2 DNA and proteins are 1.728 g/cm3 (Kieff d al., 1971) and 1.288 g/cm3 (M. R. Hall,

349

COMPLEXES

unpublished observation), respectively. From these data, a ratio of protein to DNA of approximately l.l:l can be calculated. This indicates that the HSV-2 nucleoprotein complex is about 55% protein and 45% DNA and has a molecular weight of around 2 X 107. Time Course and Extractability Nucleoprotein Complexes

of HSV-2

The time selected (12 hr postinfection) for initial experiments to extract HSV-2 NPCs was based on the fact that, in our system, complete virions were not detectable until at least 16 hr postinfection and on the assumption that putative NPCs could be precursors to virions. As virion precursors, NPCs might be expected to reach a maximum intracellular accumulation late in infection. However, it is possible that the NPCs described above are either replicative or transcriptive in function and thus might have labeling kinetics complex. different from a “structural” Therefore, we were interested in determining an optimum time for NPC labeling and extraction. In addition, since previous investigations (Goldstein et al., 1973) have TABLE

1

TIMECOURSEANDEXTRACTABILITYOFHERPES SIMPLEXVIRUSTYPE 2 NUCLEOPROTEINCOMPLEXES Labeling cycle (hr postinfection) Type of extraction

o-2

2-4

6-8

10-12

3H-cpm per extracta Nucleoprotein Hirt*

0 665

2782 1250

4600 2712

6747 4049

-a These data represent the average TCA-precipitable radioactivity of triplicate 50-4 samples analyzed from each of four separate experiments. The total volume of each extract was adjusted to 1.0 ml prior to analysis of radioactivity. *Since Hirt extracts may contain some cellular DNA, these data have been adjusted to represent only that percentage of labeled material which was represented by virus density DNA in equilibrium gradients of Hirt extracted material for each time period.

350

HALL

shown that papovavirus NPC extraction methods are relatively inefficient in recovering viral DNA from nuclei, we wished to determine the efficiency of our extraction method in removing HSV-2 DNA from nuclei. Confluent monolayers of CV-1 cells were infected with HSV-2 and labeled with rH]thymidine (5 &i/ml) from either 0 to 2,2 to 4,6 to 8, or 10 to 12 hr postinfection. At the end of each labeling period, NPCs were extracted, the extracted nuclei were retained and remaining HSV-2 DNA was recovered by the Hirt (1967) method (see Materials and Methods). The results of these studies are presented in Table 1. Although HSV-2 NPCs are not detectable in extracts from the earliest labeling cycle, extracts of the other three labeling cycles exhibit increasing amounts of label at each successively later labeling time. In addition, by comparing the counts extracted into NPCs with the remaining counts extractable by the Hirt (1967) method, it is evident that the time of extraction is not related to the efficiency of extraction of HSV-2 NPCs. In all cases, between 62 and 65% of total extracted counts were represented by NPCs.

ET AL.

IO

20 FRACTION

30

40

NUMBER

FIG. 4. Velocity sedimentation profiles of micrococcal nuclease digests of HSV-2 nucleoprotein complexes and CV-1 chromatin. HSV-2 NPCs were prepared as described (see Materials and Methods) and partially purified by centrifugation in neutral sucrose gradients (see Fig. 1). Pooled peak fractions of [3H]thymidine-labeled material from sucrose gradients were mixed with [i4C]thymidine-labeled CV-1 nuclei and treated with micrococcal nuclease. Nuclease-treated material (0.2 ml) was layered onto linear 5 to 20% sucrose gradients and analyzed as described under Materials and Methods. An arrow marks the position of 21 S SV40 Form I DNA centrifuged in separate gradients. Sedimentation is from right to left.

gradients. Pooled NPC-containing fractions from preparative sucrose gradients were utilized for nuclease digestion experControlled digestion of isolated nucleo- iments. Pooled fractions were diluted 1:5 protein complexes by micrococcal nuclease in TE buffer containing 0.15 M NaCl and has been utilized to confirm nucleosomal NPCs were pelleted by centrifugation for 5 hr at 35,000 rpm in a SW 50.1 rotor. The structure of viral chromosomes (Bellard et aL, 1976; Ponder and Crawford, 1977; pelleted [3H]thymidine-labeled NPCs were suspended in 150 ~1 of TBSS, mixed with Hidaka et aL, 1980). Chromatin fragments 150 ~1 of TBSS containing uninfected produced by limited nuclease digestion consist of a series of multimers of the unit [14C]thymidine-labeled CV-1 nuclei, and treated with micrococcal nuclease. Nunucleosome (monosomes, disomes, trisomes, etc.). The various fragments can be clease-treated mixtures were centrifuged resolved by sedimentation in sucrose gra- to remove undigested nuclear material, dients in which mononucleosomes char- and the supernatant fluids were analyzed by velocity centrifugation in 5 to 20% neuacteristically sediment at approximately 11 S (Noll, 1977; Kornberg, 1977). We uti- tral sucrose gradients (Fig. 4). The nulized micrococcal nuclease as a probe to clease digestion fragments of NPCs (closed circles) are represented by a pattern of determine whether HSV-2 nucleoprotein resistant material which extends from the complexes contain nucleosomal structure. HSV-2 nucleoprotein complexes were iso- top of the gradient to a position below the lated as described above and partially pu- 21 S marker (arrow). In contrast, the patrified by centrifugation in neutral sucrose tern of digestion fragments from CV-1 cell

Micrococcal Nuclease Digestion of HSV-2 Nucleoprotein Complexes

HSV-2 NUCLEOPROTEIN COMPLEXES

FIG. 5. Equilibrium centrifugation of DNA extracted from nuclei prepared from HSV-e-infected CV-1 cells. CV-1 cells were prelabeled with [3H]thymidine, chased, infected with HSV-2, and labeled with [32P]phosphate from 4 to 12 hr postinfection. At 12 hr postinfection, nuclei were prepared (see Materials and Methods) and an aliquot was removed for DNA isolation. DNA was extracted from isolated nuclei by treatment with Pronase and Sarcosyl followed by digestion with ribonuclease. Each DNA sample was adjusted to a final density of 1.716 g/cm3 prior to centrifugation.

nuclei (open circles) contains a series of peaks characteristic of nucleosomal structure. In addition the upper peak sedimenting at 11 S relative to the 21 S marker is characteristic of nucleosome monomers (Noll, 197’7; Kornberg, 1977). When purified [14C]HSV-2 DNA was added to a reaction mixture containing CV-1 nuclei and micrococcal nuclease as described above, labeled virus DNA was digested to TCAsoluble fragments within 15 min. Micrococcal nuclease digestion of HSV2 NPCs under similar conditions to those described above, but without incorporation of CV-1 nuclei also led to the production of a heterogeneous pattern of digestion fragments extending throughout the gradient (data not shown). Thus, it appears that HSV-2 NPCs do not contain a nucleosomal structure analogous to that of eucaryotic cell chromatin and papovavirus chromosomes. Digestion of Intranuclear HSV2 DNA by Micrococcal Nuclease The data presented in Table 1 indicate that approximately 40% of the HSV-2

351

DNA present in infected nuclei is in a form which is resistant to extraction by our standard nucleoprotein extraction method. We were, therefore, interested in determining the characteristics of this material. Monolayers of CV-1 cells were prelabeled with [3H]thymidine for 70 hr, chased for 4 hr with unlabeled thymidine, infected with HSV-2, and labeled for 10 hr with carrier-free [32P]phosphate. At 12 hr postinfection, nuclei were prepared, an aliquot was removed for DNA isolation and the remaining sample was treated with ribonuclease and micrococcal nuclease. The aliquots removed prior to micrococcal nuclease treatment of nuclei were deproteinized, treated with ribonuclease, and centrifuged to equilibrium in neutral CsCl gradients. Figure 5 shows the resolution of viral and cellular DNA obtained in typical CsCl density gradients. It is evident that the majority (>90%) of the 32Plabel has banded at the density of HSV-2 DNA, while all of the 3H label is associated with a peak at the density of CV-1 DNA. The nuclease digestion mixtures were analyzed by centrifugation in linear 5-20% sucrose gradients (Fig. 6). Within 15 min (Fig. 6A), both cellular (3Hlabeled) and viral (32P-labeled) nucleoprotein had been cleaved into a series of fragments the smallest of which sedimented at approximately 11 S as expected for nucleosomes. Figure 6B clearly shows the progression of digestion, as by 30 min, peaks of fragment sizes larger than 11 S decline while the 11 S fragment peak increases. Figure 6C shows that by 60 min of digestion mainly 11 S fragments or smaller pieces remained. Attempts were made to determine if the 11 S material contained viral and cell DNA by equilibrium centrifugation of deproteinized samples in CsCl gradients. However, the radioactive profiles of these gradients, particularly the 32P-labeled material, exhibited very broad heterogeneous peaks which did not produce definitive separation of light (cellular) DNA from heavy (viral DNA). This is probably due to the known distribution of GC- and AT-rich regions within HSV genomes (Reischig et al, 1975; Delius and Clements, 1976).

352

HALL ET AL.

Hybridization Anal@s of Micrococcal Nuchase Digestion Fragments To determine whether the nucleosomal fragments observed in Figure 6 contained viral DNA, nuclei from uninfected and HSV-2-infected cells were treated for 30 min with micrococcal nuclease and the nuclease digests analyzed by velocity sedimentation in sucrose gradients. Samples (two fraction pools) across each gradient were fixed to nitrocellulose filters and hybridized to a 32P-labeled HSV-2 viral DNA probe. It is evident that micrococcal nuclease digestions of either infected (Fig. 7A, closed circles) or uninfected (Fig. 7B, closed circles) CV-1 nuclei produced a se-

FRACTION

NUMBER

FIG. 6. Velocity sedimentation profiles of micrococcal nuclease digests of nuclei prepared from HSV2-infected cells. Nuclear samples, labeled and prepared as described in the legend to Fig. 5, were digested with micrococcal nuclease for 15 min (A), 30 min (B) or 66 min (C). Digestion mixtures were treated with RNase, layered onto linear sucrose gradients, and analyzed as described under Materials and Methods. Sedimentation is from right to left.

I

I

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20 30 NUMBER

FIG. 7. Velocity sedimentation profiles and filter disk hybridization patterns of micrococcal nuclease digests of nuclei prepared from HSV-2-infected (A) and uninfected cells (B). CV-1 cells were prelabeled with r3H]thymidine, infected with HSVJ, and labeled with rH]thymidine from 2 to 12 hr postinfection. At 12 hr postinfection, nuclei were prepared and digested with micrococcal nuclease for 30 min. Digestion mixtures were layered onto linear sucrose gradients and analyzed as described under Materials and Methods. Samples for hybridization were prepared by pooling pairs of consecutive fractions across the gradient and fixing the pooled material to nitrocellulose filter disks. Fixed DNA was hybridized to a 32P-labeled HSV-2 viral DNA probe. Sedimentation is from right to left.

ries of fragments characteristic of nucleosomes as described above (see Fig. 6). Significantly however, it is apparent that the viral DNA probe hybridized predominantly to fractions in the nucleosomal regions of the micrococcal nuclease digests of HSV-2-infected CV-1 nuclei (Fig. 7A, open circles). In contrast, hybridization of HSV-2 probe DNA to fractions from uninfected cells was barely above background and did not exhibit a nucleosomal pattern (Fig. 7B, open circles). These data, analyzed in conjunction with that presented in the previous section, indicate that at least a fraction of the intranuclear chromatin-associated HSV-2 DNA is arranged structurally in a nucleosomal form similar to that of cellular chromatin. Metrizamide Banding of Chrmatin from HSV-.%Ifected Cells Since it appeared that some intranuclear HEW-2 DNA contained nucleosomal

HSV-2 NUCLEOPROTEIN

I 1

1.18

353

COMPLEXES

material contained viral DNA, peak fractions from the metrizamide gradient were pooled, deproteinized, and centrifuged to equilibrium in neutral CsCl gradients. Figure 9 shows that material from the single metrizamide peak banded as two peaks in CsCl. Furthermore, the denser of these two peaks (3H-labeled DNA) has a density characteristic of HSV-2 DNA, while the lighter peak (‘4C-labeled DNA) is characteristic of CV-1 DNA. DISCUSSION

FRACTION

NUMBER

FIG. 8. Metrizamide density gradient analysis of chromatin prepared from HSV-2-infected CV-1 cells. Cultures were prelabeled with [“Clthymidine, chased, infected with HSV-2, and labeled with [‘Hlthymidine from 4 to 8 hr postinfection. Chromatin samples, prepared as described (Axel, 1978),were layered onto preformed 10 to 40% metrizamidegradients and centrifuged to equilibrium. Determinations of the density distribution across the gradients were calculated from the refractive indices of selected samples as previously described (Birnie et al., 1973).

structure, it was of interest to determine whether this material could be isolated by extraction methods utilized to isolate chromatin. Therefore, monolayers of CV1 cells were prelabeled with [14C]thymidine for ‘70 hr, chased for 4 hr with cold thymidine, and infected with HSV-2. Four hours after infection, cultures were labeled for 8 hr with [3H]thymidine, after which chromatin was extracted as described (Materials and Methods). Chromatin fractions were layered onto preformed 10-40s metrizamide gradients and centrifuged to equilibrium. The metrizamide gradient profile (Fig. 8) clearly shows the presence of a single major peak at a density of 1.18 g/cm3. The density value of 1.18 is characteristic of previously reported values determined for eucaryotic chromatin banded in metrizamide gradients (Birnie, 1978). Since the 3H label banded at the same density as chromatin in metrizamide, it appears that HSV-2 DNA which coextracts with eucaryotic chromatin is in a complex with the same density as chromatin. In order to show that the density 1.18 g/cm3

Over a period of several years there have been sporadic reports describing experiments which indicated that the DNA of various members of the herpesvirus group is present in infected cells as a DNA: protein complex (Kierszenbaum and Huang, 19’78;Pignatti et aZ.,1979a, b, Hyman et aZ., 1979; Shaw et a.& 1979). Recently, Pignatti and Cassai (1980) have reported extraction of 200 S NPC, from HSV-l-infected cells, which contains viral DNA and some viral capsid proteins. These authors suggest that the 200 S NPC is an intermediate in HSV-1 replication, which precedes complete encapsidation of viral DNA but follows the formation of a DNA replication complex which they have previously described (Pignatti et ah, 1979a).

10 FRACTIOth.lBER

30

FIG. 9. Equilibrium centrifugation of DNA extracted from chromatin prepared from HSV-2-infected CV-1 cells. Pooled fractions from the peak region of the metrizamide gradient shown in Fig. 7 were dialyzed, deproteinized, and centrifuged to equilibrium in neutral CsC1gradients.

354

HALL

In general, particularly with reference to the timing of synthesis of various NPCs during the virus replication cycle, our findings support those of previous investigations. However, in specific instances, such as the sedimentation rates of NPCs, some discrepancies are apparent. Early in the virus replication cycle (4 hr or less), HSV-2 DNA-protein complexes were not extractable under the conditions utilized in these investigations. Similar results have been described (Pignatti and Cassai, 1980) for HSV-1. However, using slightly modified extraction techniques soon after infection, Pignatti et al. (197913) isolated an HSV-1 DNA-protein complex containing endogenous viral DNA polymerase activity which they suggested was the initial virus DNA replication complex. Data presented here (Table 1) show that HSV-2 DNA was being synthesized in nuclei soon after infection (O-2 hr). However, since this DNA was resistant to extraction as NPC by our methods, and copurified with cellular chromatin, these data suggest that the early HSV-2 DNA replication complex is chromatin-associated as has been proposed by Knopf (1979). This proposal is also supported by the presence of nucleosomal structure in chromatin-associated DNA, since it has been shown that replicating eucaryotic and simian virus 40 DNAs maintain a nucleosomal form (Weintraub et ab, 1976; Klempnauer et aL, 1980). The apparent discrepancy in sedimentation velocity of HSV NPCs described in this investigation and those of others (Pignatti and Cassai, 1980) is difficult to reconcile. In each instance, the NPCs appear to contain unit length viral DNA associated with approximately an equal weight of protein. In addition, contrary to the known nucleosomal structure of papovavirus chromosomes, neither HSV-1 or HSV-2 NPCs were organized in a nucleosomal structure. Thus, with so many common characteristics shared by HSV-1 and HSV-2 NPCs, we suggest that their configuration, compact or extended, may be responsible for apparent differences in sedimentation velocity. Since it has previously been shown that relatively minor

ET AL.

alterations in the proteins of polyoma virus NPCs lead to significant changes in their sedimentation characteristics (Goldstein et al, 1973), an attractive speculation is that differences in protein composition account for the differing sedimentation properties of HSV-1 and HSV-2 NPCs. There is evidence that HSV-1 NPCs contain exclusively viral capsid proteins (Pignatti and Cassai, 1980). However, our preliminary studies indicate that HSV-2 NPCs contain host-coded proteins as well as virus capsid proteins (Shapiro and Hall, 1982, in preparation). Although an estimate of the amount of HSV-2 DNA which is organized into nucleosomes in CV-1 nuclei cannot be made from the experimental data presented in Fig. 6, an estimate of the maximum amount of intranuclear HSV-2 DNA which could be nucleosomal can be derived from the data in Table 1. Thus, for the lo- to 12-hr postinfection labeling cycle, 63% of intranuclear HSV-2 DNA is extractable as an NPC. This fraction of the total intranuclear HSV-2 DNA was not organized as nucleosomes (Fig. 4). Therefore, the maximum amount of HSV-2 intranuclear DNA which could be organized in nucleosomes between 10 and 12 hr postinfection is around 37%. This value lies between recently published values of 0% (Mouttet et ah, 1979) and less than 10% (Leinbach and Summers, 1980) nucleosomal organization for intranuclear HSV-1 DNA and 80% nucleosomal organization for episomal Epstein-Barr virus DNA (Shaw et al., 1979) in nuclei of an infected but nonvirusproducing Ragi cell line. The fact that Mouttet et aL (1979) found no evidence for nucleosomal organization of intranuclear HSV-1 DNA may be explained by their relatively early labeling and nuclease digest times. In the two reported instances of detection of HSV DNA in nucleosomal form (this manuscript and Leinbach and Summers, 1980), cells were labeled from 10 to 12 or 12 to 13 hr postinfection, while Mouttet et al. (1979) labeled from 6 to 7 hr postinfection. Therefore, it seems possible, as was previously suggested (Leinbach and Summers, 1980), that progeny HSV DNA does not become

HSV-2

NUCLEOPROTEIN

organized into nucleosomes until intermediate or late times during the viral replicative cycle. Similarly, the three- to fourfold difference in relative amounts of intranuclear nucleosomal DNA between HSV-1 (Leinbach and Summers, 1980) and our studies with HSV-2 may simply reflect differences in the timing of formation of nucleosomal DNA structure during the replicative cycles of HSV-1 and HSV-2. Finally, it should be emphasized that, in this investigation, the NPC which was characterized was extracted at 12 hr postinfection. If HSV NPCs are structural precursors of complete virions as has been suggested (Pignatti and Cassai, 1980), it might be expected that as they “mature” toward the structure of a complete virion their protein complement, molecular configuration, and structural organization would change. Therefore, it will be important to characterize various classes of intracellular HSV NPCs extracted from infected cells at selected time intervals throughout the HSV replicative cycle. ACKNOWLEDGMENTS

This research was supported by Public Health Service Grant CA-28029 from the National Cancer Institute to S.C.S.J. and by grants from the Reno Cancer Center Inc. and the University of Nevada Research Advisory Board to M.R.H. REFERENCES ALONI, Y., WINOCOUR,E., SACHS,L., and TORTEN,J.

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