Differential association with cellular substructures of pseudorabies virus DNA during early and late phases of replication

VIROLOGY

139, 205-222 (1984)

Differential Association with Cellular Substructures of Pseudorabies Virus DNA during Early and Late Phases of Replication’ TAMAR BEN-PORAT,2 RUTH ANN VEACH, MAYME L. BLANKENSHIP, AND ALBERT S. KAPLAN Department of b&robiologg, Vanderbilt University School of Medicine, NashviUe, Tennessee x??s.s’ Received July S, 1984 accepted August 14, 1984 Pseudorabies virus DNA synthesis can be divided into two phases, early and late, which can be distinguished from each other on the basis of the structures of the replicating DNA. The two types of replicating virus DNA can also be distinguished from each other on the basis of the cellular substructures with which each is associated. Analysis by electron microscopic autoradiography showed that during the first round of replication, nascent virus DNA was found in the vicinity of the nuclear membrane; during later rounds of replication the nascent virus DNA was located centrally within the nucleus. The degree of association of virus DNA synthesized at early and late phases with the nuclear matrix fractions also differed; a larger proportion of late than of early nascent virus DNA was associated with this fraction. While nascent cellular DNA only was associated in significant amounts with the nuclear matrix fraction, a large part (up to 40%) of all the virus DNA remained associated with this fraction. However, no retention of specific virus proteins in this fraction was observed. Except for two-virus proteins, which were preferentially extracted from the nuclear matrix, approximately 20% of all virus proteins remained in the nuclear matrix fraction. The large proportion of virus DNA associated with the nuclear fraction indicated that virus DNA may be intimately associated with some proteins. Indeed, protease-treated, “purified” DNA preparations contained two proteins (15K and 10K) with histone-like properties which were protected by the DNA from deproteinization, probably by virtue of being at the center of the concatemeric tangles of virus DNA. It is possible that these proteins play a role in anchoring virus DNA to the nuclear matrices. o 1~ Academic

Press, Inc.

INTRODUCTION

istics of replicating virus DNA might shed some light on this problem, we have studied for the last few years the mechanism of replication of the DNA of pseudorabies (PrV) (Herpesvirus suis, type 1). We have found that virus DNA replication may be divided into two phases during which the structures of the replicating DNA differ. During the first phase, replication of virus DNA is initiated on circular unit size molecules, and Cairn’s type structures are observed (Jean et c& 1977; Ben-Porat and Tokazewski, 1977; Ben-Porat and Veach, 1980). At later stages, replicating virus DNA is associated with rapidly sedimenting structures (Ben-Porat et aL, 1976). The major characteristics of the concatomeric tangles of replicating herpesvirus DNA

Rearrangements occur in the DNA of some herpesviruses leading to the formation of molecules in which various parts of the genome are present in different orientations relative to one another. The inverted repeated sequences present in the genomes of these viruses probably play a role in generating these inversions; however, the biological implications of these rearrangements are unknown (BenPorat, 1982). With the hope that an understanding of the structural character’ This investigation was supported by a grant (AI10947) from the National Institutes of Health. z Author to whom requests for reprints should be addressed. 205

0042-6822/&i Copyright All rights

$3.00

Q 1994 by Academic Press. Inc. of reproduction in any form reser vzd.

206

BEN-PORAT

are the following: (1) They consist, at least in part, of complex tangles with densely aggregated centers (Ben-Porat et aL, 1976; Jacob and Roizman, 1977). (2) They are not disrupted by treatment with detergents, proteases, or RNase (Hirsch et aL, 19’76; Ben-Porat et aL, 1976). (3) They consist of virus DNA molecules in tandem head-to-tail alignment (Ben-Porat and Rixon, 1979; Jacob et uL, 1979). (4) The average number of unit-size molecules in the linear arrays is on the average only three to five; the sedimentation characteristics of the structures are, however, those expected of much longer linear doublestranded DNA molecules (Ben-Porat and Rixon, 1979). Jacob et al. (1963) have postulated that in bacterial cells initiation of DNA synthesis occurs in association with some supporting structure, an association that should facilitate the replication of the DNA, as well as its partitioning into daughter molecules. This has been shown to be the case for several bacterial species. An association of nascent eukaryotic DNA with the nuclear membrane has been extensively looked for and while the results have been somewhat controversial, it is now generally agreed that nascent eukaryotic DNA is not associated with the nuclear membrane. On the other hand, the association of nascent eukaryotic cell, as well as nascent virus, DNA with the nuclear matrix, i.e., the fast sedimenting residue obtained from nuclei after their extraction with nonionic detergents and high salt, as well as after controlled DNase treatment, is by now well documented (Berezney and Coffey, 1975; Dijkwell et aL, 1979; Pardoll et aL, 1980; BucklerWhite et aL, 1980; Hunt and Vogelstein, 1981; Younghusband and Maundrell, 1982). Furthermore, the association of some virus proteins [including herpes simplex virus (HSV) proteins (Chin and Maizel, 1977; Buckler-White et al, 1980; Bibor-Hardy et aL, 1982; Knipe and Spang, 1982; Ben-Zeev et al, 1983)] with the nuclear matrix fraction has also been reported. In this paper, we report on experiments which show that the two phases of DNA synthesis of PrV can be distinguished

ET AL.

from each other on the basis of the substructures with which each is associated. Nascent virus DNA synthesized at the beginning of the first round of replication appears to occur in the vicinity of the nuclear membrane; the synthesis of virus DNA during the later stages occurs throughout the nucleus. Furthermore, whereas the nuclear matrix fraction is enriched for nascent DNA during both early and late DNA replication, a larger proportion of the late than of the early nascent DNA is found in this fraction. Interestingly, the association of virus DNA with the nuclear matrix is not confined to nascent DNA, a large proportion of total virus DNA synthesized by the infected cells (but not of cellular DNA) is associated with this fraction. Approximately 20% of all the nuclear virus proteins (except for two which are preferentially extracted) also are retained with the nuclear matrix fraction. These proteins include two histone-like proteins that remain associated with deproteinized concatemeric tangles of virus DNA. These concatemeric tangles of virus DNA are strongly associated with the two proteins, an association that is resistant to extraction with high salt solutions. These results suggest that these proteins may play a role in anchoring the virus DNA to the nuclear matrix. MATERIALS

AND

METHODS

Virus and cell culture. The preparations of PrV and cultivation of primary rabbit kidney (RK) cells have been described previously (Kaplan and Vatter, 1959). Media and solutions. EDS: Eagle’s synthetic medium plus 3% dialyzed bovine serum. EDS S PO& EDS without POr. EDS-FU EDS plus 5-fluorouracil (FU) (20 pg/ml) and thymidine (10 pg/ml). RSB: 0.1 M Tris, pH 7.4, 0.01 M KCl, 0.0015 M MgClz. RSB-2% SDS: RSB plus 2% sodium dodecyl sulfate (SDS). RSB-Triton: RSB plus 1% Triton X-100. PBS: 0.15 M NaCl, 2.6 mM KCl, 8 mM Na2HP04, 1 mM KHzP04, 20 mM MgClz, 1.8 mM CaClz (pH 7.0). ilfatrix In&&r: 10 mM Tris, pH 7.4, 0.2 mM MgClz, 1 mM phenyl-methyl-sulfonyl fluoride, and 1% Triton.

ASSOCIATION

OF PrV

DNA

Enzymes and chemicals Restriction endonucleases were purchased from the Bethesda Research Laboratories, DNase I, RNase A, and protease K from Worthington Biochemical Corporation, pH]thymidine (46 Ci/mmol), [14C]thymidine (59 mCi/mmol), and [‘Hlleucine (55 Ci/mmol) from Schwarz/Mann, and nuclease-free pronase from Calbiochem. i4C-labeled protein markers were purchased from Amersham. Treatment of ceils to inhibit ceUu.larDNA synthesis. In all experiments dealing with the synthesis of virus DNA, the cultures were pretreated for 24 hr before the experiment with EDS-FU. This treatment completely and irreversibly inhibits cellular DNA synthesis in RK cells without affecting virus DNA synthesis or production of infectious virus (Kaplan and BenPorat, 1961). Extraction of DNA. This was carried out as follows: Sodium sarkosinate (final concentration, 2%) was added to the samples which were heated (60” for 15 min) and digested with nuclease-free pronase (1 mg/ml) for 2 hr. The DNA was then extracted four times with phenol/chloroform/isoamyl alcohol (25:24:1) and dialyzed against buffer (0.01 M Tris, 0.001 M EDTA, pH 7.6). Restriction enz2/me digestion and gel ekctrophtwesis of DNA fragments. Digestion and agarose gel electrophoresis of PrV DNA were carried out as described by Ben-Porat and Rixon (1979). Filter strips to which restriction fragments of PrV DNA were fixed were prepared by the method of Southern (1975). Labeling of po@eptides and polyacrylamide gel electrophor& (PAGE) of proteins. These were performed as described previously (Ladin et & 1982). Commercial markers, as well as the proteins of adenovirus type 2 and herpes simplex virus, type 1, were used to determine the molecular weights of the PrV proteins. Preparation of nuclear mtties. These were prepared essentially as described by Pardoll et al. (1980). The cell monolayers were washed once with cold PBS, scraped into RSB-Triton (concentration, 4 X lo6 cells/ml), homogenized 20 times with a

WITH

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SUBSTRUCTURES

207

tight-fitting Dounce homogenizer, and the nuclear fraction was collected by centrifugation at 3000 rpm for 7 min. The nuclei were resuspended in 1 ml of matrix buffer and NaCl was slowly added to give a final concentration of 2 M. DNase (40 /rg/ml) was added and the samples were incubated for 30 set at 37’ and were then left on ice for 20 min. The samples were centrifuged for 15 min at 2000 g and the pellets (nuclear matrix fractions) were separated from the supernatant (chromatin fraction). This procedure yielded the nuclear matrix fraction which contained approximately 5% of the cellular DNA and 20% of the nuclear proteins. Is~cnic centrifugation in CsCl This was performed as described previously (Ben-Porat et u& 1976). Acid extract&m of proteins. The samples were left at 0” for 30 min in 0.25 N HCl. They were then spun at 12,000 g for 30 min and the supernatants were neutralized. The proteins were concentrated by acetone precipitation. Electron microscqq autoradiography The methods used for electron microscopy have been described previously (Ladin et al, 1980); autoradiography was performed essentially as described by Caro and Tubergen (1962). Briefly, thin sections of 1000 A were cut using glass knives. The grids supporting the sections were coated with a layer of Ilford L-4 emulsion (1:4) and incubated at 4°C for l-6 months; they were developed with D-19 and observed (unstained) in a Philips 301 electron microscope. RESULTS

Localization by Autoradiography of Nascent Virus DNA S~ntbsized During Early and Late Phases of DNA Rep lication Cellular DNA synthesis is irreversibly inhibited in RK cells by treatment with FU and thymidine (Kaplan and Ben-Porat, 1961); FU-treated cells, however, support virus growth and no differences in various aspects of virus macromolecular synthesis and virus growth can be detected between untreated cells and cells which have been

208

BEN-PORATET AL.

ASSOCIATION

OF PrV

DNA

pretreated with FU. The lack of synthesis of cellular DNA in FU-treated cells allows one to detect by autoradiography the synthesis of virus DNA in these cells. To determine whether PrV DNA synthesized during early and late phases of DNA synthesis is associated with similar cellular substructures, we first ascertained whether it occurs at similar locations within the infected cells. To this end, the silver grain distribution over the nuclei of infected cells, pulse-labeled at early and late times of the infective process, was determined by electron microscopy autoradiography. At the multiplicity of infection used (2 PFU/cell), virus DNA synthesis starts in RK cells at about 2 hr 20 min after infection. We have previously estimated that the first round of virus DNA replication is completed within 45 min after initiation (Ben-Porat et aL, 1977). Thus, one can estimate that in these cells the first round of virus DNA replication takes place up to approximately 3 hr postinfection. Figures 1 and 2 illustrate the distribution of grains over representative cells pulse-labeled during early, as well as late, phases of DNA synthesis. Table 1 summarizes the distribution of the grains over the cells at these times after infection. At early times after infection (2 hr 40 min), silver grains were found in most cases (15 out of 20) near the periphery of the nucleus in the vicinity of the nuclear membrane. The typical appearance of such nuclei is illustrated in Fig. 1. By 4 hr postinfection, the silver grains were centrally located in all cells (Fig. 2). FUtreated, uninfected cells showed occasional grains which were randomly distributed over the cells; no clusters of silver grains were observed over the nuclei of the uninfected cells.

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203

Association with the Nuclear Matrix Fraction of Nascent Virus DNA Synthesized at Various Times c&w Infection The fact that the distribution of nascent virus DNA within the nuclei appeared to differ at early and late phases of DNA synthesis prompted us /to determine whether differences could be detected at these times after infection in the degree of association of newly synthesized DNA with the nuclear matrix. The results of a representative experiment are illustrated in Table 2. The nuclear matrix fractions prepared from uninfected cells (M.I., non-FUtreated) were, as expected, enriched for newly, synthesized DNA and the proportion of this DNA that was associated with the nuclear matrices decreased after a chase. These results are similar to those reported previously by several investigators (Dijkwell et aL, 1979; Vogelstein et aL, 1980; Hunt and Vogelstein, 1981) who concluded on the basis of these results that cellular DNA is synthesized in association with the nuclear matrix. In FU-treated, uninfected cells little rH]thymidine was incorporated into DNA; the small, residual amount of DNA that was synthesized by these cells was found in the chromatin fraction. This is mitochondrial DNA, the synthesis of which is not inhibited by treatment with FU (unpublished results). Mitochondrial DNA is also synthesized in the infected cells but it represents a small fraction only of the total DNA synthesized by these cells. During the first round of virus DNA replication (2 hr 20 min and 2 hr 40 min), approximately 12% of the virus DNA synthesized during a short pulse (but only 5% of the prelabeled cellular DNA that served as an internal control) were associated with the nuclear matrix fraction.

FIG. 1. Electron autoradiomicrograph of infected cells pulse-labeled at 2 hr 40 min postinfection. FU-treated cells were infected (m.o.i., 2 PFU/cell) and incubated at 37” for 2 hr 40 min. They were then labeled for 3 min in EDS containing [‘Hlthymidine (106 pCi/ml). The cells were harvested and prepared for electron microscopy, as described under Materials and Methods. The grids were coated with photographic emulsion and exposed for 6 months. Examination of sequential sections through the same cells revealed similar patterns of grain distribution. Magnification: 12,350X.

210

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ET AL.

FIG. 2. Electron autoradiomicrograph of infected cells pulse-labeled at 4 hr postinfection. cells were treated and infected as described in the legend to Fig. 1 but were pulse-labeled min at 4 hr postinfection. Magnification: 11,600X.

The for 3

ASSOCIATION

OF PrV

DNA

WITH

TABLE

CELL

211

SUBSTRUCTURES

1

DISTRIBLITION OF NASCENT VIRUS DNA WITHIN THE NUCLEI OF INFECTED CELLS AT VARIOUS TIMES AFTER INFECXION’

Time after infection

Number of nuclei examined

2 hr 40 min 4 hr Uninfected

306 220 626

Nuclei with sitver grains 20 20 Ob

Distribution of grains over nucleus Peripheral 15 0 -

Central 5 20 -

“FU-pretreated cells were infected (m.o.i., 2 PFU/cell) and labeled for 3 min with FHjthymidine (106 &i/ml) at the indicated time of infection. The cells were prepared for electron microscopy autoradiography, as described under Materials and Methods. The films were developed after 6 months exposure for the 2 hr 40 min postinfection time point (and for the uninfected cells) and after 1 month exposure for the 4-hr time point. The total number of cells in each case with more than 3 grains per nucleus and the distribution of these grains over 20 nuclei was determined. b An occasional grain was seen over the nuclei of uninfected cells; no uninfected nucleus with more than 2 grains was observed.

These results suggested that the matrix fraction is enriched for nascent virus DNA, as it is for nascent cell DNA. At later times after infection, the proportion of newly synthesized virus DNA associated with the matrix fraction increased (28%); in some experiments more than 40% of this DNA remained associated with the nuclear matrix fraction (data not shown). In contrast to cellular DNA, which is associated with the nuclear matrix only during replication and released thereafter, virus DNA synthesized both during early and late phases of DNA replication was not displaced from the nuclear matrix fraction after a chase. Thus, it appears that the association of virus DNA with the nuclear matrix is not strictly comparable to that of nascent cellular DNA and that a large proportion of the total virus DNA in the nuclei is anchored to the matrix structure. We have attempted to determine whether specific sequences along the molecules were involved in anchoring the DNA. None was detected; the virus DNA that was released from, as well as the DNA that was retained with, the nuclear matrix fraction was complementary to all restriction fragments generated from PrV DNA that were tested (more than 60) (data not shown).

The results described above show that a relatively large proportion of virus DNA is present in the cell in a state in which it is resistant to salt extraction and limited digestion by DNase. To determine whether this virus DNA (which is known to be in the form of concatemeric tangles after the early rounds of DNA replication) is indeed associated with the nuclear matrices or whether it is part of structures that cosediment with the nuclear matrix, we have examined these structures by electron microscopy autoradiography. Examples of typical structures seen in the nuclear matrix fractions are illustrated in Fig. 3. Because most of the silver grains in the samples were distributed over structures which are similar to those previously described as nuclear matrices (Berezney, 1980; Berezney and Coffey, 1977), we conclude that the virus DNA is indeed retained in the nuclear matrices. Association of Virus Proteins with the Nuclear Matrices Because a large proportion of virus DNA remains associated with the nuclear matrix fraction (Table 2), it was of interest to ascertain the behavior of various virus proteins in this respect. Table 3 shows

906 (12)

1 (1)

106’ (12)’ 255 (21) 6800 GW

Matrix

‘H-virus

36 (5) 91 (5)

‘3,544

36 (5) 42 (5) 42 (5)

Matrix

“C-cell

120

795 960 17,643

Chromatin

DNA

Pulse

1860

610

735 863 736

Chromatin

DNA

17,005

190

2 (1) 1,036 (6)

1,825 ND 25,558

Chromatin

DNA

mo (W NDd 13,421 (34)

Matrix

‘H-virus

Chase

88 (4)

31 (4)

41 (5) ND 42 (5)

Matrix

“C-cell

1,950

680

860 ND 790

Chromatin

DNA

“Primary RK cells were labeled for 24 hr with EDS containing [%]thymidine (0.005 &i/ml). The cells were then washed extensively and further incubated in EDS or EDS-FU (to inhibit further cellular DNA synthesis) for 16 hr. FU-treated cells were infected (m.o.i., 12 PFU/cell) and at the indicated times, labeled with [8H]thymidine (100 &i/ml) for 3 min. Mock-infected cells, either FU-treated or non-FU-treated, were similarly labeled. Part of the cultures was harvested immediately after the labeling period (pulse); the remainder was incubated in EDS containing thymidine (100 pg/ml) and deoxycytidine (5 pg/ml) for 1 hr (chase) and then harvested. Nuclei were isolated and the nuclear matrix fractions were separated from the chromatin fractions, as described under Materials and Methods. The number of acid-precipitable counts associated with each fraction was determined. b cpm X lo-* per 8 X lo6 cells (two 90-mm petri plate cultures). “The numbers in parentheses represent the percentage of total acid-precipitable radioactivity (chromatin plus nuclear matrix fraction) in the nuclear matrix fraction. d ND, not determined. e MI., mock-infected.

MI. (Non-FU-treated)

M.I.” (FU-treated)

2 hr 20 min 2 hr 40 min 4 hr

Time after infection

2

ASSOCIATION OF NASCENT DNA WITH THE NUCLEAR MATRIX AT VARIOUS STAGES OF INFECTION“

TABLE

2

F

H 1 g

ASSOCIATION

OF

PrV

FIG. 3. Electron autoradiomicrograph The cells were treated and infected as postinfection with [SH]thymidine (100 isolated, as described under Materials coated with photographic emulsion and

DNA

WITH

CELL

of nuclear matrix structures isolated from infected described in the legend to Fig. 1 and pulse-labeled at &i/ml) for 5 min. The nuclear matrix fractions and Methods, and fixed. Thin sections were made exposed for 1 month. Magnification: 7660X.

the distribution of labeled proteins between the cytoplasm, the chromatin, and the nuclear matrix fractions of infected

213

SUBSTRUCTURES

cells. 4 hr were and

and uninfected cells immediately after a 20-min labeling period, as well as after a chase of 1 hr. The overall distribution of

214

BEN-PORAT TABLE

3

DISTRIBUTION OF PROTEINS BETWEEN THE CYTOPLASMIC, CHROMATIN, AND NUCLEAR MATRIX FRACTIONS OBTAINED FROM INFECTED AND UNINFECTED CELLS0 Infected

cells

M.I.

cells

Fraction

Pulse

Chase

Pulse

Chase

Cytoplasmic Chromatin Nuclear matrix

25.33* 7.25

22.67 13.13

46.30 13.70

42.10 16.41

1.25

3.06

3.15

3.00

% total cpm in nuclear matrix

3.7

7.7

5.0

4.9

% nuclear cpm in nuclear matrix

14.7

21.5

13.7

15.5

“RK cells were infected (m.o.i., I2 PFWcell) or mock-infected (MI.) and incubated in EDS. At 4 hr postinfection the cells were incubated in EDS S AA containing [sH]leucine (100 &i/ml) for 20 min. Part of the cultures was harvested immediately thereafter (pulse) and part was further incubated for 2 hr in EDS 2X AA and then harvested. Nuclei were isolated and the nuclear matrix fractions were separated from the chromatin fractions, as described under Materials and Methods. The number of acid-precipitable counts in each fraction was determined. *cpm X lo-‘.

the labeled proteins did not differ dramatically between the infected and uninfected cells; however, the proportion of total labeled proteins after the chase that was retained with the nuclear matrix was larger in infected than in uninfected cells. Analysis of the proteins by PAGE (Fig. 4) revealed the following: (1) Although some cellular nuclear proteins were not detectably synthesized by the infected cells, for example, the 36K, 17K, and 13K proteins (compare lanes AN with BN), most of the cellular nuclear matrix proteins, for example, the 63K to 45K proteins were synthesized (compare lanes AM with BM). Thus, a differential degree of inhibition of various cellular nuclear proteins was observed after infection, the nuclear matrix proteins being less affected than other. proteins. (2) In contrast to unin-

ET

AL.

fected cells, in which specific nuclear proteins were associated with the nuclear matrix fraction, in infected cells most virus nuclear proteins, including for example the major capsid proteins, were present in the nuclear matrix fraction. Only two virus proteins appeared to have been preferentially extracted from the matrix fraction: the 136K major DNAbinding protein (Ben-Porat et aL, 1982b) and the 23K protein. Thus, although the methods that we have used yielded nuclear matrix fractions from uninfected cells that were, as expected, considerably enriched for several proteins (the nuclear matrix proteins 63K-45K), similar preparations from infected cells, though enriched for these cellular matrix proteins, were not significantly enriched for any specific virus protein. With the exception of the 136K and 23K proteins, which were preferentially removed from the nuclear matrix fraction, a relatively small proportion [approximately 20% (see Table 3)] of most of the nuclear virus proteins remained associated with the nuclear matrix fraction. Association DNA

of Proteins

with Concateme-ric

The large proportion of virus DNA that remains associated with the nuclear matrix fraction suggested that the virus DNA molecules may be anchored to the nuclear matrix structure (and possibly to each other) by specific proteins. Indeed, deproteinized concatemeric virus DNA sediments more rapidly than expected on the basis of its length, as determined by its relative number of free ends (Ben-Porat and Rixon, 1979), indicating that it may have an unusual structural configuration and may be complexed with some other macromolecules. We had previously considered this possibility and had subjected the deproteinized virus DNA to a variety of treatments including digestion with proteolytic enzymes, RNase, treatment with SDS and Triton, as well as extraction with 4 M NaCl, to test for the presence of “linker” molecules (Ben-Porat et cd, 1976 and unpublished results). None of

ASSOCIATION

OF

A

N

PrV

DNA

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CELL

C

6

M

N

SUBSTRUCTURES

M

C

215

D N

C

N

1 1

FIG. 4. Autoradiogram of the polypeptides synthesized by infected and uninfected cells present in the nuclear and nuclear matrix fractions isolated from these cells. RK cells were infected (A and C) or mock infected (B and D) and incubated for 90 min (A and B) or for 99 min (C and D) in EDS g AA containing [8H]leucine (100 &i/ml). The cells were harvested, the cytoplasmic, nuclear, and nuclear matrix fractions were isolated and the proteins were electrophoresed in 10% acrylamide gels, as described under Materials and Methods. N, nuclear fraction; M, nuclear matrix fraction; C, cytoplasmic fraction.

these treatments affected the sedimentation rates of the “purified” concatemeric DNA preparations. To investigate further the possibility that proteins may be present in the preparations of concatemeric DNA, infected cells were labeled between 4 and 6 hr postinfection with either [3H]leucine and [‘4C]arginine or [3H]leucine and [14C]thymidine. The DNA was extracted and the amount of labeled proteins that remained associated with preparations of deproteinized concatemeric DNA was determined. Table 4 shows that 0.3% of the total [3H]leueine incorporated into the infected cells during the labeling period remained associated with the purified DNA preparations. A large proportion (60%) of these proteins was acid soluble; furthermore, these proteins were rela-

tively rich in arginine (compared to the bulk of proteins synthesized by the infected cells), as ascertained from their leucine-to-arginine ratio. Thus, the proteins that remained associated with the concatameric DNA fraction have histonelike properties; they are acid soluble and are relatively rich in arginine. The fact that some proteins remained associated with the DNA preparation, despite extensive digestion with proteases and repeated deproteinization, indicated that these proteins may be present in the preparations in a protected state. That this is indeed the case is shown by the results of the experiment summarized in Table 5. In this experiment, the DNA from [‘Hjleucine-labeled infected cells was purified. Part of the preparation was treated with DNase and part was acid-

216

BEN-PORAT

ET

TABLE PROTEINS

ASSOCIATED

WITH DNA [“Clthymidine (WI

A Total acid-precipitable radioactivity B Recovery after phenol extraction and dialysis C Recovery after pronase digestion, phenol extraction, and dialysis D Recovery after acid extraction of c

AL.

4

EXTRACTED

FROM INFECTED [SHJeucine 6)

CELLS’ Ratio [‘H]leucine/[“C]arginine

100.0

100.00

10.8

83.7

0.29

5.1

86.1

0.30

4.6

0.3

0.19

3.5

D RK cells were infected and labeled in EDS S AA between 3-6 hr postinfection with either [“Clthymidine (0.05 &i/ml) and [8H]leucine (25 &i/ml) or with [“Clarginine (0.1 &i/ml) and [%I]leucine (25 nCi/ml). The cells were harvested, a portion was acid precipitated to determine the amount of label incorporated into the cells (A), and the DNA was extracted, as described under Materials and Methods, and extensively dialyzed (B and C). Part of the dialyzed samples was treated with 0.25 N HCl on ice for 30 min and centrifuged for 30 min at 15,OOOg. The supernatant was neutralized and the number of counts in it were determined.

extracted. Half of each sample (as well as of an untreated control sample) was digested with pronase; the samples were dialyzed extensively and the amount of radioactive protein that remained undiTABLE EFFECT OF PRONASE PROTEIN COMPLEX

A-Untreated

C-Acid

TREATMENT ON THE DNAAFTER ITS DISSOCIATION Pronase digestion

Treatment

B-DNase

5

treated soluble

+ + + -

cpm/sample 3220 4130 410 4410 320 2680

“Cells were infected and labeled with [‘Hlleucine (100 &i/ml) between 3 and 6 hr postinfection. The DNA was extracted, as described under Materials and Methods, and dialyzed extensively. The DNA preparation with the residual proteins associated with it was either used as such (A), treated with DNase (10 pg/ml) (B), or extracted with acid (C). Part of the samples was then pronase-treated (500 pg/ml). The samples were dialyzed and the number of counts that remained nondialyzable was determined.

alyzable in each sample was determined. As expected from the results described above, the ‘H-labeled proteins present in the untreated DNA-protein complex were not susceptible to pronase digestion. After DNase treatment or after acid extraction, however, the proteins became susceptible to pronase digestion, as indicated by the fact that the [3H]leucine-labeled material had become dialyzable. We conclude from these results that proteins that remain associated with concatemeric DNA are probably in the center of the tangles of DNA and are thereby protected. Proteins Remaining in the DNA Preparation Are Associated with Vim, Not Cell, DNA Figure 5 shows that the proteins that remain in the deproteinized preparations of DNA are associated with virus, not cell, DNA. In this experiment, FU-treated infected cells were labeled with [3H]leucine and [14C]thymidine, the DNA was extracted, and the DNA preparation was treated with glutaraldehyde to crosslink the proteins to the DNA. The samples were then subjected to isopycnic centrifugation in CsCl to separate cellular from

ASSOCIATION

OF

PrV

DNA

WITH

CELL

SUBSTRUCTURES

FIG. 5. The proteins that remain in the DNA preparation are associated with virus, not cellular, DNA. FU-treated cells were infected (m.o.i., 20 PFU/cell) and incubated at 37”. At 4 hr postinfection, the cultures were incubated for 60 min in EDS S AA containing [‘Hlleucine (100 &i/ml) and [“Cjthymidine (0.05 &i/ml). The cells were harvested, lysed, treated with pronase, and the DNA was extracted and dialyzed extensively. The samples were fixed with glutaraldehyde (0.01% for 15 min at room temperature) and centrifuged to equilibrium in CsCl. Samples were collected dropwise and the amount of radioactivity associated with each was determined.

virus DNA. The [3H]leucine-labeled proteins in the samples were associated with virus (14C-labeled) DNA only (Fig. 5); none was associated with cellular DNA (uv absorbing peak). If the DNA preparations were subjected to isopycnic centrifugation without prior glutaraldehyde treatment, the labeled proteins became dissociated from the virus DNA and were found at the top of the gradient (data not shown). We conclude from these results that proteins are associated with deproteinized virus DNA; that they are not covalently linked to the DNA since they are normally removed from the DNA during isopycnic centrifugation but that they can be crosslinked to the virus DNA by treatment with glutaraldehyde. PAGE ProjEles of the Proteins Remaining Associated with the Concatemeric complexes The proteins remaining associated with the DNA preparations were analyzed by PAGE; the autoradiograms are illustrated in Fig. 6. Two protein bands (15K and

10K) were detected. Similarly migrating proteins were detected in nuclear preparations of infected, but not of uninfected, cells indicating that these are virus proteins. In the DNA complex, the 15K protein was usually present in much larger amounts than the 10K proteins. On the other hand, the 10K protein was more abundant in the nuclear fraction of infected cells; the 10K (but not the SK) protein is a structural virus protein (see first lane, Fig. 6). Figure ‘7 illustrates an autoradiogram of the proteins present in the nuclei of the cells infected with wild-type or a ts mutant which is DNA- at the nonpermissive temperature. Interestingly, the 10K (and to a lesser extent the 15K) protein is drastically underrepresented in the nuclei of infected cells arrested during early stages of infection, i.e., under DNA- conditions. The 10K protein is also consistently not detectable in cells labeled at early stages of infection or under DNAconditions (in cells infected with DNAts mutants or in the presence of phosphonoacetic acid). The 10K protein can be

218

BEN-PORAT

Vir

Inf

Con

MI

ET

AL.

MI

con

Inf

FIG. 6. Autoradiogram of the proteins associated with “purified” DNA preparations. Cells were infected (m.o.i., 20 PFU/cell) or mock infected and incubated in EDS S AA containing [‘Hlleucine (100 &i/ml) between 3 and 6 hr postinfection. The cells were harvested, lysed, treated with pronase, and subjected to several cycles of phenol-chloroform extraction to obtain “purified” DNA preparations. The DNA preparations were treated with DNase, dialyzed, and the samples were electrophoresed in a 12% acrylamide gel to identify the proteins associated with concatemeric virus DNA (Con). The proteins present in the nuclear fractions, isolated from infected (Inf) and mock-infected (M.I.) cells were similarly electrophoresed. To identify the structural and nonstructural proteins, purified [aH]leucine-labeled virus (Vir) was also electrophoresed. A and B represent the results of two different experiments.

classified as a late PrV protein script in preparation).

(manu-

DISCUSSION

The replication of the DNA of PrV occurs in two phases: early and late (BenPorat, 1982). During the early phase, replicating DNA is mainly in the form of unit-size Cairn’s type molecules. Thereafter, replicating DNA is associated with concatemers in which unit-size DNA molecules are in head-to-tail tandem alignment. These structures may be generated

by a rolling circle mechanism but their exact nature is difficult to clarify because of the large size and tangled structure of the molecules, and because of the tendency of replicating PrV DNA to fragment upon isopycnic centrifugation in CsCl, a procedure necessary for its separation from cellular DNA (Ben-Porat and Tokazewski, 1977). Thus, the detailed structure of replicating late DNA remains to be ascertained. As part of an attempt to gain a more detailed understanding of the processes involved in early, as well as in late, phase

WT

ASSOCIATION

OF

TsC

Vir

MI

PrV

DNA

FIG. ‘7. Autoradiogram of the nIlclear proteins present in cells infected with a DNAmutant. Cells were infected at 41’ with wild-type virus (WT), or a DNAts mutant (tsC), or were mock infected (M.I.), and were labeled between 4 and 5 hr postinfection in EDS S AA containing PH]leucine (100 &i/ml). The nuclear fractions were isolated and the proteins present were electrophoresed in an acrylamide gel (10%). The proteins present in preparations of purified [JH]leucine-labeled virions (Vir) were also electrophoresed.

DNA replication, we have determined whether virus DNA is associated with different nuclear substructures during each of these phases of replication. Our results show that while virus DNA synthesized during early phase appears to occur mainly in the vicinity of the nuclear membrane, virus DNA synthesized during late phase becomes centrally located. Rixon et al. (1983) have also recently reported that HSV DNA accumulating in the cells (during 2-hr labeling periods) at early stages of infection appear to be located in close proximity to the nuclear membrane while at late stages the DNA is located throughout most of the nucleus.

WITH

CELL

SUBSTRUCTURES

219

Our results show that nascent virus DNA (synthesized during a 3-min pulse) behaves similarly. On the other hand, Bibor-Hardy and Simard (1980) have reported that virus DNA is synthesized near the periphery of the nucleus, even at late stages of infection. The basis for the discrepancy between their results and those reported by Rixon et al (1983), as well as our results (this paper), is unclear. Evidence has accumulated in recent years that nascent DNA of eukaryotic cells is associated with the nuclear matrix, i.e., with the skeletal residual structure that remains sedimentable after treatment of nuclei with DNase and extraction with high salt (Hunt and Vogelstein, 1981; Pardoll et aL, 1980). Nascent polyoma DNA, as well as adenovirus DNA, have also been found associated with the nuclear matrix fractions (Buckler-White et aL, 1980; Younghusband and Maundrell, 1982). We show here that the DNA of a herpesvirus (pseudorabies) is also nuclear matrix associated. Of interest is the finding that the degree of association of virus DNA with the nuclear matrix fraction differed between early and late phases of replication. A much larger proportion of late than of early nascent virus DNA was associated with the nuclear matrix fractions, as indicated by the fact that the percentage of nascent virus DNA relative to that of prelabeled cellular DNA was always higher at the late stage than at the early stage of DNA replication. Thus, there appears to be a difference in the degree of association with the nuclear matrix of nascent virus DNA synthesized during early and late phases of DNA replication, as well as a difference in the localization of nascent virus DNA within the nucleus. The nuclear matrix fractions that we have isolated from uninfected cells were enriched for newly synthesized DNA which, after a chase period, was released. Thus, these nuclear matrix fractions have characteristics similar to those previously isolated and more thoroughly characterized by others (Kaufmann et al, 1981; Pardoll et aL, 1980).

220

BEN-PORAT

The method that we have used to prepare the nuclear matrix fractions yielded preparations containing about 20% of the total nuclear proteins (5% of the total cellular proteins) and about 5% of the total cellular DNA. However, a relatively large proportion of virus DNA was associated with the nuclear matrix fractions not only immediately after its synthesis but also after a chase. The association of virus DNA with the nuclear matrix is not related to nucleocapsid formation; it occurs in cells infected at the nonpermissive temperature with a mutant (tsN) in which nucleocapsid formation is blocked (unpublished results). An analysis of the distribution of nuclear proteins between the chromatin and the matrix fractions revealed that the percentages of the proteins that were retained in the nuclear matrix fraction were somewhat larger in infected than in uninfected cells. However, while the nuclear matrix fractions from both infected and uninfected cells were enriched for several cellular matrix proteins (63K, 6OK, 57K, 53K, as well as 45K), no enrichment for specific virus proteins in the nuclear matrix fraction was observed. Evidence that HSV capsids and HSV capsid proteins are associated with the nuclear matrix has been presented (Bibor-Hardy et aL, 1982; Tsutsui et uL, 1933). Our results confirm the fact that virus capsid proteins are associated with the nuclear matrix. However, we did not observe any preferential retention of capsid proteins in the matrix fraction; approximately the same percentage (20%) of all nuclear virus proteins [with the exception of two proteins (136K and 23K) that were preferentially extracted] remained associated with this fraction. Two conformational forms of the major DNA binding protein of HSV-1 have been described previously (Knipe et al, 1982); two conformational forms of the 136K (136Ka and b) major DNA binding proteins of PrV can also be distinguished (Fig. 4). The two forms of the major DNA binding protein cannot be distinguished antigenically using monospecific sera against HSV-1 DNA binding protein

ET

AL

(Knipe et aL, 1982; T. Ben-Porat, unpublished results). It is of interest that one of the forms of the 136K protein of PrV (136Kb) appears to be preferentially extracted from the nuclei during the preparation of the nuclear matrix fraction. Thus, form “a” is more firmly bound to the nuclear matrix than is form “b.” Whether this reflects a functional difference between the two forms of the 136K cannot, however, be ascertained from these results. The concatemeric tangles of virus DNA are associated with two proteins, 10K and 15K. These proteins have histone-like properties, are relatively rich in arginine (Table 4), and are DNA binding proteins (T. Ben-Porat, unpublished results). They are protected from protease digestion by the virus DNA with which they are associated. Thus, after the DNA-protein complex has been dissociated (for example, by digestion of the DNA with DNase), the proteins become susceptible to digestion with proteases. It seems likely therefore that these proteins are present in the center of the concatemeric “tangles” and are thereby protected from proteolytic attack, as well as from other deproteinizing agents. It may be premature to speculate about the role of these proteins. However, because the concatemeric tangles are strongly associated with two proteins (an association that is not disrupted by treatment with 2 M NaCl, the procedure used to prepare the matrix fractions) the possibility that these proteins may be involved in anchoring the DNA to the matrix suggests itself. Furthermore, the fact that the 10K protein is a late protein (Fig. 8 and manuscript in preparation); i.e., is not present in significant amounts during early phases of DNA synthesis when a smaller proportion of the replicating virus is matrix associated, also lends credence to a possible role of this protein in anchoring the virus DNA to the nuclear matrix. It is interesting to note that recombination (and probably inversion of the short unique sequence of the genome also) occurs mainly at early stages of infection

ASSOCIATION

OF PrV DNA WITH

(Ben-Porat et aL,1982a) prior to the massive association of the virus DNA with the nuclear matrices. The firm association of late DNA with the nuclear matrix may be one of the factors that restrict most recombinational events to the early phase of infection. REFERENCES BEN-P• RAT, T. (1982). Organization and replication of herpesvirus DNA. In “Organization and Replication of Viral DNA” (A. S. Kaplan, ed.), pp. 14’7172. CRC Press, Boca Raton, Fla. BEN-P• RAT, T., BLANKENSHIP, M. L., DEMARCHI, J. M., and KAPLAN, A. S. (1977). Replication of herpesvirus DNA. III. Rate of DNA elongation. J. ViroL 22, 734-741. BEN-P• RAT, T., BROWN,L., and VEACH, R. A. (1982a). Recombination occurs mainly between parental genomes and precedes DNA replication in pseudorabies virus-infected cells. J. Vird 44, 134-143. BEN-P• RAT, T., KAPLAN, A. S., STEHN, B., and RUBENSTEIN, A. S. (1976). Concatemeric forms of intracellular herpesvirus DNA. Virology 69.547-560. BEN-P• RAT, T., and RIXON, F. J. (1979). Replication of herpesvirus DNA. IV. Analysis of concatemers. Virology 94, 61-70. BEN-P• RAT, T., and TOKAZEWSKI, S. (1977). Replication of herpesvirus DNA. II. Sedimentation characteristics of newly-synthesized DNA. Virology 79, 292-301. BEN-P• RAT, T., and VEACH, R. A. (1980). Origin of replication of the DNA of a herpesvirus (pseudorabies). Proc NatL Acad. Sci. USA 77, 172-175. BEN-P• RAT, T., VEACH, R. A., and HAMPL, H. (1982b). Functions of the major nonstructural DNA binding protein of a herpesvirus (pseudorabies). Virology 124,411-424. BEN-ZEEV, A., ABULAFIA, R., and BRATASIN, S. (1983). Herpes simplex virus and protein transport are associated with the cytoskeletal framework and the nuclear matrix in infected BSC-1 cells. lrirdogy 129,501-507. BEREZNEY, R. (1980). Fractionation of the nuclear matrix. J. Cell BioL 85, 641-650. BEREZNEY, R., and COFFEY, D. S. (1975). Nuclear protein matrix association with newly synthesized DNA. Science (Washington, D. C.) 189,291-293. BEREZNEY, R., and COFFEY, D. S. (1977). Nuclear matrix. Isolation and characterization of a framework structure from rat liver nuclei. .J. CeU BioL ‘73, 616-637. BIBOR-HARDY, V., POUCHELET, M., ST. PIERRE, E., HERZBERG, M., and SIMARD, R. (1982). The nuclear matrix is involved in herpes simplex virogenesis. Virology 121, 296-306.

CELL SUBSTRUCTURES

221

BIBOR-HARDY,V., and SIMARD, R. (1980). Viral nucleic acid synthesis in relation to the proliferation of nuclear envelope in HSV-infected cells. Bid CeU. 39, 191-196. BUCKLER-WHITE, A. J., HUMPHREY, G. W., and PIGIET, V. (1980). Association of polyoma T-antigen and DNA with the nuclear matrix from lytically infected 3T6 cells. Cell 22, 37-46. CARO, L. G., and TUBERGEN, R. P. (1962). High resolution autoradiography. J CeU BioL 15, 173188. CHIN, W. W., and MAIZEL, J. V. (1977). The polypeptides of adenovirus. VIII. The enrichment of E3 (11,000) in the nuclear matrix fraction. virology 76,79-89. DIJKWEL, P. A., MULLENDERS, L., and WANKA, F. (1979). Analysis of the attachment of replicating DNA to a nuclear matrix in mammalian interphase nuclei. NucL Acids Res. 6, 219-230. HIRSCH, I., ROUBAL, J., and VONKA, V. (1976). Replicating DNA of herpes simplex virus type 1. Intervirology 7, 155-175. HUNT, B. F., and VOGELSTEIN, B. (1981). Association of newly replicated DNA with the nuclear matrix of Physarum polycephalum. NucL Acids Res. 9, 349-363.

JACOB, F., BRENNER, S., and CUZIN, F. (1963). On the regulation of DNA replication in bacteria. Cold Spring

Harbor

Sgmp.

Qunnt.

BioL 28.329-348.

JACOB, R. J., MORSE, L. S., and ROIZMAN, B. (1979). Anatomy of herpes simplex virus DNA. XII. Accumulation of head-to-tail concatemers in nuclei of infected cells and their role in the generation of the four isomeric arrangements of viral DNA. J. ViroL

29, 448-457.

JACOB, R. J., and ROIZMAN, B. (1977). Anatomy of herpes simplex virus DNA. VIII. Properties of the replicating DNA. J. ViroL 23, 394-411. JEAN, J.-H., BLANKENSHIP, M. L., and BEN-P• RAT, T. (1977). Replication of herpesvirus DNA. I. Electron microscopic analysis of replicative structures. Virology 79, 281-291. JEAN, J.-H., and BEN-P• RAT, T. (1976). Appearance in vivo of single-stranded complementary ends on parental herpesvirus DNA. Proc NatL Ad Sci USA 73, 2674-2678.

KAPLAN, A. S., and BEN-P• RAT, T. (1961). The action of 5-fluorouracil on the nucleic acid metabolism of pseudorabies virus-infected and noninfected rabbit kidney cells. Virology 13. 78-92. KAPLAN, A. S., and VATTER, A. E. (1959). A comparison of herpes simplex and pseudorabies viruses. Virology 7. 394-407. KAUFMANN, S. H., COFFEY, D. S., and SHAPER, J. H. (1981). Considerations in the isolation of rat liver nuclear matrix, nuclear envelope and pore complex lamina. Exp. Cell Res. 132, 105-123.

222

BEN-PORAT

D. M., QUINLAN, M. P., and SPANG, A. E. (1982). Characterization of two conformational forms of the major DNA binding protein encoded by HSV-1. J. Wol. 44, 736-741.

KNIPE,

D. M., and SPANG, A. E. (1982). Definition of a series of stages in the association of two herpesviral proteins with the cell nucleus. J. Viral 43, 314-324.

KNIPE,

LADIN, B. F., BLANKENSHIP, L. M., and BEN-P• RAT, T. (1980). Replication of herpesvirus DNA. V. The maturation of concatemeric DNA of pseudorabies virus to genome length is related to capsid formation. J. vird 33,1151-1X4. LADIN, B. F., IHARA, S., HAMPL, H., and BEN-P• RAT, T. (1982). Pathway of assembly of herpesvirus capsids: An analysis using DNA+ temperaturesensitive mutants of pseudorabies virus. VirdoQy 116,544~561.

ET AL. PARDOU, D. M., VOGELSTEIN, B., and COFFEY, D. S. (1989). A fixed site of DNA replication in eukaryotic cells. CeZl 19, 527-536. RIXON, R. J., ATKINSON, M. A., and HAY, J. (1983). Intranuclear distribution of herpes simplex type 2 DNA synthesis in examination by light and electron microscopy. J. Gen Wol 64, 2087-2092. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. iUol Biol 98, 503-517. TSUTSUI, Y., NISHIYAMA, Y., YOSHIDA, S., MAENO, K., and HASHINO, M. (1983). Role of the nuclear matrix in the growth of HSV-2. Arch. V&l i’7,27-38. VOGELSTEIN, D., PARDOLL, D. M., and COFFEY, D. S. (1980). Supercoiled loops and eukaryotic DNA replication. Cell 22, ‘79-85. YOUNGHUSBAND,H. B., and MAUNDRELL, K. (1982). Adenovirus DNA is associated with the nuclear matrix of infected cells. J. Viral 43, 705-713.