Characterization of defective interfering viral particles present in a population of pseudorabies virions

VIROLOGY

60, 29-37

(1974)

Characterization of Defective Interfering Viral Particles in a Population of Pseudorabies Virions’ TAMAR Department

BEN-PORAT, of Microbiology,

JEAN Vanderbilt

M. DEMARCHI, University Accepted

School April

AND

ALBERT

of Medicine,

Nashville,

Present

S. KAPLAN Tennessee

37232

16, 1974

Repeated passage of undiluted pseudorabies virus in rabbit kidney cells resulted in a decrease in the yield of infectious virus; by passage 49 (Pr-49) the titer was reduced by approximately 99%. The lower yields of infectious virus produced by the cells are due to interference by defective viral particles which possess sedimentation and antigenic properties similar to those of standard pseudorabies virions. The viral DNA synthesized by cells infected with Pr-49 virus and the DNA encapsidated into particles produced by these cells have a lower buoyant density in CsCl than does the DNA of standard Pr virus. This DNA contains viral DNA sequences with varying degrees of reiteration frequencies. Cellular DNA sequences could not be detected in Pr-49 viral DNA. INTRODUCTION

Evidence has accumulated in recent years that some members of the herpesvirus group, which are normally cytocidal, may cause cell transformation provided virus infectivity is inactivated prior to infection (Rapp and Jerkofsky, 1973). The frequency of cell transformation by inactivated herpesviruses is quite low, however, and the transforming capacity of these viruses cannot be detected without prior inactivation because the lytic functions of the viruses are expressed. To study the process of transformation by the herpesviruses, it would clearly be desirable to isolate particles with a deletion in that part of their genome which specifies the lytic functions. The isolation and characterization of such particles is the ultimate aim of the experiments described in this paper. Defective viral particles are known to appear in large numbers in virus populations as a result of serial passage at high multiplicity (see review by Huang, 1973). We have passed serially, undiluted pseudorabies (Pr) virus in rabbit kidney (RK) cells and have isolated defective, interfer‘This investigation the National Institutes

was supported by a grant of Health (AI-10947).

from

ing (DI) particles with the hope that some of these particles may have the desired deletions in their genomes. In the present communication we describe the characteristics of populations of the defective particles. The Pr DI particles contain DNA with a buoyant density significantly lower than that of the DNA from standard virus. Furthermore, the DNA from the DI particles contains some sequences which have been amplified considerably. MATERIALS

AND

METHODS

Virus and cell culture. The properties of Pr virus and the cultivation of RK monolayer cultures were described previously (Kaplan, 1957, 1969a). Primary RK cells were grown in 90-mm petri dishes in ELS; the experiments were performed after the cells had reached confluence (approximately 4 x lo6 cells per culture). The medium was changed to Eagle’s medium 16 hr before the experiments were performed. Standard virus is a Pr virus stock which has been carried for the last 15 years in our laboratory. It has been passaged 24 times at a multiplicity of 1 PFU or less/ cell. Plaque-purified virus was obtained by picking plaques obtained from standard

30

BEN-PORAT,

DEMARCHI

virus (4 plaques/plate). Virus obtained from each plaque was used to infect one culture (4 x lo6 cells) and the virus released into the medium was used to infect new cultures at a multiplicity of 0.5 PFU/ cell to prepare a virus stock for DNA analysis. Media and solutions. ELS: Earle’s saline containing 0.5% lactalbumin hydrolyzate and 5% bovine serum. Eagle’s medium: Eagles synthetic medium (Eagle, 1959) plus 5% bovine serum. SSC: Saline citrate (consists of 0.15 MNaCl + 0.015 Msodium citrate). PB: Equimolar amounts of Na,HPO, and NaHJ’O,. TBSA: A buffer containing the same salts as PBS (Dulbecco and Vogt, 1954), except that the phosphate is replaced by Tris .HCl, 0.01 M, pH 7.5, plus 1% crystalline bovine albumin. Chemicals and radiochemicals. Ribonuclease A was purchased from the Worthington Biochemical Corporation, Pronase (nuclease-free) from Calbiochem, and hydroxyapatite (Bio-Gel HT) from Bio-Rad. Thymidine- 91 (specific activity, 53 Ci/mmole) and thymidine- ‘C (specific activity, 57 mCi/mmole) were purchased from SchwarzlMann. Serial undiluted passage of Pr virus. RK cells were infected (multiplicity, 250 PFU/ cell) and incubated in 3 ml of Eagle’s medium for 20 hr. The culture fluids were collected and centrifuged to remove cellular debris. One milliliter of the fluid was used undiluted to infect new cultures, and the remainder was quick-frozen and stored at - 70”. This procedure was repeated 50 times. At periodic intervals, the culture fluids were assayed for infectious virus by the plaque method. Purification of virions. Culture fluids were collected without disrupting the cells and clarified by centrifugation at 5000 rpm for 10 min in a Sorvall SS-1 rotor. The virus in the supernatant fluids was sedimented on a 30% sucrose cushion by centrifugation at 13,500 rpm for 1 hr in a Spinco SW-25.1 rotor. The virus was collected, layered onto a linear sucrose gradient (1530% sucrose in TBSA), and centrifuged in a Spinco SW-25.1 rotor at

AND

KAPLAN

10,000 rpm for 140 min. Samples of 1 ml were collected from the bottom of the tube. The contents of the tubes containing the virus were pooled, 6 volumes of TBSA were added slowly to dilute the sucrose, and the virus was collected by centrifugation in a Spinco No. 30 rotor at 13,500 rpm for 1 h. Extraction and sonication of cellular DNA. DNA was extracted by the sodium

dodecyl sulfate (SDS)-chloroform-isoamyl alcohol method, essentially according to Marmur (1961). The extracted DNA was precipitated with alcohol, redissolved in SSC, treated with RNase (50 pg/ml), which had been boiled for 10 min, and then with nuclease-free Pronase (200 pg/ml), and extracted as above. After alcohol precipitation, the DNA was dissolved in 0.1 x SSC and sonicated 10 min in a Raytheon sonic oscillator at maximum setting. The resulting fragments had a sedimentation value of 6-7 S in neutral sucrose gradients, determined as described previously (Rakusanova et al., 1971) using ribosomal RNA as markers. The DNA was dialyzed with 2 changes against 1000 volumes of 0.1 x SSC and stored at -70”. The concentration of DNA was determined by UV absorption or calorimetrically by the diphenylamine reaction (Burton, 1956). Preparation

of

3H-labeled viral

DNA.

The following two methods were used: (1) Cells were infected and incubated for 20 hr in Eagle’s medium containing thymidine9H (20 &i/ml). The viral particles produced by the cells were purified and the DNA extracted as described for cellular DNA. (2) The infected cells, labeled as in Method (l), were harvested and treated with SDS. Thymidine‘C-labeled cellular DNA was added as a marker and the samples were centrifuged in CsCl gradients, as described previously (Kaplan, 1969b). Samples were collected dropwise, w-viral DNA was clearly separated from ‘“C-cellular DNA, and the w-labeled viral DNA was sonicated and dialyzed as described for cellular DNA. Denaturation of DNA. DNA in 0.1 x SSC was heated for 10 min at 103” and quick-cooled. DNA-DNA

reassociation

in solution.

INTERFERENCE

BY DEFECTIVE

Heat-denatured ?-I-labeled viral DNA was allowed to reassociate at 67” in 0.12 M phosphate buffer in the presence or in the absence of unlabeled heat-denatured cellular or viral DNA. Samples were removed at various times and analyzed for the presence of single- or double-stranded DNA by fractionation on hydroxyapatite as follows: Two grams of hydroxyapatite were suspended in 10 ml of 0.15 M PB containing 0.4% SDS and loaded onto a water-jacketed column at 60”. The samples containing the DNA were diluted at least 20-fold with 0.15 M PB-0.4% SDS and loaded onto the column. Single-stranded DNA was obtained by washing the column with 50 ml of 0.18 M PB-0.4% SDS. Double-stranded DNA was obtained by washing the column with 50 ml of 0.5 M PB-0.4% SDS. The samples were acidified with trichloroacetic acid (7%), the precipitated DNA was collected on Millipore filters, and the percentage of %-labeled viral DNA reannealed at each point was calculated after correction for the amount of double-stranded DNA present at T, [usually approximately 5% of the total counts in denatured DNA (T,) were found in the double-stranded fraction]. The results were plotted as a function of Cot (Britten and Kohne, 1968). DNA-DNA reassociation on filters. This was carried out essentially according to Denhardt (1966). DNA was fixed to filters as described previously (Rakusanova et al., 1971). The filters were preincubated at 67 o for 6 hours in Denhardt’s solution and then incubated at 67 o for 20 hr in 1 ml 4 x SSC in Denhardt’s solution containing Wlabeled DNA. The filters were washed on both sides with warm (67 “) 4 x SSC in a suction apparatus, dried, and counted in a liquid scintillation spectrometer.

VIML

31

PARTICLES

250 PFUlcell. Culture fluids containing the virus produced by these cells were pooled and used undiluted to infect new cells, as described in Materials and Methods, a procedure repeated 50 times. By undiluted passage 30, the yield of infectious virus produced by the cells was reduced by more than 99%. There was thereafter a fluct.uation in infectious virus titer. The experiments to be described were performed with passage 49 virus, referred to as Pr-49. This preparation had a titer of 2.1 x IO7 PFU/ ml, approximately go-fold lower than standard virus (1.9 x 10s PFUlml). Formation of Noninfectious Particles by Cells Infected with High Passage Virus The following experiments were performed to determine the relative “specific infectivity” of the virions (i.e., the ratio of noninfectious to infectious virus) produced by cells infected with Pr-49. Figure 2 shows that cultures infected with Pr-49 virus yielded particles (containing thymidine- 9I labeled DNA) which possess sedimentation characteristics in sucrose gradients which are similar to those of standard Pr virions. However, the size of the peaks (note the different scales) of virus produced by the cells infected with the two types of virus populations differed

RESULTS

Effect of Undiluted Passage of Pr Virus on the ~oduction of Infectious Virus Figure 1 illustrates the effect of serial passage of undiluted standard Pr virus stock upon the formation of infectious virus by RK cells. At the start of the experiment the multiplicity of infection (m.o.i.) was

UNDILUTED

FIG. 1. Effect the production of in a serial fashion different passage in Materials and

VIRUS-

PASSAGE

~UMSER

of undiluted passage of Pr virus on infectious virus. Pr virus was passed in RK cells and the virus yield at levels was determined, as described Methods.

BEN-PORAT,

32

DEMARCHI 5

5

PR 49 YIRVS

STANDARD“lR”S

4

4

* 43 : :: 2

3i 2a ”

l-lif!L 5

IO

I5

20 FRACTION

5

IO

15

20

NUMBER

FIG. 2. Formation of virions by Pr-49 virusinfected cultures. Two RK cultures (8 x lo6 cells) were infected (m.o.i., 5 PFU/cell) with either standard virus or with Pr-49 virus. (Pr-49 was used undiluted and standard virus was diluted in Eagle’s medium to give the desired multiplicity). The cultures were incubated for 20 hr with thymidineJH (20 &i/ml), the virions were purified and centrifuged in sucrose gradients, and the amount of radioactivity associated with the virus peak was determined, as described in Materials and Methods.

significantly in that the peak of particles from cells infected with Pr-49 virus was approximately 12% of that produced by standard virus. This experiment was performed with a multiplicity of 5 PFU/cell for both Pr-49 and standard virus. However, the size of the peak of virus obtained from cells infected with standard virus was independent of multiplicity of infection and was the same whether the cells were infected with a m.o.i. of 5 or of 100 PFU/cell. Uninfected cells treated identically showed no detectable radioactivity in the position in the sucrose gradient where the virus bands. In the experiment illustrated in Fig. 2 the amount of labeled DNA that becomes associated with virions produced by cells infected with Pr-49 is approximately 12% that of the population of virions produced by cells infected with standard virus. (In different experiments values between 15% and 7% were obtained,) The reduction in the number of PFU produced by cells infected with Pr-49 as compared to standard virus was, however, considerably greater (approximately 99%). Thus, if the specific activity of the viral DNA synthesized by Pr-49 virus- and by standard virus-infected cells is the same, it is clear that the specific infectivity of the virus

AND

KAPLAN

produced by cells infected with Pr-49 virus is lower than that of standard virus. That this is indeed the case is illustrated in Table 1, which shows that the ratio of infectious to noninfectious viral particles in the preparation of purified standard virions was approximately 1: 18, whereas the ratio in the population of virions produced by cells infected with Pr-49 virus was 1: 177. Thus, the specific infectivity of Pr-49 virus was approximately lo-fold lower than that of standard virus. It should be pointed out that the differences in ratios of infectious standard

and

particles in populations are

to noninfectious Pr-49 virus

calculated on the basis of the assumption that the amount of DNA per particle is the same. Preliminary evidence indicates that this is indeed the case. If the defective particles contained less DNA than the standard virions, the difference in the specific infectivity between the two populaESTIMATION PARTICLES

___~ Virus

Standard Pr-4g ___.

OF THE PRODUCED STANDARD

TABLE

1

SPECIFIC

INFECTIVITY

BY CELLS AND Pr-49

INFECTED VIRUS”

OF VIRAL WITH

PartiCkS/d” in virus peak (x 10 ‘)

103.20 7.80

“RK cultures (20/sample, -8 x 10’ cells) were infected with either undiluted Pr-49 virus (m.o.i., 5 PFU/cell) or standard virus (m.o.i., 5 PFU/cell). After 1 hr unadsorbed virus was removed and the cultures were incubated for 20 hr in Eagle’s medium containing thymidine-3H (20 &i/ml). The virus which had been released into the culture fluids was purified, as described in Materials and Methods, and the number of PFU, as well as the amount of radioactivity associated with the peaks of purified virus obtained in the sucrose gradients was determined. The viral DNA present in the cells was purified by density gradient centrifugation in CsCl, as described in Materials and Methods (preparation of 3H viral DNA, method 2), and its specific activity (cpm/Fg) was determined. b The number of particles was calculated using the specific activity of the DNA (column 2) and the estimation that 6 x lo9 particles (containing DNA with a molecular weight of 100 x lo6 daltons) should contain 1 pg of DNA.

INTERFERENCE

tions still.

of viral

particles

BY DEFECTIVE

TABLE

Standard

Passage No.

1 2 1 2

Inoculumundiluted Yield (PFU/ml) 1.0 6.5 8.0 9.2

Sample

A B C

3

VIRUS TO INTERFERE WITH THE OF STANDARD Pr VIRUSJ Inoculum

Eagle’s medium

Pr-49 Virush

Standard Pr Virus’

0.1 ml 0.9ml

0.9 ml 0.9ml -

0.1 ml 0.1 ml

Titer PFU/ culture x 10-s

1.41 0.60 28.50

‘Cultures (4 x lo6 cells) were inoculated as described in the Table. Sixty minutes later unadsorbed virus was removed by washing; the cultures were incubated in 3 ml of Eagle’s medium for 20 hr, and the titer of infectious virus was determined. * Inoculum: Pr-49, 2.1 x lo7 PFU/ml. ’ Inoculum: Standard Pr virus, 2 x lOa PFU/ml. TABLE

4

EFFECT OF INCUBATION WITH Pr VIRUS ANTISERUM THE INTERFERING ABILITY OF PURIFIED Pr-49 PARTICLES” Treatment Infection Pr Pr-49 Pr Pr Pr

ON

Virus titer PFU/ml

Superinfection Pr-49 Pr-49 + Antiserum Pr + Antiserum

8.1 6.4 1.8 7.3 9.5

x x x x x

lOa lo8 10’ 108 108

2

EFFECT OF DILUTION OF Pr-49 AND STANDARD Pr VIRUSES ON THE FORMATION OF INFECTIOUS VIRUS”

Pr-49

TABLE CAPACITY OF Pr-49 REPLICATION

Table 2 shows the effect of dilution of Pr-49 virus on the yield of PFU. After the second diluted passage of Pr-49 virus the titer of the infectious virus was approximately 70-fold higher than when Pr-49 virus was passaged in undiluted form. The titer of infectious virus produced by cells infected with standard virus was not affected appreciably by the multiplicity of infection. This experiment indicates that preparations of Pr-49 virus contain a factor that interferes with the formation of the infectious virus. Table 3 shows that the addition of Pr-49 virus to a standard virus inoculum reduced the yield of infectious virus by approximately 95%, thus demonstrating again that Pr-49 virus preparations contain a factor which interferes with the production of infectious virus. The interfering factor present in preparations of Pr-49 virus is not soluble; sedimentation of Pr-49-containing culture fluid (25,000 g for 1 hr) results in its removal.

33

PARTICLES

would be greater

Evidence for the Presence of Interfering Particles in Pr-49 Virus Populations

Virus

VIRAL

x x x x

107 lo6 10” 108

Inoculumdiluted 1: 20 Yield (PFU/ml) 1.8 4.7 1.0 9.1

x x x x

lo8 108 10” 108

n Passage No. 1: Cultures (4 x lo6 cells) were infected with either concentrated (m.o.i., 5 PFU/cell) or diluted (1: 20) Pr-49 virus. Cultures were also infected with undiluted standard virus (m.o.i., 250 PFU/cell) or with a 1:20 dilution of the same virus. Passage No. 2: After a 20-hr incubation period, the yield from each culture of passage No. 1 was used to infect new cultures either in undiluted form or after a 1:20 dilution and the cultures incubated for 20 hr. The number of PFU produced by cultures infected with passages No. 1 and No. 2 was determined.

u Cells were infected with standard Pr virus (m.o.i., 2 PFWcell) and after being incubated for 1 hr (to allow adsorption) were superinfected with the purified viral particles obtained from cultures infected with Pr-49 (10 PFU/cell), as well as with these particles after incubation for 1 hr at 37” with a 1: 20 dilution of standard Pr viral antiserum. The cells were washed to remove unadsorbed virus, as well as Pr antiserum, and were incubated for 20 hr at 37”, when the titer of infectious virus produced by the cells was determined.

Thus, the interfering factor is not interferon. Evidence that Pr viral particles themselves constitute the interfering factor is presented in Table 4. In this experiment viral particles produced by cells infected with Pr-49 virus were purified, as described in Materials and Methods, and their ability to suppress the growth of standard Pr virus was tested. Addition of purified Pr-49 viral particles to cells infected with stan-

BEN-PORAT,

34

DEMARCHI

dard virus inhibited the production of infectious virus approximately 40-fold. Exposure of these viral particles to antiserum prepared against standard Pr virus inhibited their interfering ability. Thus it is clear that interference with the synthesis of infectious Pr virus by Pr-49 culture fluids resides in particles which have sedimentation and antigenic properties similar to Pr virions. It is safe to assume therefore that the interfering factor is defective interfering Pr viral particles which have been enriched in the viral particle population during repeated passage at high multiplicity. Synthesis of Viral DNA in Cells Infected with B-49 Virus

Table 5 shows that incorporation of thymidine into DNA by Pr-49 virus-infected cells was almost the same as by cells infected with standard virus. Infection of cells with Pr-49 virus does not stimulate the incorporation of thymidine into cellular DNA (see below), and therefore Pr-49 virus-infected cells incorporate approximately as much thymidine into viral DNA as do cells infected with standard virus.

INCORPORATION

OF

TABLE 5 THYMIDINE-$H INTO WITH Pr-49 Emus”

Infection

3.5

5

Since the specific activity of the viral DNA produced by cells infected with the two types of virus populations is approximately the same (Table 1, column 2), we conclude that viral DNA synthesis occurs at approximately the same rate in cells infected with Pr-49 virus as with standard Pr virus. The reduced number of viral particles (see Fig. 2 and Table 2) produced by Pr-49-infected cells is thus not due to a lack of accumulation of viraf DNA in the cells. Buoyant Density in G&l DNA

Viral

The experiments described below were designed to characterize the DNA synthesized by cells infected with Pr-49 virus, as well as the DNA incorporated into the viral particles produced by these cells. Figure 3

50 FRACTION

6.5

of B-49

INFECTED

Time after infection (hr) 2

Pr-49 virus Standard virus Uninfected

CELLS

AND KAPLAN

9

11

7.4” 31.4 56.7 66.7 85.3 62.3 5.2 43.8 86.6 87.3 75.0 59.4 0.1 0.4 0.3

(‘RK cultures were incubated in Eagle’s medium containing 5-fluorouracil (10 rglml) to suppress cellular DNA synthesis (Kaplan and Ben-Porat, 1961) for 16 hr. The cultures were then infected either with undiluted Pr-49 (5 PFU/cell) or standard virus (7 PFU/cell) and incubated furtber in Eagle’s medium. At various times, the medium of different cultures was changed to Eagle’s medium containing thymidine-W (0.5 &Z/ml), and the cultures were incubated for 60 min. The cells were harvested, acidified, and acid-washed to remove acid nonprecipitable label and the amount of thymidine-‘% incorporated into the cells of each culture was determined. * Cpm x 10 Vculture (4 x lo6 cells).

60

70

NUMBER

FIG. 3. Buoyant density in CsCl of Pr-49 viral DNA. (A) RK cultures were infected with either standard virus or Pr-49 virus (m.o.i., 5 PFU/cell). Four hours after infection the cells were incubated with Eagle’s medium containing either thymidine- “H (IO &i/ml) or thymidine- ‘C (0.2 &X/ml) for 4 hr. Actively growing, uninfected cells were also labeled with thymidine- W for 4 hr. The cells were collected in SSC containing 3% SDS, and the DNA was analyzed by equilibrium sedimentation in CsCl. “C-Labeled cells infected with Pr-49 virus (filled circles) were mixed with W-labeled cells infected with standard virus (open circles) and with V-labeled uninfected cells (open circles). (B) SH-Thymidine labeled purified Pr-49 viral particles (open circles) were mixed with ‘C-thymidine labeled purified standard virus (filled circles), treated with SDS, and the DNA was analyzed by equilibrium sedimentation in C&l. The arrows indicate the position of standard viral DNA.

INTERFERENCE

BY DEFECTIVE

shows the distribution in C&l of ?-I- and ‘*C-differentially labeled standard viral and Pr-49 viral DNA. pifferentially labeled DNAs may differ in their buoyant densities, the ‘C-labeled DNA having a slightly higher density than the H-labeled DNA (Cassai and Bachenheimer, 1973).] To avoid this problem, the specific activity of the ‘C-labeled thymidine was kept relatively low and under these conditions coincidence of standard viral DNA labeled with thymidine- ‘C or with thymidine- 3H was obtained). Figure 3A shows the banding pattern of a mixture of thymidine-SH labeled DNA synthesized by cells infected with standard virus (Pr viral DNA marker), thymidine- H-labeled DNA synthesized by uninfected cells (cellular DNA marker), and thymidine- ‘C-labeled DNA synthesized by cells infected with Pr-49 virus. Most of the DNA synthesized by Pr-49 virus had a buoyant density in CsCl about 0.005 g/cm3 lower than the DNA of standard virus. ‘C-label did not appear in the region of cellular DNA, indicating that this DNA is not synthesized by Pr-49 virus-infected cells. Figure 3B illustrates the distribution in the density gradient of DNA obtained from purified standard virus labeled with thymidine- ‘“c and purified Pr-49 virus labeled with thymidine-SH. The density of the DNA contained in Pr-49 viral particles was the same as that synthesized by cells infected with this virus. Thus, the low density viral DNA synthesized by Pr-49 virus-infected cells becomes encapsidated into Pr viral particles. Reannealing Kinetics Synthesized by Pr-49 Virus

of the Viral Cells Infected

VIRAL

35

PARTICLES

Pr viral DNA and Pr-49 viral DNA were compared to test for the presence of sequences which may have been amplified. We also tested whether cellular DNA sequences are associated with Pr-49 viral DNA. Figure 4 shows the reannealing kinetics in solution of Pr-49 viral DNA and of plaque-purified Pr viral DNA. The DNA of plaque-purified Pr virus reanneals with second-order kinetics with a Cot,, of approximately 0.30 which is in the range expected of DNA of similar complexity (approximate molecular weight, 100 x lo6 daltons), most of the sequences of which are present as one copy per viral genome (Britten and Kohne, 1968). The DNA of Pr-49 virus, however, reanneals differently and some sequences reanneal more than 100 times faster than do the sequences of normal Pr viral DNA. The kinetics of reannealing of this DNA do not fit the theoretical curve for a second-order reaction; the curve is shallow, reflecting the presence in the populations of DNA molecules of sequences with varying degrees of reiteration frequencies. Figure 5 shows the reannealing kinetics of 3H-labeled Pr-49 viral DNA in the presence of unlabeled standard viral DNA or of I

“““‘I

“-

‘-“““1

““‘T

“‘7

DNA with

The relatively low buoyant density of the DNA in Pr-49 viral particles could be either due to the integration of some sequences of cellular DNA into most of the viral DNA molecules or to the amplification of some viral sequences of low guanine + cytosine (G + C) content at the expense of sequences of high G + C content. To distinguish between these two alternatives the reannealing kinetics of plaque-purified

FIG. 4. Reassociation kinetics of Pr-49 virus and plaque-purified Pr virus DNA. The viral DNAs were obtained by Method 2, as described in Materials and Methods, and heat-denatured. Each reaction mixture contained 1 pglml of W-labeled DNA (specific activity of Pr-49 DNA, 7 x 10’ cpm/pg; specific activity of plaque-purified DNA, 8.1 x lO’cpm/pg) in 0.12 MPB and was allowed to reassociate at 67”. Samples were removed at intervals and single-stranded DNA was separated from double-stranded DNA, as described in Materials and Methods. Open circles, Pr-49 viral DNA; filled circles, plaque-purified Pr viral DNA.

36

BEN-POST,

DEMAR~HI

AND KAPLAN

tions a decrease in titer of infectious virus occurred, with a concomitant change of the DNA of the virus to a higher buoyant density. 5 ' 40. Our experiments show that serial unY _ diluted passage ‘of Pr virus results in a :2 GOconsiderable decrease in the amount of infectious virus produced (approximately ii 80 99%). The decrease in the production of P aw100 infectious virus is caused by particles i 103 IO’ which interfere effectively with the syntheIO’ I02 TIUE (MINUTES) sis of standard Pr virus. That these partiFro. 5. .Reassociation of %I-Pr-49 viral DNA in the cles are defective Pr virions is indicated by particles have presence of unlabeled standard Pr viral DNA and the fact that the interfering unlabeled cellular DNA. The W-labeled Pr-49 viral sedimentation and antigenic characterisDNA was the same as that used in the experiment tics of Pr virions. illustrated in Fig. 4. The reassociation kinetics of this The properties of the DNA in the virus DNA were determined as in Fig. 4, either without population containing the defective virions added DNA (open circles) or in the presence of 22 (Pr-49) have been partially characterized. &ml of sonicated, heat-denatured standard Pr viral The main points to emerge from these DNA (filled circles) or in the presence of 1 mg/ml of experiments are the following: (I) Most of sonicated, heat-denatured RK cellular DNA (filled the DNA in the Pr-49 virions differs from triangles). the DNA of standard virus in that its cellular DNA. This experiment shows that buoyant density in C&l is 1.727 g/cm’ 0.005 g/cm5 lower than practically all the Pr-49 viral DNA frag- (approximately ments contain viral sequences, since in the standard Pr viral DNA). (2) Hybridization presence of standard Pr viral DNA the studies show that Pr-49 DNA consists reassociation kinetics of all of this DNA mainly of Pr viral DNA. This is deduced were significantly increased. The presence from the fact that Pr-49 viral DNA does not to cellular DNA and of cellular DNA did not affect the rate of anneal detectably reannealing of Pr-49 viral DNA, indicating that standard viral DNA drives the hybridthat the latter does not contain sequences ization of Pr-49 viral DNA to completion. which anneal to cellular DNA (under the We cannot exclude on the basis of these conditions used, only repetitive sequences results the possibility that. small fragments of cellular DNA (smaller than the DNA of cellular DNA would be detected). Table 6 shows that cellular DNA sequences also fragments analyzed in these experiments) cannot be detected in Pr-49 viral DNA, TABLE 6 using the technique of hybridization on REASSOCIATIONOF 3H-L~~~~~~ STANDARDPr V~HAL filters.

s o44 20.

--I

DISCUSSION

In most virus systems in which this phenomenon has been studied, appearance of defective interfering particles following serial undiluted passage of virions has been found (Huang, 1973). The experiments in this paper show that Pr virus, one of the herpesviruses, is no exception. That interfering particles appear after undiluted passage of herpes simplex virus has been previously reported by Bronson et al. (1973), who showed that under these condi-

DNA OR Pr-49 VIRAL DNA WITH CELLULAR AND STANDAMI Pr VIRAL DNA FIXED TO FILTERS Viral DNA

Input

3H-DNA Reacting with I%‘i

Standard Pr-49

4490” 1980

2.2” 1.0

DEA

Blank filters

956’ 586

0 1.5

” Cpm/vial. ” Cpm annealed to filter to which 20 gg cellular DNA was fixed. / Cpm annealed to filters to which 10 pg viral DNA was fixed.

INTERFERENCE

BY DEFECTIVE

are covalently linked to viral DNA. Upon hybridization of Pr-49 viral DNA to standard viral DNA, tails of cellular DNA would be associated with the doublestranded molecules and would be retained on the hydroxyapatite. These tails would consist of nonreiterated sequences of cellular DNA only, since no hybridization to cellular DNA was detected under conditions that should have revealed the presence in the DNA preparations of reiterated cellular sequences. (3) Pr-49 viral DNA consists of sequences of varying degrees of reiteration. We do not know the organization within the DNA molecules of these sequences, i.e., whether some DNA molecules contain only reiterated sequences or whether the reiterated sequences are present in varying degrees in DNA molecules which also contain unique sequences of viral DNA. In general, one of the features of DI particles is the deletion of a segment of the genome; the deleted portion may be replaced by other reiterated viral sequences. The experiments described here indicate that this may well be the case in the population of defective Pr virions which has been enriched by undiluted serial passage. The relatively low buoyant density of the Pr-49 viral DNA could thus be the result of amplification of sequences of the viral genome relatively low in their G + C content. Although we have not been able to detect cellular sequences in Pr-49 viral DNA, we cannot exclude the possibility that small segments of cellular nonrepetitive sequences have been inserted into the viral DNA molecules at several different locations and confer upon this DNA its lower buoyant density.

ACKNOWLEDGMENTS We gratefully acknowledge the excellent assistance of Betty Lonis and Linda Brown.

technical

VIRAL

PARTICLES

37

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