Viral interference of HSV-1: Properties of the intracellular viral progeny DNA

VIROLOGY

120,205-214

(1982)

Viral Interference G. KffMEL,’

of HSV-1: Properties of the Intracellular Progeny DNA

Viral

B. HENNES-STEGMANN, C. H. SCHRODER, K. W. KNOPF, H. C. KAERNER

AND

German Cancer Research Center, Institute for Virus Research, 280 Im Neuenheimer Feld, 6900 Heidelberg, Germany Received January 5, 1982;accepted March 22, 1982 Intracellular progeny DNA was isolated and characterized from cells infected with standard herpes simplex virus or from cells coinfected with standard virus and with a virus stock obtained by serial passages at high multiplicity of infection (HP virus). The latter was shown to contain an excess of variant virus particles interfering with the production of infectious progeny virus. The ratio of plaque-forming to interfering virus in the HP virus stock used in this study was determined to be l/10. In both infections similar amounts of unit length viral DNA were synthesized. Restriction endonuclease digestion of intracellular viral progeny DNA yielded fragment patterns showing that the majority of the DNA molecules present in standard and HP HSV DNA yield the same restriction fragments. That the typical end fragments could be demonstrated suggests a correct processing of concatemeric precursor DNA into HSV unit length DNA. Despite the obvious similarity of the two DNA species the infectivity in transfection assays of progeny DNA formed in HP virus-infected cells was by three log lower than that of standard DNA. As shown by controls involving cotransfections with standard viral DNA and heterologous DNA, this difference can be attributed to the presence in HP DNA of HSV DNA molecules that interfere with the plaque formation by standard DNA. An alteration in various steps of DNA processing such as DNA methylation and incorporation of ribonucleotides into the DNA was demonstrated not to correlate with interference. Both in HP and standard virus DNA preparations methylation was below the level of detection of 10 5-methylcytosine residues per unit length of viral DNA. Low values were also obtained for the uridine content of the DNA. Almost 100% of the radioactivity incorporated could be recovered as deoxyribonucleosides and less than 0.07% as uridine.

sists of repeats of restricted regions of the standard genome (Frenkel et al., 1975, In high multiplicity Passages of herpes 1976). AS has been reported earlier, the simplex virus (HSV) variant virus parti- latter virus particles cannot be the only cles arise (I particles) that restrict the cause for the phenomenon of interference replication of infectious standard virus in the system HSV-1 ANG and RC-37 cells @ronson et al., 19% Frenkel et & 1975; (Stegmann et al., 1978; SchriSder and UrMurray et al., 1975; Schroder et al., 1975/ baczka, 1978). The present study is con1976; Wagner et al., 1974). This phenom- cerned with the characterization of DNA enon, well known in other animal virus f rom HP virus and of progeny DNA obsystems, has been termed viral interfertained upon coinfection with standard and ence (Huang and Baltimore, 1977). The I Hp virus. The data presented suggest that particles are not able to replicate in the interference is caused by virions containabsence of infectious helper virus. In this ing a genome of high complexity that rerespect they resemble another type of de- sembles the standard genome rather than fective particles, the genome of which con- the well-characterized repetitive defective genomes. It has been shown earlier that i To whom reprint requests should be addressed. packaging of progeny DNA into virus parINTRODUCTION

205

0042-6822/82/090205-10$02.00/0 Copyright Q 1982 by Academic Prese.. Inc. All righta of reproduction in my form reserved.

206

KtiMELETAL.

titles is affected whereas the yield of viral DNA remains approximately the same regardless of whether cells have been infected with standard virus alone or together with I particles (Stegmann et al., 1978). Similar data have been obtained earlier by Ben-Porat and Kaplan (1976) with pseudorabies virus. To answer the question whether DNA maturation or modification might specifically be affected in the interfering system we compared the progeny DNA obtained in infection with standard virus (standard progeny DNA) to that obtained in coinfection with standard virus and HP virus containing I particles (HP progeny DNA). Specifically we looked for methylation of, and incorporation of ribonucleotides into the DNA. Methylation had been reported to occur transiently during the infectious cycle (Sharma and Biswal, 1977). Covalent linkage of ribonucleotides into DNA of HSV has been claimed by Hirsch and Vonka (1974). Both the presence of 5-methylcytosine and of uridine in progeny DNA might indicate the degree to which progeny DNA has been processed (Kumel et al., 1981). Certain features of processing of the progeny DNA can also be studied by restriction endonuclease analysis. Random cleavage of concatemeric precursor DNA during the infectious cycle (Hirsch et al., 1976) for example would lead to altered DNA fragment patterns. As an ultimate criterion for the correct maturation of progeny DNA its infectivity was studied in transfection assays. METHODS

CeUs.African green monkey kidney cells, RC-37 (Italdiagnostic Products, Rome, Italy), were routinely grown either in glass bottles or plastic petri dishes in a humidified 5% (v/v) COz incubator. Basal Eagle’s medium (1955) containing a twofold standard concentration of amino acids and 7% (v/v) fetal bovine serum served as growth medium. BSC-1 cells (ATTC) were grown in the same medium except for the addition of nonessential amino acids. Virus. A DNA plaque-purified HSV-1

stock of the strain ANG (Schroder et al., 1975/1976; Darai and Munk, 1976) that had been passaged three times at low m.o.i. (0.01 PFU/cell) on RC-37 cells served as standard virus. The preparation of virus stocks has been described elsewhere (Schriider et al., 1975/1976). High-multiplicity passages of the virus were performed as previously described (Stegmann et al., 1978). Plaque-forming virus was titrated as described by Russell (1962). Preparation of viral wogenv DNA from inJected cells. RC-37 cells, 1.5 X 10’ cells/ 145-mm petri dish, were infected as indicated under Results. [6-3H]Uridine, 3 &i/ ml, was added to the medium M 199 (Morgan et al., 1950) that was used for the incubation of infected cells. Twenty-four hours after infection the cells were washed twice with phosphate-buffered saline (PBS) and subsequently lysed by the addition of 20 ml per dish of 0.01 MTris-HCl (pH 7.5), 0.1 MNaCl, 5 X 10e4MEDTA, and 1% sarcosyl. The lysate was incubated with 300 pg Pronase/ml overnight at room temperature. Finally viral progeny DNA was purified by two cycles of banding in neutral CsCl equilibrium density gradients after addition of CsCl directly to the lysate (1.71 g/cm3). Viral DNA obtained in this way usually had a specific radioactivity of 26,000 cpm/pg. Restriction enzyme analysis was performed as described earlier (Kaerner et al., 1979). SV 40 RF1 DNA was kindly provided by Dr. W. Waldeck. Transfectim assag. For the analysis of the infectivity of viral DNA in transfection experiments total intracellular DNA was prepared and precipitated by the calcium-phosphate technique as described by Graham et al. (1973). BSC-1 cells were trypsinized, washed with Hepes buffer, and pelleted. A pellet of 2 X lo6 cells was resuspended in the suspension of DNAphosphate precipitate in l-ml volumes and shaken for 45 min at 37”. Subsequently 5 X lo6 RC-37 cells in 20 ml of basal Eagle’s medium supplemented with 1% (w/v) carboxymethylcellulose (CMC) and 5% (v/v) fetal bovine serum were added. The resulting mixture of transfected BSC-1 cells and RC-37 indicator cells was seeded into four 60-mm petri dishes. The formation of

INTRACELLULAR

207

VIRAL PROGENY DNA OF HSV-1

plaques was monitored after 4 days at 37”. Linearity between the amount of viral DNA added and the number of plaques obtained was established in each transfection experiment. Velocity sedimentation. For the analysis of size distribution of DNA molecules the DNA samples were sedimented into a lo30% sucrose gradient in 0.01 Tris-HCl (pH 7.5) for 4 hr at 39,000 rpm at 20” in a Beckman SW41 rotor. For the analysis of single-strand interruptions identical sedimentation conditions were used except that Tris-HCl was omitted and the sucrose gradient was superimposed with a 0.240.35 N NaOH gradient. Analysis of viral DNA for uridine and 5-methyl cytosine. Viral progeny DNA, pu-

rified by two cycles of CsCl density centrifugation was RNase treated and enzymatically hydrolyzed to 5’-mononucleotides by incubation with pancreatic DNase (20 pg/ml, Worthington) for 2.5 hr at 3’7” in 0.1 M sodium acetate, 0.05 M magnesium acetate (pH 6.5). The pH was then changed to 8.6 by adding ammonia. Snake venom phosphodiesterase (Worthington) was added (20 units/ml) and the incubation continued for 4 hr. In some cases the resulting nucleotides were adsorbed to activated charcoal for further purification, washed with distilled water, and eluted with 50% ethanol, 0.5% ammonia. PEI thin-layer chromatography: The procedure of Randerath and Randerath (1967) was used with minor modifications. The solvent systems were 1 M acetic acid, 0.3 M LiCl for the first, and a solution of 6 g NazB407* lOHz0,3 g H3B03, and 30 ml ethylene glycol in 70 ml water for the second dimension. Cellulose thin-layer chromatography: An aliquot of the DNA digest, usually 10 pg of nucleotides in l-10 ~1, was spotted on 40 X 20-cm thin-layer plastic sheets coated with unmodified cellulose together with appropriate marker nucleotides. Development in the first dimension was with isobutyric acid-water-ammonia (66:20:1) with the solvent front moving 35 cm and with saturated ammonium sulfate1 M sodium acetate-isopropanol (80:18:2) (Gtinthert et al., 1976). Ultraviolet-absorb-

g06

0.2 MOI OF HP& W?“S

y&U

/CELL)

FIG. 1. Quantification of the fraction of interfering virus particles in HP virus. The reduction in the yield of infectious progeny virus of RC-37 cells infected with standard HSV-1 ANG was determined after coinfection with increasing amounts of HP virus.

ing spots were excised and directly counted in a standard scintillation cocktail. The remainder of the plate was cut into l-cm squares which were analyzed for background radioactivity, tailing effects, and the possible occurrence of extra spots. RESULTS

Quanti$cation of I Particles in a High-Multiplicity

Passage of HSV-1

HSV-1 ANG was passaged on RC-37 cells four times at a m.o.i. of 10. The resulting fourth passage virus stock (HPvirus) yielded low amounts of infectious progeny upon infection of cells, indicating the presence of I particles. The number of I particles in relation to the number of infectious particles was determined in coinfection experiments with standard virus at a constant m.o.i. of 0.2 and with HP4 virus at m.o.i. ranging from 0.01 to 1.0. The resulting titers of infectious progeny determined 24 hr after infection are shown in Fig. 1. The yield obtained in the coinfection experiment at a m.o.i. of 0.1 of the HP virus was determined to be one-third of the yield in the control culture infected with standard virus alone. For the interpretation it has to be considered that according to the Poisson distribution, infection with a multiplicity of one I particle per cell would mean that one-third of the cells are not infected by an I particle. For

KtiMEL ETAL.

208

TABLE 1 INFECWITY OF INTRACELLULARPROGENYDNA AND VIRUS YIELD Intracellular

ST HSV-1 ANG

HSV-1 ANG

Yield per lo6 cellsb 6%)

2 2

2

3 2

m.o.i. (PFWcell)

progeny DNA”

InfectivityC (PFU/pg DNA) A

B

C

Progeny virus yieldb (PFU/106 cells)

4 x lo4 50

1.3 x 10’ 40

4.6 X l@ 110

3 x lo* 6 x lo6

‘Total intracellular DNA prepared as described by Graham et al. (1973) was assayed for infectivity. amount of viral DNA was determined in CsCl density gradients. bData are the means of three independent experiments. ’ Results of three independent experiments (A, B, C) are given.

convenience, it is assumed that the yield of progeny virus would be reduced to zero in an infected cell if hit by a single I particle. The observed reduction of progeny virus yield to one third at an m.o.i. of 0.1 of the HP virus (Fig. 1) consequently corresponds to a multiplicity of one I particle per cell, a multiplicity that leaves onethird of the cells unaffected by I particles whereas virus production would be completely suppressed in the other two-thirds of the cells. Thus the results of the coinfection experiment (Fig. 1) together with the assumption mentioned above results in an estimate of 10 I particles per one infectious particle in the HP virus tested. This figure must be taken as a minimum estimate, since a single I particle in a cell certainly does not reduce the virus yield to zero. Recycling of infectious progeny virus may occur but does not influence greatly the virus yields determined 24 hr after infection (Schroder and Urbaczka, 1979). Infectivity of Intracellular HSV-1 Progeny DNA in a Transfection Assay We tried to apply the calcium-.phosphate technique (Graham et al., 1973) for the assay of infectivity of .HSV DNA to RC-3’7 cells which had been routinely used as host cells for the study of HSV interference. Despite great effort the use of the above cells proved to be impracticable and thus BSC-1 cells were used instead. For the preparation of intracellular viral prog-

The

eny DNA monolayer cultures of RC-37 were infected with (i) standard HSV-1 ANG alone or with (ii) standard virus together with HP virus, each at an m.o.i. of 2 PFU/cell. Total intracellular DNA was isolated from the infected cells 24 hr p.i. following the procedure of Graham et al. (1973). The infectivity of the two DNA samples thus obtained was determined in transfection assays using BSC-1 cells as primary host and RC-37 as indicator cells. For the calculation of the infectivity defined as number of plaques obtained per microgram of DNA, the amount of viral DNA in the two DNA preparations was measured after purification by two cycles of banding in CsCl density gradients. Approximately the same amounts of progeny DNA,,were obtained in infection with standard virus alone and in coinfection with standard virus plus HP virus; this finding confirms previous results on the surprisingly efficient formation of progeny DNA in the presence of I particles (Stegmann et al., 1978). The infectivity of intracellular standard progeny DNA determined in individual.experiments ranged from 2 X lo3 to 4 X lo4 PFU/pg DNA. Comparable values were obtained for DNA isolated from mature HSV-1 virus particles (data not shown). The infectivity of intracellular HP progeny DNA was found to ,be three log units lower (Table 1). The difference in the infectivity of the DNA preparations was even. higher than “the difference determined for the respective yields of infectious progeny virus.

INTRACELLULAR

VIRAL PROGENY DNA OF HSV-1

These results could be explained in two ways. The reduced infectivity of HP progeny DNA might either reflect a reduction in the synthesis of infectious DNA or the presence of interfering genomes. The latter argument is based on the finding of Wigler and co-workers that copies of all classes of DNA present in a calcium phosphate precipitate are taken up in a transfection competent cell (Wigler et al., 1980). In order to discern between these two possibilities cotransfection experiments were carried out using purified viral DNA samples. Upon transfection of BSC-1 cells with standard DNA together with equal amounts of HP progeny DNA a reduction in plaque formation by a factor of 10 was determined (Table 2). Two types of control experiments were performed to show that this reduction was due to the presence of interfering viral genomes in HP DNA. BSC-1 cells were cotransfected with standard DNA mixed with (i) an excess of pBR 322 DNA or (ii) XbuI fragments of viral standard DNA. The results in Table 2 clearly point to the involvement of interfering viral genomes: First, pBR 322 DNA does not reduce the plaque-forming capacity of standard DNA even when given in a threefold excess. Second, XbaI fragments of HSV-1 ANG DNA do not significantly reduce plaque formation in (1:l) cotransfections with standard DNA. As indicated in Table 1, the total amount of newly synthesized viral progeny DNA remained the same, regardless whether I TABLE 2 INTERFERENCEOF HP PROGENYDNA IN TRANSFECTIONEXPERIMENTS Transfecting DNA” Standard b&9 0.3 0.3 0.3 0.3

Cotransfection

(649

No. of plaques (%)

None HP DNA X&z1 fragments of standard DNA pBR-322

0.2 0.3

100 11 70

1

96

a DNA samples purified by banding in CsCl density gradients were used.

209

FIG. 2. Size distribution of DNA molecules of infectious standard HSV-1 ANG DNA and HP progeny DNA. Sedimentation into lo-3096 sucrose is from right to left. 0, 3H-labeled HSV-1 ANG DNA; A, 14Clabeled HP progeny DNA.

particles were present or absent in the infecting virus while the transfection data indicate differences in the biological activity between standard and HP progeny DNA. Such differences could also reflect imperfect or incorrect DNA maturation or additional steps of DNA modification in the interfering system. Consequently we compared the two samples of viral progeny DNA with respect to their size, number of single strand interruptions, restriction endonuclease cleavage patterns, their degree of methylation, and their uridine content. The Sizing and Restriction Eru&muclease Cleavage Patterns of IntraceUular HSV-1 ANG Progeny DNA Velocity sedimentation through neutral sucrose gradients shows that infective standard progeny DNA cosediments with the HP progeny DNA (Fig. 2). Sedimentation through alkaline sucrose gradients together with a mixture of SV40 form I and form II DNA as sedimentation markers reveals that the distribution of singlestrand interruptions is approximately the same in both DNA preparations. A mean number of 6-10 ss interruptions per unit length DNA molecule can be estimated from the sedimentation profile given in Fig. 3. A comparative analysis of the structure of intracellular HP DNA and standard

KthfEL

10 FRACTION

20 NO.

FIG. 3. Distribution of single-strand interruptions in HSV-1 ANG standard and HP progeny DNA. Sedimentation into alkaline 1040% sucrose is from right to left.

ET AL.

Frenkel, 1980). It was beyond the scope of this study to attribute each of the additional bands to a certain type of defective viral DNA. The majority of defective DNA species present in the HP progeny DNA preparations studied here proved to be HpaI resistant (Fig. 4~). The HWI-resistant DNA was cleaved with Hind111 and yielded additional bands between the fragments J and K in the Hind111 pattern of HP progeny DNA (Fig. 4e). Apart from the band representing this resistant DNA moiety, both HP and standard DNA H@aI fragment patterns are identical. For the interpretation of the results of the restriction enzyme analysis it has to be considered that HP virus contains an overwhelming excess of interfering over infectious standard virus (Fig. 1) and that the infectivity of the two progeny DNA preparations differs by three orders of magnitude (Table 1). However, from the data in Fig. 4, it can be derived that the majority of the molecules in both DNA preparations are of the same complexity. The same reasoning applies for the comparison of DNA isolated from standard and from HP virus, the DNA fragment patterns of which are shown in Fig. 5. On the other hand, identical cleavage patterns do not exclude minor structural variations or other types of DNA modification such as methylation or replacement of deoxyribonucleotides by ribonucleotides to be responsible for the reduced infectivity of HP progeny DNA.

DNA preparations by restriction endonuclease cleavage with HpaI, HindIII, and BumHI is shown in Fig. 4. All standard HSV DNA fragment bands are clearly present in the respective HP HSV fragment ,patterns. Notably, the DNA frag- DNA Methp!ation and Uricline Content of ments representing the termini of stanViral Progeny DNA dard HSV-1 DNA are present in the DNA methylation and the presence of patterns of HP progeny DNA suggesting ribonucleotides in progeny DNA were a proper cleavage of concatemeric precursor DNA in the course of processing of studied as possible causes of interference. viral DNA (Hirsch et al., 1976). Represen- The DNA was hydrolyzed using pancreatic DNase and snake venom phosphodiestertative in this respect is the L-terminal HpuI L fragment (Kaerner et al., 1979) ase and the resulting mononucleotides were analyzed by 2D thin-layer chroma(Fig. 4). The restriction fragment patterns of HP HSV DNA besides individual band tography to identify both Fi-methylcytidine and uridine monophosphate. The DNA intensity differences show some additional bands as compared to standard HSV DNA. had been labeled by the addition of [6This can be attributed to the presence of 3H]uridine to the culture media of infected variable amounts of various classes of re- cells 3.5 hr after infection. Since [Siterative defective DNA (for a review, see 3H]uridine is readily converted to dCTP

INTRACELLULAR

211

VIRAL PROGENY DNA OF HSV-1

AB --/Cl -D,E -F -/LO Z

--IvK3

J

-L ;NM -Cd

7.7

‘b ,K2 N3 N

Kl

7

-03 -0

-K rh - I

-T -x2

-u -v

-N -

a

Y,B’

d

b

F‘IG. 4. Comparison of the structure of intracellular progeny HSV-1 ANG standard (b, d, f) and HP DNA (a, c, e) by cleavage with Hind111 (a, b), HpaI (c, d), and BarnHI (e, f).

-La -c

-def,

-F GH --K4

+C,D

--IJ -KIN -M

-K drvl3

Q3

-J

-0

-P,cl, --R -x3 ST -Xi! -u I -v

-K -L -M -N

‘N3

2L

--NI -04

-I

-M2 -MI

,K2

-N

01

w,xi -Y -A’.B’ a

C

d

e

f

FIG. 5. Analysis of standard (b, d, e) and HP 4 HSV-1 ANG DNA (a, c, e) isolated from virus particles by cleavage with Hind111 (a, b), HpaI (c, d), and BumHI (e, f).

Z

212

KOMEL

ET AL.

TABLE

3

METHYLATION AND CONTENT OF RIB~IWCLEIXIDES Radioactivity

OF INTRACELLULAR VIRAL PROGENY DNA

(%l cpm) associated with nucleotide spots in 2D CIC

Infecting virus

dCMP

C-MdCPM

UMP

dTMP

dAMP

dGMP

CMP

AMP

GMP

LP 5 HP4 None

14726 31441 8421

1 1 98

17 21 28

4166 8736 2273

1 1 0

3 4 2

8 7 13

2 2 3

4 3 5

No&. Viral DNA from [6-%ljuridine-labeled infected cells was isolated, purified, enzymaticly cleaved to 5’-mononucleotides, and analyzed in two-dimensional cellulose thin-layer chromatography together with marker substances as described under Methods. The last line shows the results for total cellular DNA from uninfected cells.

and dTTP, both the uridine residues suspected in HSV DNA and the cytosine residues can be labeled. The use of cytidine or uridine labeled in the 5 position is impossible because of their chemical instability (at low pH hydrogen exchange occurs) and because the label would be removed during the methyl group transfer (Glinthert et al., 1976; Fink and Fink, 1962). The reason for labeling the DNA with a pyrimidine nucleoside tritiated in the 6 position in a study of DNA methylation is that the bases are methylated in the 5 position by specific methyl transferases after incorporation into nascent DNA. Thus the specific radioactivity of cytosine equals that of the methylated cytosine (Gtinthert et al., 1976) and the comparison allows to calculate the number of 5-methylcytosine residues per unit length progeny DNA molecule. Such an estimate is not possible for the respective number of ribonucleotides due to insufficient data about the pool sizes after infection with HSV. After labeling the uridine pool, it can be assumed that the specific activity of the incorporated uridine is equal to or higher than that of the incorporated cytosine. Contamination of the DNA samples by free RNA was excluded by two cycles of buoyant density centrifugation and RNase treatment. The identity of the nucleotides on the chromatography plates was ascertained by use of two independent techniques of two-dimensional thin-layer chromatography. The data comprised in Table 3 demonstrate that there is no difference in DNA methylation as well as in uridine content in the progeny DNAs an-

alyzed. Almost 100% of the radioactivity label incorporated could be recovered as deoxyribonucleosides and less than 0.07% as rH]uridine both in standard progeny DNA and in HP progeny DNA. Methylation was below the level of detection of ten 5-methylcytosine residues per unit length DNA molecule in both DNA preparations. The results listed in Table 3 thus indicate that methylation or incorporation of ribonucleotide stretches are not involved in interference. DISCUSSION

Homologous interference in HSV-1 ANG replication exerted by viral I particles has been reported to be in part due to reduced encapsidation of newly synthesized viral DNA whereas the yield of progeny DNA proved not to be affected (Stegmann et al., 1978). Similar observations were reported for pseudorabies virus by Ben-Porat and Kaplan (1976). Further studies concerning aberrations in the viral replication seemed necessary since the extent to which the encapsidation of progeny DNA is reduced in the interfering system can only in part explain the reduction in the yield of infectious progeny virus. UV irradiation data proved the interference to be caused by an intracellular process due to a genome function of the I particles (Schrbder and Urbaczka, 1979). Therefore, we studied parameters of DNA synthesis and of DNA maturation in cells infected with a mixture of I particles and standard virus in comparison with intracellular progeny DNA from cells infected with standard HSV-1 ANG alone.

INTRACELLULAR

VIRAL

HP progeny DNA proved to be of HSV unit length and renders S- and L-terminal restriction fragments identical to those of standard DNA indicating that it is similarly processed as viral standard DNA. In the HP virus stock investigated in this study repetitive defective DNA represents only a minor constituent (Fig. 5~). It appears unlikely that the presence of this DNA moiety alone is responsible for the observed high ratio of interfering to infectious virions (Fig. 1). An alteration in the pattern of DNA methylation obviously is not the cause of the change in the biological activity of HP progeny DNA, since both standard and HP progeny DNA contain less than 10 5-methylcytosine residues per genome length molecule. Similarly the incorporation of ribonucleotides appears not to be the cause for the observed reduction of infectivity. It should be mentioned that the uridine content, which turned out to be less than 0.07% of the total radioactivity incorporated when cells are labeled with [63H]uridine, is much lower than reported by other authors (Hirsch and Vonka, 1973). Thus the presence of ribonucleotide stretches in HSV-1 DNA at least does not appear to be a general phenomenon, although the degree to which uridine is incorporated into HSV-1 progeny DNA might vary for different host cells and with the virus strain used. The marked reduction of the infectivity of HP progeny DNA in transfection assays as compared to standard progeny DNA could be explained by the presence both of interfering DNA molecules and of noninfectious standard genomes that have been replicated and processed like infectious progeny DNA but lack infectivity due to additional minor structural variation. The nature of these variations of the standard DNA which are suspected to cause the low infectivity of HP progeny DNA and its interfering capacity in transfection assays remains to be resolved. Minor variations in certain regions of the genomes of individual HSV strains have recently been reported to occur frequently (Lonsdale et al., 1980; Podbielski, 1982) and

PROGENY

DNA OF HSV-1

213

sometimes appear to be associated with severe alterations of the biological properties of the virus (Kaerner et al., 1981). It is noteworthy that the HP4 stock described here apparently contains a new class of repetitive defective HSV-1 ANG (HSV-ANG dDNA 3) which is HpaI resistant and Hind111 sensitive. Isolated dDNA 3 could be shown (Schriider and Gray, unpublished data) to originate from IRS of the prototype isomer of HSV-1 ANG standard DNA. The structural similarity of HP progeny DNA to viral standard DNA, on the other hand, suggests that its low infectivity and interfering capacity is rather due to DNA molecules of high complexity than to repetitive DNA molecules of low complexity. Alternatively interference could be exerted by repetitive defective genomes exclusively provided interference occurs even at very low ratios of standard to defective genomes. Considering results reported earlier (Schroder et al., 1975/19’76; Stegmann et al., 1978; Schroder and Urbaczka, 1978) that the kinetics of accumulation of repetitive defective genomes do not coincide with the cyclic fluctuations of infectious virus yields in serial passages of HSV-1 ANG at high multiplicity, we prefer, however, the idea that a major fraction of interfering genomes is not of the reiterated type. REFERENCES

BEN-P• RAT, T., and KAPLAN, A. S. (1976). A comparison of two populations of defective, interfering pseudorabies virus particles. virdogy 72, 471-479. BRONSON,D. L., DREESMAN,G. R., BISWAL, N., and BENYESH-MELNICK,M. (1973). Defective virions of herpes simplex viruses. Intenrirology 1,141-153. DARAI, G., and MUNK, K. (1976). Neoplastic transformation of rat embryo cells with herpes simplex virus. Int. J. Cancer l&469-481. EAGLE, H. (1955). The minimum vitamin requirements of the L and HeLa cells in tissue culture, the production of specific vitamin deficiencies, and their cure. J. Exp. Med 102, 595601. FINK, R. M., and FINK, K. (1962). Relative retention of ‘H and ‘“C labels of nucleosides incorporated into nucleic acids of Neurospora. J. Bid Chem 237, 2889-2891. FRENKEL,N., JACOB,R. J., HONESS,R. W., HAYWARD, G. S., LOCKER,H., and RCIIZMAN,B. (1975). Anatomy of herpes simplex virus DNA. III. Character-

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