Early events required for induction of chromosome abnormalities in human cells by herpes simplex virus

VIROLOGY44, 544--55~ (1971)

Early Events Required for Induction of Chromosome Abnormalities in Human Cells by Herpes Simplex Virus F. J. O'NEILL

ANn F. RAPP

Department of Microbiology, The Milton S. Hershey Medical Center of The Pennsylvania State University, Hershey, Pennsylvania 17033 Accepted February 2, I97I Studies on chromosome abnormalities induced in human diploid cells by herpes simplex virus type 2 (tISV-2) were undertaken to determine the period of the viral replication cycle during which these abnormalities occur. When cells pretreated for 16-18 hr with human interferon were inoculated with HSV-2 for 4 hr, chromosome abnormalities did not occur except at low levels found in control cultures. Thus, HSV-2 induced chromosome damage occurs following virM uneoating and requires some translation of the viral genome. Other experiments were performed to determine whether these abnormalities occur prior to synthesis of viral DNA. Cytosine arabinoside (ara-C), which is known to inhibit herpes simplex viral DNA synthesis, was used in these experiments. Although ara-C produced a 99.9% inhibition of IISV-2 multiplication, it did not prevent induction of chromosome damage by HSV-2. These results indicate that HSV-2 induced chromosome damage occurs in the absence of extensive viral DNA replication. In fact, ara-C acted synergistically with IISV-2 in the induction of multiple chromosome breaks. The nature of this synergism is unknown. INTRODUCTION Chromosome abnormalities have been identified in mammalian cells inoculated with a variety of animal viruses. Among the DNA viruses, herpes simplex (I-Iampar and Ellison, 1961; Stieh et al., 1964), herpes zoster (Benyesh-Melnick et al., 1964), adenovirus type 12 (MacKinnon et a/., 1966; Stich and 5~ohn, 1965; Stolz el al., 1967; zur Hausen, 1967, 1968), adenovirus types 4 and 18 (Cooper et al., 1967) and SV40 (Wolman eta/., 1964) have been reported to break chromosomes. Certain RNA viruses including measles (Nichols et al., 1965), Rous sarcoma virus (Nichols et al., 1964) and yellow fever virus (Harnden, 1964) also appear to possess this property. Chromosome abnormalities have also been noted in cells transformed in vitro or in cells of tumors induced by certain oncogenie viruses including SV40 (Yerganian et al., 1962; Moorhead and Saksela, 1963) and adenovirus type 12 (Stich and Yohn, 1965). With SV40, similarities have been noted in the types of abnormalities

induced directly b y the virus and those found in cells transformed b y SV40 (Wolman et al., 1964). I t has also been known for some time t h a t h u m a n tumors contain significantly higher levels of chromosome abnormalities t h a n do normal cells (Ishihara et at., 1963; Makino et al., 1964; Miles 1967a,b). Some of the abnormalities found in various h u m a n tumors appear similar to those found in 8V40 transformed h u m a n cells (Miles et at., 1966; Spiers and Baikie, 1967) and in h u m a n cells infected with herpes simplex virus (O'Neill and Miles, 1969). I n spite of the relatively large literature illustrating chromosome abnormalities following virus inoculation, little is known of the mechanism b y which viruses produce this effect. Allison (1966) has suggested t h a t during viral uneoating, accomplished b y lysosomal digestion and during later viral events, certain lysosomal enzymes are released which might act to produce chromosome damage. I t has also been suggested t h a t some expression of the viral genome is necessary for 544

HSV I~EPLICATION AND CHROMOSOME DAMAGE induction of chromosome abnormalities by HSV (Rapp and Hsu, 1965). Recently, it has been shown that chromosome abnormalities induced by HSV can be detected as early as 4 hours after virus inoculation of human cells (O'Neill and ~$iles, 1969). Thus, this phenomenon should be amenable to detailed analysis by treatment of virus infected ceils with certain antimetabolites without seriously affecting the ability of these cells to enter mitosis. The present report offers evidence that chromosome abnormalities induced by herpes simplex virus occur at a period preceding viral DNA synthesis but take place after viral uncoating and require some transcription or translation of the viral genome. These conclusions are based on experiments showing inhibition of viral DNA synthesis without inhibition of chromosome damage, and experiments showing inhibition of chromosome damage in human cells by pretreatment of cultures with human interferon. MATERIALS AND METHODS

Cell culture. Diploid human embryonic lung (HEL) cells were obtainecl commercially (HEM Laboratories, Rockville, L\,iaryland) and propagated in Eagle's basal medium containing 10% fetal bovine serum, penicillin, and streptomycin, in 8-ounce prescription bottles. Cultures were used before the 20th subeultivation. Viruses. Herpes simplex virus type 2 (HSV-2) strains 332 and 333 were obtained from Dr. W. E. Rawts (Baylor College of Medicine, Houston). Strain 333PP is a substraln of 333, plaque-purified twice in human erabryonic kidney cell cultures. Stocks of HSV-2 were obtained by inoculating monolayers of HEL with an input multiplicity of about 1 plaque-forming unit (PFU) of virus per cell. T h e virus was harvested after cytopathic effects became pronounced by disrupting the cells with two cycles of freezing and thawing, and then titrated on monolayers of rabbit kidney cells in 60-ram petri plates under a methyleellulose overlay as described by Rapp (1963). The virus titer is expressed as PFU/ml. Stocks of Newcastle disease virus (NDV) were prepared by inoculating HeLa cell

545

monolayers with the virus at an input of about 1 PFU per cell. The virus was harvested as above, and titrated on monolayers of HEL in 60-ram petri plates under an agar overlay. Stocks of vesicular stomatitis virus (VSV) were also obtained in HeLa cells. Again, the virus was harvested by disrupting the cells by cycles of freezing and thawing, and titrated on HeLa monolayers in 60-ram petri plates under an agar overlay. Interferon preparations. Interferon was induced and extracted by a modification of the method of Desmyter et al. (1968). Cultures of HEL were inoculated with 1 PFU/cell of NDV in medium without serum. Three days later, the fluids were harvested and titrated to pH2 with 1/20 N HC1. The fluids were then refrigerated at 4 ° for 4-5 days. The pH was brought back to neutrality with 1 N NaOH. The interferon was assayed by making 2-fold dilutions of the fluids in Eagle's medium containing 2 % fetal bovine serum. Monolayers of HEL in 60-ram petri plates were then inoculated with the diluted interferon (2 plates per dilution) overnight at 37 °. Each interferon treated plate and control plates were then inoculated with 50 PFU of VSV. The virus was adsorbed for 1 hr and the plates overlayed Mth medium containing agar. Plaques were read 2 days later; the interferon titer is expressed as the reciprocal of the dilution resulting in a 50 % reduction of VSV plaques. The titer of the interferon extracts employed in the experiments reported here was approximately 100 units/ml. Purified human interferon ldndly supplied by Dr. K. M. Fantes (Glaxo Research Ltd., London) was also used in some experiments; the titer was 10,000 units/ml against Sindbis virus in vervet kidney cells. Chromosome preparations. Chromosome preparations were made by a modification of the methods of Moorhead et al. (1960) and Miles and O'Neill (1966). Briefly, cells to be prepared for chromosome analysis were incubated with 0.05 t~g/ml of Colcemid (CIBA, Summit, New Jersey) for the final 90 rain of culture. The cells were then harvested with 0.1% pronase and sedimented in conical centrifuge tubes at 500 g. The cells were resuspended in 1 ml of fetal bovine serum conraining 100 units of heparin, and then 4 ml

54:6

O'NEILL AND RAPP

of double distilled water was added. After gentle mixing with a Pasteur pipette, the cells were allowed to stand at room temperature for 15 rain. This hypotonie t r e a t m e n t caused the cells to swell resulting in subsequent spreading of chromosomes. The hypotonized cells were then resedimented and fixed with a solution of 3 parts methanol to 1 part glacial acetic acid, b y gently overlaying the cell pack with the fixative. After standing at room temperature for a further 15 rain, the cells were very gently resuspended in the fixative. T h e fixative was then changed. The cells were again resuspended but in a small volume of fixative. Approxim a t e l y 0.05-0.1 ml of the suspended cells was then gently spread over a wet micro-

scope slide and allowed to air dry. The slides had been previously cleaned with acetone and then dipped in a dry-ice water b a t h before the fixed cells were added. The cells were stained with Giemsa and mounted in Permount. RESULTS

Effect of Human Interferon on HSV-2 Induced Chromosome Abnormalities in Human Cells These experiments were undertaken to determine the relationship between viral uncoating, transcription of the viral genome, and virus-induced chromosome abnormalities.

FIG. 1. Metaphase of human embryonic lung cell from a culture inoculated with HSV-2, strain 332 for 4 hr. Arrows indicate chromosomes with accentuation or attenuation of secondary constrictions on chromosomes No. 1, 9, and 16 (long narrow arrows). Also note accentuation or stretching of satellites on the aerocentric chromosomes (short wide arrows).

HSV REPLICATION AND CHI~OMOSOME DAMAGE Logarithmically growing cultures of H E L were incubated overnight (16-18 hr) with 10 u n i t s / m l of the etude interferon or 100 u n i t s / m l of the purified interferon, in a total volume of 20 ml of growth medium. Experiments M t h higher dilutions of interferon were not performed. The fluids were then decanted and the I-IEL cells were inoculated with 2 or 5 P F U / e e l l of HSV-2 strain 332 for 1 hr at 37 °. After the 1-hr adsorption period, growth medium was added and the cultures were incubated for an additional 3 kr. Controls were treated in the same manner, b u t with the omission of the interferon. All cultures were then harvested for chromosome analysis. The slides were coded, as they were in all the experiments in this report, and the frequency of chromosome damage was tabulated for each slide. At least 3 slides

547

from each of the t r e a t m e n t schedules were analyzed. The types of abnormalities scored in these studies were cells containing one break, cells containing 2 breaks, cells containing 3 or more breaks, the n u m b e r of accentuated secondary constrictions on chromosomes No. 1, 9, and 16 (Fig. 1), and cells with eroded chromosomes (Fig. 2). The secondary constrictions were considered to be accentuated if they extended beyond the width of a metaphase eln'omosome. The results of these experiments appear in Table 1. Virus-treated cultures showed a significant increase in the number of cells with chromosome or ehromatid breaks when compared to control (untreated) preparations. Cells with 3 or more breaks were rare in control preparations but fairly common in virus-treated

FIG. 2. Metaphase of human embryonic lung cell 4 hr after HSV-2 (332) inoculation illustrating erosion of nearly all chromosomes.

548

O'NEILL AND RAPP TABLE 1

EFFECT OF HUMAN INTERFERON ( I F ) ON H S V - 2 (332) INDUCED CHROMOSOME ABNORMALITIES IN I~UM3_N EMBRYONIC LUNG CELLS

1

tISV-2a tISV-2 -~ IF ~ I-ISV-2b ItSV-2 + IF b Control

12 4 16 4 5

No. of cells with breaks 2 3 or more

0 1 5 0 1

10 0 13 0 0

Secondary constrictions

Erosion

31 1 38 0 1

5 0 13 0 0

Total cells scored

300 300 225 225 200

Input multiplicity was 2 PFU/eell of HSV-2. Prior to HSV-2 inoculation, cultures were incubated for 16-18 hr with approximately 10 units/ml of the crude interferon preparation. Input multiplicity was 5 PFU/cell of HSV-2. Prior to HSV-2 inoculation, cultures were incubated or 16-18 hr wi~h approximately 100 units/ml of purified interferon. preparations appearing at an incidence of up to 6 or 7% (10/300 and 13/225). Chromosome erosion (Fig. 2), which was undetectable in control preparations, also appeared in about 6% of virus-treated cultures. Marked accentuation of secondary constrictions on chromosomes 1, 9, and 16 (Fig. 1), as noted in an earlier report (O'Neill and Miles, 1969), were also fairly common in virus-treated cultures but rare in controls. T h e incidence of all types of abnormalities seemed to be in proportion to the dose of virus with cultures inoculated with 5 P F U / cell of virus showing more abnormalities than those inoculated with 2 P F U / c e l l (Table 1). Cultures pretreated with human interferon, either the crude extract or the purified preparation, and then inoculated with HSV-2, did not show chromosome damage except at low levels found in urfinoculated control cultures (Table 1).

Effect of Cytosine Arabinoside (ara-C) and Iododeoxyuridine on HSV-2 Induced Chromosome Abnormalities These expeirments were performed to determine whether the HSV-2 induced abnormalities would occur regardless of the absence of certain events in the virus replication cycle. Two compounds were employed for this purpose, cytosine arabinoside (ara-C) and iododeoxyuridine. Both iododeoxyuridine (25 or 100~g/ml) and ara-C (10 ~g/ml), when added to H E L cultures 24 hr after a 1 hr virus adsorption period, produced more than a 99.9 % inhibition in virus multiplica-

tion. In addition, ara-C produced an apparent complete inhibition of D N A synthesis when added for 3 hr after virus adsorption. This was shown b y the failure of such treated cultures to incorporate tritiated thymidine during the final hour of incubation (O'Neill and Rapp, 1971). Rapidly dividing I-IEL cells were inoculated with 2 or 5 PFU/cell of HSV-2 (332 or 333PP), and the virus was adsorbed for 1 hr at 37 °. Ara-C (10 ~g/ml) or iododeoxyuridine (25 ~g/ml and 100 ~g/ml) was then added, and the cultures were incubated for an additional 3 hr as described in the previous experiments. After chromosome preparation, the cells were analyzed as described in the interferon experiments. The results show that neither ara-C nor iododeoxyuridine inhibit any of the HSV-2 induced chromosome abnormalities (Tables 2 and 3). However, ara-C and a plaque purified strain of 333 virus appear to act synergistically to produce a large number of cells with multiple breaks (Table 2). Similar observations were made with ara-C and other strains of HSV-2 (O'Neill and Rapp, 1971). It is noteworthy that iododeoxyuridine at either concentration did not have this effect (Table 3). Except for the synergistic effect observed with ara-C on the production of cells with 3 or more chromosome breaks, the incidence of all the types of chromosome abnormalities studied was not reduced in virus-inoculated cultures treated with inhibit.or. However, there seems to be a slight increase in the incidence of secondary constrictions in both ara-C and

HSV I:LEPLICATION AND CHROMOSOME DAMAGE

549

TABLE 2 EFFECTS

OF ARA-C

ON HSV-2

(333 PP) INDUCED CIIROMOSOME LUNG CEI&S

No. of cells with breaks

Ara-C +333 333 PP Ara-C Control

PP

ABNORMALITIES

IN HU~'[&N EMBRYONIC

2

3 or more

Secondary constrictions

Erosion

1

Total cells scored

25 10 40 5

8 6 7 1

60 5 4 0

55 43 0 0

Ii 12 0 0

225 225 225 200

TABLE 3 EFFECTS

OF

IODODEOXYURIDINE (IUDR) ON HSV-2 (333 PP AND 332) ABNORMALITIES IN HUMAN EMBRYONIC LUNG CELLS No. of cells with breaks 1

IUDI~ HSV-2 a b HSV-2 alone a b I U D R Mone a b Control

15 21 8 22 10 6 3

2

3 or more

4 3 3 5 0 0 0

5 15 3 16 0 0 0

INDUCED

Secondary constrictions

Erosion

35 56 27 48 2 5 1

12 16 15 18 0 0 0

CHROMOSOME

Total cells scored 225 225 225 225 225 225 150

IUDR was used at a concentration of 25 ~g/ml. For virus-treated cultures, strain 333 PP of HSV-2 was used. IUDR was used at a concentration of 100 ~g/ml. Strain 332 of HSV-2 was employed. iododeoxyuridine-treated virus-infected cultures. DISCUSSION The mechanism of action of iododeoxyuridine on herpes virus multiplication is not well understood. Roizman et al. (1963) reported that iododeoxyuridine produced a complete inhibition of HSV replication in human cells at 5 ug/ml. When added only 2-4 hours after HSV infection, iododeoxyuridine produced a delay in the appearance of newly formed infectious virus. These investigators also reported that iododeoxyuridine was not incorporated into viral DNA. Thus, it was suggested that iododeoxyuridine inhibits synthesis of viral DNA (Roizman et at., 1963). However, other investigators working with other herpes viruses have reported that iododeoxyuridine is incorporated into viral DNrA (Kaplan and J~en-Porat, 1966; Prusoff, 1967). This, along with other lines of evidence, suggests that iododeoxy-

uridine affects a later event, possibly causing production of defective late proteins (Prusoft, 1967). Beyond showing a marked inhibition of HSV-2 multiplication, the experiments reported here were not designed to elucidate the mechanism of iododeoxyuridine action. Thus, aside from the experiments which show that iododeoxyuridine has no inhibitory effect on HSV-2 induced chromosome damage, it can only be concluded that late events are not required for the induction of this damage. The antiviral action of ara-C is more dearly understood. Levitt and ]?ecker (1967) have shown that ara-C at 20 ~g/ml produces a complete inhibition of HSV DNA synthesis with the prevention of subsequent virion formation. These investigators also showed that ara-C also completely inhibited cellular DNA synthesis in both virus-infected and control cultures. The results of the autoradiographic experiments reported here are

550

O'NEILL AND RAPP

in accord with the findings of Levitt and Becker (1967). Thus, since ara-C does not prevent HSV-2 induced chromosome abnormalities, it can be concluded that these abnormalities occur in the absence of viral DNA synthesis and prior to this event. Waubke et al. (1968) showed that HSV induced chromosome abnormalities in hamster cells 4 hr after virus inoculation. When thymidine-3H was added during the final hour of culture, they were unable to detect extrachromosomal DNA synthesis in mitotic cells with virus-induced chromosome abnormalities. From these results these investigators concluded chromosome abnormalities occurred in the absence of HSV DNA synthesis. However, more recently, Hummeler et al. (1969) in an electron microscopic study showed, in these same cells, evidence for the production of new virus progeny as early as 3 hr after inoculation with HSV. Thus, it may be concluded that viral DNA synthesis can occur as early as 3-4 hr after virus inoculation. Nevertheless, the results of our experiments agree with the conclusions of Waubke et al. (1968) that HSV induced chromosome damage occurs at a period preceding viral DNA synthesis. Moreover, we show this in human diploid cells. However, since human interferon effectively inhibits the chromosome effects of HSV-2, it is indicated that translation of the viral genome is necessary. This conclusion requires acceptance of the theory that interferon inhibits translation of viral mRNA. Although the mechanism of action of interferon is unknown or in question, it would appear that its effect occurs at a very early event in virus replication since it has been shown to prevent virus-induced "shuttingoff" of host cell RNA synthesis, also considered to be a very early event (Levy, 1964; Levy et al., 1966). Since interferon inhibits multiplication of both RNA and DNA viruses, it has been argued that its most probable mode of action is at the earliest event after viral uneoating and is probably at a translational event (Joklik, 1965). It has also been shown that there is a decreased affinity of interferon-treated ribosomes for viral mRNA, and that even when such combinations occurred, translation is markedly

inhibited (Marcus and Salb, 1966; Carter and Levy, 1968). Therefore, it has been suggested that interferon inhibits viral replication by mediating the production of a substance called translation inhibitory protein (TIP), which prevents translation of viral mRNA early in the viral replication cycle (Marcus and Salb, 1966). However, this hypothesis has been opened to question since Kerr et al. (1970) were unable to confirm the reports of Marcus and Salb (1966) and Carter and Levy (1968). Allison and Mallucci (1965) suggested that virus-induced chromosome abnormalities may be mediated by breakdown of lysosomes with subsequent release of DNase. Since it has been suggested that viral uncoating could be accomplished by the action of lysosomal enzymes following lysosomalvirion fusion (Allison, 1966), it would appear that this step could allow for release of these catabolic enzymes with subsequent chromosome damage. More recently in a study of chromosome damage induced by measles virus and adenovirus type 12, Aula and Nichols (1968) reported no detectable lysosomal breakdown in replicate cultures showing virus-induced chromsome breakage. These investigators were also unable to prevent these chromosome abnormalities by the addition of cortisone acetate, a substance known to stabilize lysosomal membranes. Since our experiments suggest that HSV-2 induced chromosome damage occurs after viral uncoating, it would appear that lysosomal labilization during viral uncoating is not a factor in the production of these effects. However, our results do not rule out the possible action of lysosomal DNases during events in the HSV-2 replication cycle subsequent to viral uncoating. Investigations from a number of laboratories, when correlated, also suggest that chromosome abnormalities induced in Syrian hamster cells by adenovirus type 12 (adeno 12) occur during early events in the adeno 12 replication cycle. Kitamura et al. (1964) have shown that adeno 12 does not replicate in Syrian hamster cells. However, MacKinnon et al. (1966) and zur Hausen (1968) were able to produce chromosome abnormalities in these cells with adeno 12.

HSV REPLICATION AND CHROMOSOME DAMAGE MaeKinnon et al. (1966) also noted the inability of adeno 12 to replicate and to produce viral structural antigens in these cells. I n adeno 12 infected B H K 2 1 cells (a continuous line of Syrian hamster cells), Doerfler (1969) was unable to detect synthesis of viral D N A but Strohl (1969) has reported extensive chromosome breakage. I t thus appears that adeno 12 induced chromosome damage occurs in the absence of detectable viral D N A synthesis. Although chromosome damage induced b y some viruses seems to occur during early events in the viral replication cycle, the mechanism or factor(s) responsible for this damage remains unknown. Association of chromosome damage-with lysosomal DNases seems doubtful in light of the work of Aula and Nichols (1968). One possibility is that the virus genome codes for a cellular DNase early in infection. Recently, Burlingham and Doerfler (1969) found an endonuelease associated with adenovirus virions b u t it has not been determined whether this endonuclease is synthesized during adeno 12 abortive infection of hamster cells. I t has been shown that chromosome abnormalities occur in higher than normal frequencies in mammalian cancers (Ishihara et al., 1963; Makino et al., 1964; Stieh and Yohn, 1965; Miles et al., 1966; Miles 1967a, b). If these neoplasms have a viral etiology, then the recurrent chromosome abnormalities found in these tumors could be the result of the persistence of an early viral function. Recently, Fujinaga and Green (1970) have demonstrated the presence of an early viral m R N A in adenovirus type 2 transformed hamster cells. A late viral m R N A , found in productively infected cells, was not found in the transformed cells. If this should prove to be a general phenomenon, there m a y be an association between early viral events, subsequent recurrent chromosome changes, and oncogenesis. ACKNOWLEDGMENTS This study was conducted in part under Contract No. 70-2024 within the Special Virus-Cancer Program of the National Cancer Institute, NIH, PHS, and under Postdoctoral Fellowship Number 5 FO2 CA44381-02to Frank J. O'Neill.

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REFERENCES ALLISON, A. C. (1966). The role of lysosomes in pathology. Proc. Roy. Soc. Med. 59, 867-871. ALLISON,A. C., and M~LLUCCI,L. (1965). Histochemical studies of lysosomes and lysosomal enzymes in virus-infected cell cultures. J. Exp. Med. 121,463-476. A~-LA,P., and NichoLs, W. W. (1968). Lysosomes and virus induced chromosome breakage. Exp. Cell Res. 51, 595-601.

BENYESH-MELNICK,

M., STICH, H. F., RAPP, F.,

and I-Isu, T. C. (1964). Viruses and mammalian chromosomes. III. Effect of herpes zoster virus on human embryonal lung cultures. Proc. Soc. Exp. Biol. Med. 117, 546-549.

BURLINGHAM,

B. T., and DOERFLER, W. (1969). endonuclease associated with adenovirus type 2 and 12. Fed. Proc. Fed. Amer. Soc. Exp. Biol. 28, 434.

An

CA~TER, W. A., and LEVY, H. B. (1968). The recognition of viral RNA by mammalian ribosomes. An effect of interferon. Biochim. Biophys. Acta 155, 437--443. COOPER, J. E. F., STICH, I-I. F., and YOIIN, D. S. (1967). Viruses and mammalian chromosomes. VIII. Dose response studies with human adenovirus types 18 and 4. Virology 33,533-541. DESMYTER, J., MELNICK, J. L., and R_~wLs, W. E. (1968). Defectiveness of interferon production and of rubella virus interference in a line of African green monkey kidney cells (Vero). J. Virol. 2, 955-961.

DOEnFLEn, W. (1969). Nonproductive infection of baby hamster kidney cells (BHK21) with adenovirus type 12. Virology 38, 587-606. FVJINAGA,K., and GREEN, M. (1970). Mechanism of viral carcinogenesis by DNA mammalian viruses. VII. Viral genes transcribed in adenovirus type 2 infected and transformed cells. Proc. Nat. Acad. Sci. U. S. 65, 375-382. HAMPAa, B., and ELLISON,S. A. (196t). Chromosomal aberrations induced by an animal virus. Nature (London) 192, 145-147. HARNDEN, D. G. (1964). Cytogenetic studies on patients with virus infections and subjects vaccinated against yellow fever. Amer. J. Human Genet. 16,204-213. HUMMELE~, K., TOMASSINI,N., and Z_~JAc, B. (1969). Early events in herpes simplex virus infection: a radioautographic study. J. Virol. 4, 67-74. ISHIH~RA, T., KIKUC~II,Y., and SANDBEnG,A. A. (1963). Chromosomes of twenty cancer effusions : correlation of karyotypic, clinical and pathologic aspects. J. Nat. Cancer Inst. 39, 1303-1361. JOKLII;, W. K. (1965). The molecular basis of the viral eclipse phase. Progr. Med. Virol. 7, 44-96.

55 9,

O'NEILL AND RAPP

•APLAN, A. S., and BEN-PORAT, T. (1966). Mode of antiviral action of 5-iodouracil deoxyriboside. J. Mol. Biol. 19,320-332. KERR, I. M., SONNA]3END,J. A., and MARTIN, E. M. (1970). Protein-synthetic activity of ribosomes from interferon-treated cells. J. Virol. 5, 132-144. KITnMVRA, I., VAN HOOSIER,JR., G., SAMPLE,L.,

TAYLOR, G., and TRENTIN,J. J. (1964). Characteristics of human adenovirus type 12 induced hamster tumor cells in tissue culture. Proe. Soc. Exp. Biol. Meal. 116, 563-568. LEVITT, J., and BECKER, Y. (1967). The effect of cytosine arabinoside on the replication of herpes simplex virus. Virology 31, 129-134. LEVY, I~I. B. (1964). Studies on the mechanism of interferon action. II. The effect of interferon on some early events in mengo virus infection in L cells. Virology 22, 575-579. LEVY, H. B., SNELLBAKER, L. F., and BARON, S. (1966). Effect of interferon on RNA synthesis in Sindbis virus infected cells. Proc. Soe. Exp. Biol. Med. 121, 630-632.

MACKINNON, E., KALNINS, V. I., STICH, H. l~., and YOHN, D. S. (1966). Viruses and mammalian chromosomes. VI. Comparative karyologic and immunofluorescent studies on Syrian hamster and human amnion cells infected with human adenovirus type 12. Cancer Res. 26, 612-618. MAKINO, S., S~SAKI, M. S., and TONOMURA, A. (1964). Cytological studies of tumors. XL. Chromosome studies of fifty-two human tumors. J. Nat. Cancer Inst. 32, 741-777. M,%ncus, P. I., and SALB, J. M. (1966). Molecular basis of interferon action; inhibition of viral RNA translation. Virology 30, 502-516. MILES, C. P. (1967a). Chromosome analysis of solid tumors. I. Twenty-eight nonepithelial tumors. Cancer 20, 1253-1273. MILES, C. P. (1967b). Chromosome analysis of solid tumors. II. Twenty-six epithelial tumors. Cancer 20, 1274-1287. MILES, C. P., and O'NEILL, F. (1966). Prominent secondary constrictions in a pseudodiploid human cell line. Cytogenetics 5, 321-324. MILES, C. P.j GELLEI~,W., and O'NEILL, F. (1966). Chromosomes of Hodgkin's disease and other malignant lymphomas. Cancer 19, 1103-1116.

MOORHEAD, P. S., and SAKSELA, E. (1963). Nonrandom chromosomal aberrations in SV40transformed human cells. J. Cell. Comp. Physiol. 62, 57-83.

MOORHEAD, P. S., NOWELL, P. C., MELLMAN, W. J., BATTleS, D. M., and HUNGEREORD,D. A. (1960). Chromosome preparations of leukocytes cultured from human peripheral blood. Exp. Cell Res. 20, 613-616.

NICHOLS, W. W., LEVAN, A., AVLA, P., and NO~RBY, E. (1965). Chromosome damage associated with the measles virus in vitro. Hereditas 54, 101-118.

NICHOLS, W. W., LEVAN, A., CORIELL, L. L., GOLDNER, H., and AttLSTROM, C. G. (1964). Chromosome ~tbnormalities in vitro in human leukocytes associated with Schmidt-Ruppin Rous sarcoma virus. Science 146, 248-250. O'NEILL, F. J., and MILES, C. P. (1969). Chrmo-o some changes in human cells induced by herpes simplex, types 1 and 2. Nature (London) 223, 851-852. O'NEILL, F. J., and RAPP, F. (1971). Synergistic effect of herpes simplex virus and cytosine arabinoside on human chromosomes. J. Virol., in press. PRVSOFF, W. It. (1967). Recent advances in chemotherapy of viral diseases. Pharmaeol. Rev. 19, 209-250. RAPP, F. (1963). Variants of herpes simplex virus: isolation, characterization, and factors influencing plaque formation. J. Bacteriol. 86, 985991. RAPt,, F., and HstL T. C. (1965). Viruses and mammalian chromosomes. IV. Replication of herpes simplex virus in diploid Chinese hamster cells. Virology 25,401-411. ROIZMAN, B., AURELIAN, L., and RO&NE, P. R. (1963). The multiplication of herpes simplex virus. I. The programming of viral DNA duplication in HEp-2 cells. Virology 21, 482498.

SPIERS, A. S. D., and BAIKIE, A. G. (1967). Reticulum cell sarcoma. Demonstration of chromosomal changes analogous to those in SV40 transformed cells. Brit. J. Cancer 21,679-683. STICH, H. F., and YOHN, D. S. (1965). Viruses and mammalian chromosomes. V. Chromosome aberrations in tumors of Syrian hamsters induced by adenovirus type 12. J. Nat. Cancer Inst. 35, 603-615. STICH, ~-I. F., Hsu, T. C., and RAPe, F. (1964). Viruses and mammalian chromosomes. I. Localization of chromosome aberrations after infection with herpes simplex virus. Virology 22, 439-445. STOLZ, D. B., STICH, It. F., and YOHN, D. S. (1967). Viruses and mammalian chromosomes. VII. The persistence of chromosomal instability in regenerating, transplanted and cultured neoplasms induced by human adenovirus type 12 in Syrian hamsters. Cancer Res. 27,587-598. STROHL, W. A. (1969). The response of BHK21 cells to infection with type 12 adenovirus. I. Cell killing and T antigen synthesis as correlated virM genome functions. Virology 39, 642-652.

HSV REPLICATION AND CHROMOSOME DAMAGE WAUBKE, 1:~., ZUR HAUSEN, H., and I-IENLE, W. (1968). Chromosomal and autoradiographic studies of cells infected with herpes simplex virus. J. Virol. 2, 1047-1054. WOLMAN, S. R., HIRSCHHORN,K., and TOD&RO, G. J. (1964). Early chromosomal changes in SV40-infected human fibroblast cultures. Cytogenetics 3, 45-61. YERGANIAN, G., SHEIN, H. M., and ENDERS, J. F. (1962). Chromosomal disturbances observed in

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human fetal renal cells transformed in vitro by simian virus 40 and carried in culture. Cytogenetics 1, 314-324. zu~ I~IAUSEN, H. (1967). Induction of specific chromosomal aberrations by adenovirus type 12 in human embryonic kidney cells. J. Virol. 1, 1174-1185. ZUR HAUSEN, H. (1968). Chromosomal aberrations and cloning efficiency in adenovirus type 12infected hamster cells. J. Virol. 2, 915-917.