changes in nucleic acid content of hela cells infected with herpes virus

VIROLOGY 5,549-560 (1958)

Changes in Nucleic Acid Content of Hela Cells Infected with Herpes Virus’

ALISON NEWTONS AND M. G. P. STOKER

Department of Pathology, Cambridge University, England

Accepted March 11, 1958

The nucleic acid content of HeLa cells was determined at various times after exposure to a high multiplicity of herpes virus. The DNA content rose 6 to 9 hours after infection, before any increase in infective virus, which was just detectable at 12 hours. By 72 hours the cells contained nearly double their normal content of DNA. This increase in DNA was confined to the nucleus. No significant change in RNA content was observed.

INTRODUCTION

The propagation of animal viruses in tissue culture has made possible a more accurate study of the dynamics of virus multiplication in infected cells, and growth cycles have now been described for many virus-cell systems. There are, however, few reports of simultaneous studies on the chemical changes produced, except for those dependent on cytochemical techniques.

Studies on growth of herpes virus have been carried out in cultured rabbit cornea1 cells by Scott et a&. (1953). Feulgen-positive material, presumably deoxyribonucleic acid (DNA), accumulated in the nucleus during the early stages of infection, but the fully formed eosinophilic inclusion body was apparently Feulgen-negative (see also LBpine and Sautter, 1946). Ackermann and Francis (1950) have investigated the changes in gross chemical composition; including nucleic acid, of the organs of chicks infected with herpes virus. They found a rise in both DNA and ribonucleic acid (RNA) ; such observations are, however difficult to relate directly to the cycle of virus multiplication.

As part of a general investigation of herpes virus multiplication in

1 This work was carried out with financial support from the Medical Research Council.

2 Holder of the Benn Levy Studentship in Biochemistry in the University of Cambridge during part of the study.

549

550 NEWTON AND STOKER

HeLa cells, the nucleic acid content of the cells has been studied by direct chemical analysis at successive stages in the growth cycle, and the changes found are reported in this paper. A more detailed analysis of the growth cycle (M. G. P. Stoker and R. W. Ross, unpublished) and a study by cytochemical and fluorescent antibody techniques (Ross and Orlans, 1958) will be reported elsewhere.

MATERIALS AND METHODS

Ce2Z.s. Cultures of human cervical carcinoma cells (strain HeLa) were maintained in this laboratory by culture on glass; subcultures were made after suspension in 1: 20,000 sodium Versenate (Na ethylenediamine- tetraacetic acid solution), in phosphate-buffered saline (PBS) free of Ca and Mg (Dulbecco and Vogt, 1954). Continuous sheets of healthy cells, grown for 2-3 days in loo-ml or 200-ml babies’ feeding bottles were used for experiments.

Culture medium. The normal growth medium contained 20% human serum, 0.5 % la&albumin hydrolyzate, 80% Gey’s (1949) saline solution; 0.002 % phenol red, 100 units penicillin, and 100 1.18 streptomycin per milliliter were also added.

Before inoculation with virus the above medium was replaced by maintenance medium, which was similar to the growth medium except that it contained 5 % rabbit serum and additional saline in place of human serum.

Cell counts. Cell numbers were determined in a hemocytometer after dispersal of the cells in sodium Versenate to remove clumps. A total of at least 600 cells was counted in twelve l-mm squares. A variance of about 12 % was found in those counts which were analyzed.

Virus strai?z. The Melbourne egg-and-mouse-adapted line of the HF strain (HFEM) was used and was kindly supplied by Dr. P. Wildy. It was passed repeatedly in HeLa cells in this laboratory. Stock virus suspension consisted of culture media from HeLa cells infected 34 days previously with the eighth or higher passage of the virus. This medium was centrifuged to remove debris and the supernatant fluid was stored in small volumes at -70”.

Virus Assay EXPERIMENTAL

Even after thirty passages in HeLa cells the infectivity of the HFEM strain for the chick chorioallantoic membrane (CAM) remained more

NUCLEIC! ACID CONTENT OF HERPES INFECTED HELA CELLS 551

than ten times the infectivity for HeLa cells. Virus development was, therefore, studied by the pock-counting technique using a modification of the method of Nadeje el al. (1955). Pocks on four or more membranes were counted per sample, and dilutions giving an average of between 10 and 150 pocks per membrane were used (Stoker and Ross, unpublished). The standard error of the counts generally lay between 10 % and 20 %.

Though the more sensitive pock count is satisfactory for investigation of virus development during the growth cycle, it was necessary to ex- press virus input in terms of HeLa cell infectivity. Using a plaque- counting echnique in HeLa cells, A. E. Farnham (unpublished) has found that the plaque-forming titer of the HFEM strain grown in He-La celJs is 7.5 % of the pock-forming titer (egg:HeLa ratio 13: 1). It has also been shown that 80 % of the HeLa plaque-forming units are absorbed in 2 hours from a thin layer of fluid. We can, therefore, deduce that after 2 hours’ exposure 6 % of the total pock-forming virus in the inoculum can successfully initiate infection in the HeLa cells.

Growth Curves

Cell sheets in 100- or 200-ml babies’ feeding bottles were washed with PBS before inoculation of virus suspensions (pock-forming titer about 5 X lo7 per milliliter) in maintenance medium. Three milliliters of inoculum was used for 200-ml bottles containing approximately 2 to 3 million cells and 1.5 ml inoculum for loo-ml bottles with approximately 1 to 2 million cells. After 2 hours for virus adsorption at 37”, the cell sheets were washed five times with PBS, 10 ml of maintenance medium was added, and the bottles replaced at 37”. Since the cells were exposed to between 50 and 75 pock-forming units per cell, it may be deduced that the input after 2 hours’ exposure was 3 to 4.5 HeLa plaque-forming units per cell (see above). Control bottles received identical treatment except that the virus inoculum was replaced by the appropriate amount of maintenance medium.

The cells were harvested at various time intervals after infection, 3 infected and 3 control bottles being used at each point. The medium was taken and kept for virus assay. The cells were then removed from the glass in sodium Versenate solution (1: 20,000) after washing three times in PBS free from Ca and Mg. Cell nmnbers were determined and the nucleic acid content of the cell suspension estimated.

For virus assay an aliquot of the cell suspension from each bottle was pooled and diluted 1: 1000 in maintenance medium. The cells were then


disintegrated by ten cycles of freezing at -70” and thawing at 37”, a procedure which damages the cells but has no effect on free virus (Stoker and Ross, unpublished). The disrupted cell debris and also the pooled culture medium from the same original bottle were then titrated by pock counts on the CAM.

Nucleic acid estimation. Determination of the nucleic acid content of the cells was carried out by a modification of the method described by Ogur and Rosen (1950). Perchloric acid was added to the suspension to give a concentration of 0.2 iV, the whole being kept at 0”. After half an hour the acid-insoluble precipitate was removed by centrifugation. This precipitate was washed twice by resuspension and sedimentation in 0.2 M perchloric acid, the combined supernatants were saved for estimation of acid-soluble nucleotides. The insoluble residue was suspended in 0.5 M perchloric acid and heated at 70” for 20 minutes: the precipitate was removed by centrifugation. This hot acid extraction was repeated twice. The clear supernatant fluids obtained were combined and samples taken for determination of total nucleic acid and DNA. Total nucleic acid was estimated by the absorption of ultraviolet light of wavelength 260 rnp by a diluted aliquot in a silica cell of light path 1 cm using a Unicam spectrophotometer. The concentration of nucleic acid was calculated from this value assuming an absorption of 28 by a 0.1% solution of nucleotides (Gale and Folkes, 1953). The DNA content of a further sample was determined by the calorimetric method of Burton (1956), calibrated on a sample of DNA of known phosphorus content prepared from HeLa cells. Extraction of lipid by 3: 1 alcohol-ethyl ether or by the method of Reichert (1944) did not change the color production by the diphenylamine reagent in normal or infected cells.

The RNA content of the cells could be deduced from the difference between total and DNA contents, values obtained by this method agreed with determinations made on ceIIs after lipid extraction, by the cysteine method described by Dische (1955). The orcinol method could not be used because the presence of large amounts of DNA interfere with the color production (Dische, 1955).

Preparation of cell nuclei. Nuclei were prepared from infected and normal cells by the use of citric acid solutions. Although this method does not give good results for many chemical estimations, it has been recommended for the isolation of nuclei for nucleic acid determination

NUCLEIC ACID CONTENT OF HERPES INFECTEED HGLA CELLS 553

by Duunce (1955). Its use enables quantitative fractionation to be carried out on small samples.

Cells obtained by centrifugation of the suspension in sodium Ver- senate were suspended in 5 ml of 2.5 % citric acid solution and incubated at 37” for 1 hour with frequent shaking. The nuclei were separated by centrifugation at 600 g for 20 minutes. This was repeated twice but with incubation periods of 20 minutes. The combined supernatant fluids were regarded as the “cytoplasmic fraction.” The nuclei obtained were suspended in 2 ml of citric acid solution; this fraction also contained a little cell debris. Nuclear counts were carried out on this suspension in a hemocytometer, using a phase contrast microscope. The nucleic acid content of both fractions was then determined as described.

Nucleic Acid Content of Infected Cells

In all the experiments to be described the amount of nucleic acid found has been related to the cell numbers rather than to the total nucleic acid present in the culture, as it was felt that this gave a more meaningful result for the following reasons. Although the number of cells inoculated into the bottles has been carefully controlled, growth of HeLa cells in quantities adequate for chemical anaIysis is not sufficiently reproducible to give identical cell numbers in each population. Secondly, multiplication of the HeLa cells occurs in the normal and, to some ex- tent, in the infected cell cultures during the course of the experiment, and so changes in the nucleic acid content of the culture due to increas- ing cell numbers might obscure changes brought about by virus infection (see Table I).

The nucleic acid content of normal HeLa cells is close to that reported by others (Leslie et al., 1957), i.e., 17.5 ,ug DNA and 30-50 pg RPU’A per million cells. During the course of the experiment the amount of nucleic acid in the control cells fell slightly (Table 1); as the DNA content remained constant this was presumably due to a fall in the amount of RNA contained. This was possibly due to the fact that the cells were in “maintenance” rather than growth medium.

The total nucleic acid content in the herpes-infected HeLa cells was slightly greater than in control cells, but the differences observed were not significant. However the DNA content of the cells changed markedly after infection, as shown in Fig. 1 (see also Table 1). Nine hours after infection the DNA content was 40% greater than in control cells, and

TABL

E 1

THE

P~‘U

CLE

IC A

CID

C

ON

TEN

T”

OF

CEL

LS

INFE

CTE

D

WIT

H

HER

PES

VIR

US

,vea

n ce

ll no

. To

tal

nu+i

c ac

id

Hou

rs

afte

r (1

0~/t!

ottle

) (p

g/lO

e ce

lls)

DN

A (&

lo6

cells

) D

educ

ed

RN

A (to

tal

- D

NA)

(p

g/lO

e ce

lls)

infe

ctio

n C

b I”

C

I C

I

C

I z 3

0 3.

2 62

.5

f 2.

5 17

.5

f 0.

6 45

zk

3

2 6

2.3

2.8

56

ziz

3.5

59

f 4

16 i

1

20.5

f

1.2

40

* 4

39

f 4

9 3.

5 2.

7 53

f

4 63

i

3.5

17.5

A

2 24

.5

f 1.

3 35

.5

zt

3 38

.5

i 3

24

5 4.

1 3.

1 50

f

2 69

f

5.5

18.5

f

1 25

f

1.5

31.5

*

2 44

i

3 48

4.

1 5.

8 43

f

2 52

f

3 19

i

1.2

27.2

f

3 24

&

3 28

.5&

t 2

72

6.1

4.5

50

* 4

52

f 11

18

zk

2 31

zk

2

31

zk

2 32

f

2 0

96

52

f 2

50

& 5

17.5

f

2 35

.5

f 1.

3 34

.5

r!z

1 15

f

1

a Sh

owin

g st

anda

rd

erro

r. 5

C

= co

ntro

l; I

= in

fect

ed.

NUCLEIC ACID CONTENT OF HFXPES INFECTED HELA CELLS 555

40’ I

36,

32

Hours after infection

FIG. 1. DNA content of HeLa cells after infection with herpes standard error).

virus (with

then, after a stationary period, it continued to rise gradually. During this period the apparent RNA content of the cells as deduced from the difference between total nucleic acid and DNA content, did not differ significantly from the content of control cells. Cells showing gross cytopathic changes 3 or 4 days after infection contain double the DNA content of normal cells and little or no RNA.

Experiments were then carried out to determine the correlation between time of appearance of new virus in the first cycle and this observed increase in DNA synthesis. Associated studies on growth of herpes virus in HeLa cell cultures has repeatedly shown a latent period of more than 9 hours, followed by a rise in infectivity of the cell fraction at 12 hours, and release of virus into the medium after 16 hours. In several experiments.residual virus in the latent period was reduced by antiserum treatment and constituted less than one pock-forming unit per 50 cells?


Meon cell no. IO? bottle

.3 2.0 1.7 1.7 I.8 2.3 1 ) 1 8 n

0 6 12 IS 24 Hours clf:er infection

FIG. 2. Virus and DNA content of HeLa cells after infection with herpes virus. Separate DNA determination on cells from each of three bottles at various time intervals. Virus assay on pooled samples from the same three bottles.

so it was unlikely that it masked an earlier rise of virus (Stoker and Ross, unpublished).

Figure 2 illustrates the results obtained from DNA analysis and virus assay carried out on the same cell population. In this experiment the level of DNA had increased slightly at 6 hours and significantly at 9 hours following exposure of the cells to the virus. New virus was not detectable in the cells until 12 hours after infection, i.e., at least 3 hours after a significant rise in the DNA content of the cells. The release of virus from the cells into the surrounding fluids occurred even later than this. We can therefore conclude that there was a significant increase of DNA during one cycle of virus multiplication.

Nucleic Acid Changes in Cell Nuclei

As multinucleate giant cells can be detected in cultures of HeLa cells about 24 hours after exposure of the cell to herpes virus, it seemed pos-

NUCLEIC ACID CONTENT OF’ HERPES INFECTED HELA CELLS 557

‘Cytoplasm’ infected c4

Hours offer infection

FIG. 3. DNA content of “nuclear” and “cytoplasmic” fractions of HeLa cells after infection with herpes virus.

sible that the results described previously were a result of this formation of giant cells. As there is also some evidence that growth of herpes virus occurs in the cell nucleus, it seemed important to determine whether the increased DNA synthesis observed in infected HeLa cells took place in the cytoplasm or nucleus. For both these reasons, therefore, experiments were performed in which nuclear and ‘~cytop1asmic” fractions were prepared from cell suspensions obtained as in the previous experiments. The results shown in Fig. 3 indicate that the higher DNA content of the infected cell is a result of the increased amount of DNA in the cell nucleus. The small amount of DNA found in the “cytoplasm? fraction, which was probably derived from a small proportion of frag- mented nuclei, shows no significant difference to that of control cells.

DISCUSSION

It appears from these results that HeLa cells producing herpes virus are also synthesizing an excess of DNA. It seems unlikely that these results are due to some systematic error introduced into the cell-counting method. If, for example, many broken and uncounted cells had been contributing to an apparently high DNA content after infection, then this DNA should have appeared in the “cytoplasmic” fraction. Very little DNA was found in this fraction and the amount in the nuclei was


comparable to that found in whole cells. Moreover the numbers of cells obtained from infected cultures increased rather than diminished during the course of the experiment.

The results would be easier to interpret if it were known whether herpes virus contained DNA, or RNA, or both. Cytochemical observations suggest an increase in DNA in the nuclei of infected cells, but it does not necessarily follow that this DNA is all in the virus particles. Indeed, the results reported in this paper show that a surprising excess of DNA is produced by the cell; even if one assumes that the virus con- tains 10% DNA it would need nearly lo6 particles per cell to account for all the surplus DNA at 12 hours, i.e., during the first cycle of infection. The actual number of particles in infected cells is difficult to esti- mate by assay of infectivity, because of incomplete or inactivated virus but in electron micrographs of sectioned HeLa cells, kindly made by Dr. Kenneth Smith, it is very difficult to find recognizable virus particles at this time. This would suggest, either that most of the excess DNA was viral nucleic acid which was not incorporated into particles, or that it was HeLa cell DNA, normal or abnormal, which was produced as an indirect manifestation of virus multiplication.

The increased synthesis of DNA begins in the first cycle of virus multiplication, long before virus is released and several hours before new infective virus appears in the cells; it can also be detected before there is any visible alteration in the cells as observed by histochemical techniques in this laboratory (Ross and Orlans, 1958). Further studies on the release of virus from single cells (Wildy, Stoker, and Ross, unpublished) suggest that the time of release of virus is not uniform even when cells are infected almost simultaneously. If this is so the virus which can first be detected at 12 hours at the end of the apparent latent period comes from a few cells only, the majority of cells having a much longer latent period. Since the method used for DNA analysis is relatively insensitive, any significant change in the level of DNA detected is probably due to an alteration in this majority of the HeLa cell population. Thus the increased DNA synthesis in the cell may represent a relatively early change in the course of virus multiplication.

The excess DNA is located in the nucleus and is presumably the same as the Feulgen-positive material described by others. HeLa cells do not show the typical eosinophilic Feulgen-negative inclusion, found at a late stage in other herpes-infected cells, and this is probably related to the continued presence of excess DNA which is found in the HeLa cell

NUCLEIC ACID CONTENT OF HERPES INFECTED HELA CELLS 559

nucleus. The localization of the excess DKA in the nucleus adds some support to the proposal that this is the site of primary virus synthesis as put forward by Gray and Scott (1954) and supported by electron microscopy (Morgan et al., 1954) and fluorescent antibody st,udies (Lebrun, 1956; Ross and Orlans, 1958).

ACKNOWLEDGMENT

We are very grateful to Mrs. Bridget Cook for her valuable technical assist- ance.

REFERENCES

ACKERMANN, W. W., and FRANCIS, T. (1950). Some biochemical aspects of herpes infection. Proc. Sot. Exptl. Biol. Med. 74, 123-126.

BURTON, K. (1956). A study of the conditions and mechanism of the dipheny- amine reaction for the calorimetric estimation of deoxyribonucleic acid. Bio- them. S. 62, 315-323.

DISCHE, 2. (1955). Color reactions of nucleic acid components. In The Nucleic -4cids (E. Chargaff and J. N. Davidson, eds.), Vol. 1, p. 285. Academic Press, New York.

L)OWVCE. A. L. (1955). The isolation and composition of cell nuclei and nucleoli. In The Nucleic Acids (E. Chargaff and J. N. Davidson, eds.), Vol. 2, p, 93. Academic Press, New York.

DULBECCO, R., and VOGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. S. Exptl. Med. 99, 167-182.

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LEBRUN, J. (1956). Cellular localization of herpes simplex virus by means of fluorescent antibody. Virology 2, 496510.

L&PINE, P., and SAUTTER, V. (1946). Etude histochimique des lesions dues aux ultravirus. Les acides nucleiques. Ann. inst. Pasteur 72, 174-183.

LESLIE, I., FULTON, W. C., and SINCLAIR, R. (1957). The metabolism of human embryonic and malignant cells and their response to insulin. Biochim. et Bio

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MORGAN, C.: ELLISON, S. A., ROSE, H. M., and MOORE, D. H. (1954). Structure and development of viruses as observed in the electron microscope. (1) Herpes simplex virus. S. Exptl. Med. 100, 195-202.

NADEJE, T., TAMM, I., and OVERMAN, J. R. (1955). A new technique for dropping the chorioalIantdic membrane in embryonated chicken eggs. J. Lnb. Clin. Med. 46, 648-650.


OGUR, M., and ROSEN, G. (1950). The nucleic acids of plant tissues. I. The extraction and estimation of desoxypentose nucleic acid and pentose nucleic acid. Arch. Biochem. 25, 262-276.

REICHERT, R. (1944). Determination of the total lipid content of natural products. Helv. Chim. Acta 27, 961-965.

Ross, R. W., and ORLANS, E. (1958). S. Pathol. Bacterial. in press. SCOTT,T. F.McN., BURGOON,~. F., CORIELL, L.L.,and BLANK, H. (1953).The

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changes in nucleic acid content of hela cells infected with herpes virus

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