164 BIOCHIMICA ET BIOPHYSICA ACTA

S T U D Y OF SOME B I O C H E M I C A L C H A N G E S R E L A T E D TO

P O L I O V I R U S M U L T I P L I C A T I O N I N H E L A CELLS

II. RELATION B E T W E E N T H E KINETICS OF

POLIOVIRUS MULTIPLICATION, CERTAIN BIOCHEMICAL CHANGES

AND T H E EFFECT OF CHLORAMPHENICOL ON MULTIPLICATION

GUILLERMO CONTRERAS, ADELA OHLBAUM AND JOSbL TOHA

Instituto Bacterioldgico de Chile and [nstituto de Fisica y Matemdticas, Universidad de Chile,

Santiago (Chile)

(Received July 3oth, 196o)

SUMMARY

I. The kinetics of poliovirus multiplication in suspended Hela cells show an eclipse period of about 9 ° rain, a latent period of I oo rain and next a multiplication period during which the amount of virus increases, reaching a maximum about 6 h after infection.

2. There is a direct relation between the amount of eclipsed virus, VE, to that of produced virus, VP, of the form: VE ~ = VP.

3. The amount of eclipsed virus, VE, is also directly related to the early incre- ments in specific act ivi ty of ribonucleic acid, Ai, their relation being of the form: VE = Ai ~.

4. The 280/260 O.D. ratio of the infected ribonucleic acid decreases at 19o min approximating the value for poliovirus ribonucleic acid.

5. The inhibitory action of chloramphenicol on one cycle of poliovirus multi- plication is observed in the decreased amount of eclipsed virus, produced virus and the increment of specific activity of ribonucleic acid. Apparently the relations described above are also valid for the system under the influence of the antibiotic.

INTRODUCTION

There are reports about the kinetics of the multiplication of poliovirus and about some biochemical changes related to it 1, 3; but the information about the first 6o-12o min is lacking. The s tudy of the incorporation of 3~p into proteins and nucleic acids in Hela cells infected with poliovirus showed that there were several changes during the first 60 rain of the growth cyclO. I t was then decided to run such experiments, i.e., omitting the usual washes of excess virus, in the assumption that the kinetics of poliovirus could be investigated even in the presence of a rather large amount of the remaining virus, and that the early par t of the curve would be of interest.

Abbreviat ions: PFU, plate forming units; RNA, ribonucleic acid.

Biochim. Biophys. Acta, 47 U96I) I64-t71

BIOCHEMICAL CHANGES AND POLIOVIRUS MULTIPLICATION 165

The incorporation of the isotope into proteins and nucleic acids are described in the preceding paper 4. This report describes the kinetics of one complete growth cycle of poliovirus in suspended Hela cells, the relation of it with some biochemical changes and the inhibitory action of chloramphenicol over the multiplication of the virus.

MATERIALS AND METHODS

Spinner cultures of Hela cells and poliovirus type I have been used as described s, 4 The technique of preparing the experimental lots including the way of adding chloramphenicol has been described in detail in the preceding paper 4.

Except for one experiment, all the others have been run simultaneously for chemical fractionation and virus determinations. This was done from samples of about i ml that were immediately frozen. The next days, they were thawed and frozen three successive times; centrifuged for 5 min at 60o × g; the supernatant diluted in balanced salt solution; inoculated into bottles with Hela cell monolayer and overlaid with agar and nutrient medium such as described by HSIUNG AND MELNICK s. Accordingly, the virus titers shall be expressed in plaque forming units, PFU/ml. It is important to note that we shall be concerned with the total virus in the system, i.e., that in the medium, plus the intracellular one. All the virus aliquots of one or two series (chloramphenicol treated and non-treated) of each experiment were t i trated simultaneously and no less than four bottles were used per dilution.

RESULTS

The kinetics of poliovirus multiplication

The curve representing the kinetics of one cycle of poliovirus multiplication is shown in Fig. I. The virus titers, in PFU/ml, of nine experiments have been used,

.•I0 8

>

2~

Vo_

Echpsed V,rus VE ~ ~t

Prod~=,~ V=ru~ Ve=

q b ~ ltipll¢otlon PI ~iod

o , ~ 0 do t4o t~o 3~ ,7o ,~o T i m e ' i n m l n ta s s 3 7 6 8 5 N u m b e r o f exper lments

Fig. I. Kine t i cs of one cycle of po l iovi rus mul- t i p l i c a t i on in suspended He la cells. E a c h po in t r epresen t s the a r i t h m e t i c m e a n in P F U / m l for the n u m b e r of e x p e r i m e n t s shown below the abscissa. The k n o w n inocu la t ed a m o u n t is V o. The m e a n for 70, 13o and I9o min ( i6 deter- mina t ions ) is V 1 and the m a x i m u m t i t e r a t

a b o u t 6 h is Vm.

Biochim. I3iophys. .4 ~ ta, 47 (1961) 164-171

166 G. CONTRERAS, A. OHLBAUM, J. TOHA

the number contributing to each arithmetic mean is indicated below the time scale. There are three quantities of virus in the curve which interest us and we shall name them as follows: inoculated amount, Vo; amount of the latent period, i.e., between 90 and I9O min, V,; and the maximum amount attained around 6 h, Vm. Now, we can state the following facts: (a) There is a sharp decrease in the amount of virus in the first IO min after inoculation, which continues for the next 80 min at a lower speed. The missing virus at 9 ° rain, defined as the ratio : Vo/V~ = VE shall be called, eclipsed virus. (b) There is an horizontal segment of the curve between 9 ° and 19o min, i.e., the amount of virus remains unchanged. (c) After 3 h, the quanti ty of virus begins to increase, first at a slow rate, later, at a very fast rate, reaching a maximum at about 6 h. The increment in virus amount is defined as the ratio Vm/Vz = VP, and shall be called: produced virus.

There seems to be a relation between VE and VP as shown in Table I and Fig. 2, where we have plotted the logarithm of VE 2 on the abscissa and the logarithm of VP

TABLE I

R E L A T I O N B E T W E E N T H E A M O U N T OF E C L I P S E D A N D T H E A M O U N T OF P R O D U C E D V I R U S

Expt. Eclipsed VE 2 Produced X 2 number virus (VE) virus (VP)

25 1.92 3.7 2.52 0.3762 22 2.5 6.25 7.3 o.I 760 19 3.65 I3. 4 18.9 2.2574 13 4.4 I9.4 22.5 0.4948 27 4.7 22.1 23.2 0.0548 17 6.3 39.7 26.6 4.3224 14 8.9 79.2 93.o 2.4o4 ° 12 lo.6 112.o I IO.O o.o357 16 13. 3 177.o 175.o 0.0226 18 14 196.o 184.o 0.7346

IO Expts . lO.8784

0 3 o

2.5

ts

i o 2 3 4 5 6 7 891 t.s 2 LOG2'S VE2" 3

Fig. 2. Direct relation between produced virus, VP, and eclipsed virus, VE. The decimal loga-

r i thms of VP and VE ~ have been plotted.

UJ

154

Fig. 3. Direct relation between VE and the increments of infected R N A specific activity. The increments have been calculated as shown at the bo t tom of Table I I and the squares are

plotted on the abscissa.

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BIOCHEMICAL CHANGES AND POLIOVIRUS MULTIPLICATION 16 7

on the ordinates. The data of ten experiments has been used and it can be noted, that the two phenomena are fitted by a straight line that goes through the origin and has a regression coefficient of 0.96. The chi square test for the ten sets of points show that they do not significantly deviate from the straight line that fits them, indicating that there may be a relation of the form: VP = VE 2, between the two phenomena.

Relation between the increment of specific activity of RNA and the amount of eclipsed uir~ls

In the system described one would expect to find some relation between the changes observed in the chemical species described in the accompanying paper 4 and the kinetics of virus multiplication.

The relation between the increments of specific activity of ribonucleic acid, RNA, in the infected series within the first 9 ° rain and the amount of VE for nine experiments is shown in Table II and Fig. 3. The increments of the infected series, 2i, calculated as shown at the bottom of Table II, for each experiment, are presented in the first column, next are the squares of the increments, and it can be seen that they are very similar

T A B L E I I

RELATION BETWEEN THE INCREMENTS IN SPECIFIC ACTIVITY OF RIBONUCLE1C ACID AND THE AMOUNT OF ECLIPSED VIRUS

I n c r e m e n t of spec i f ic a c t i v i t y : A = speci f ic a c t i v i t y a t Tt . I n c r e m e n t of speci f ic a c t i v i t y of spec i f ic a c t i v i t y a t To

i n f e c t e d se r ies i n f e c t e d se r ies : A i =

/] n o n i n f e c t e d series"

Expt. Increment, A i Eclipsed virus Xz in specific activity Ai 2

number of RNA* (VE)

22 1.42 2.02 2. 5 o ,922 25 1.487 2.22 1.92 0 .0405 19 2.15 4.6 3.65 o.1963 27 2.42 5.9 4.7 o .3o64 14 2 .739 7.5 8.9 o .2613 17 3.1o8 9.6 6.3 1.7285 I I 3-34 11.2 lO. 5 0 .0467 18 3 .618 13.2 14 0 .0457 16 4 .1377 17.2 13. 3 1.1436

9 E x p t s . 3.8612

* Speci f ic a c t i v i t y c o u n t s / m i n / p g R N A .

to the amount of VE. The direct relation between these two phenomena is plotted in Fig. 3, where we have A~ on the abscissae and the amount of VE, on the ordinates. I t can be seen that the two set of values are fitted by a straight line that goes through the origin and has a regression coefficient of o.85. In this case also the chi square test for the nine set of points shows that they do not deviate significantly from the straight line that fits them, indicating that there may be a relation of the form: VE = Ai 2, between the two phenomena.

B i o c h i m . B i o p h y s . . 4 e t a , 47 (196I) I64-171

168 G. CONTRERAS, A. OHLBAUM, J. TOHA

Comparison of the O.D. ratios of RNA in the infected and non-infected series

I t the two phenomena described above are related, one would expect to find some structural changes in the RNA of the infected series, that could be reflected in some alteration of the O.D. ratios of this substance.

Opt ica l Density Ratios

260 260 260

.ggo • .730 .410 t

.700 .360 .930 t ~

• ..._ ~ . / ~/'~"'°~l ..&" . . . . . . . . . . .

650 310 " °"""" "" " ' i . . . • . . . . , , o . . ......... . . . \ / ; \

, \ /,, J ~ / I

/ I I . . . . . . . . 1 5 o , -

, I ~Vi . . . . . w ~ - I ~ .7go .~so4 . 2 1 o - I ~ t / r ,

J 4 X / o o l ~ !,.&" t ] ~ '"°r: ] J e<ooi .o 2go J ~ . . ~ • = _ _ _ ~-~- . ~.

~b io ,.~o do ~;o 3 ; o 3~o Time in Minutes

Fig. 4- Changes in the O.D. ratios of RNA during the observat ion period. The horizontal bars describe the absence of significant variat ion in the non-infected cells. The change observed a t

19o mid for 28o/26o and 29o/26o ratios is significant for both.

The RNA ratios: 25o/26o; 280/260 and 290/260 at pH 7 for the infected and the control series are plotted in Fig. 4. In the non-infected series, the mean values for these ratios were: 882; 647 and 309 respectively, during the 6-h observation period; and none of the observed values show a significant deviation from their respective mean.

As shown in Fig. 4 the only statistically significant changes observed between the two series are in the 28o/26o and 290/260 ratios at 19o miD.

Since the mononucleotide composition of the RNA of Hela cells and poliovirus is known1,% and the absorbancy of each mononucleotide at pH 7 is also known 7, it is possible to calculate the mean ratio for the two RNAs. In Table I I I we can see the

T A B L E I I I

28o THE O B S E R V E D - O.D. RATIO COMPARED

260 TO THE ONE CALCULATED FOR HELA CELLS AND POLIOVIRUS

The nucleotide composi t ion of Hela cells and poliovirus such as given by other au thors 1,8 has been multiplied by their respective rat ios at pH 7 (see ref. 7), added and divided by four.

Ccdcula~ed Observed

Hela cells x o.589 Non infected o.647 Poliovirus 6 o.499 Infected o.555 Difference 9o 92

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BIOCHEMICAL CHANGES AND POLIOVIRUS M U L T I P L I C A T I O N 16 9

observed value of o.647 and o.555 for the non-infected and the infected series at 19o min, respectively compared to the calculated values for the cells and the virus. It is remarkable that in both cases the 280/260 ratio of the virus (or the infected series) is lower in the same proportion.

The e~ect of chloramphenicol on the kinetics of poliovirus multiplication

This antibiotic could have an influence on the multiplication of poliovirus as well as the effect on the proteins and the nucleic acids, described in the accompanying paper.

If the amount of virus in the chloramphenicol treated and in the non-treated series are compared, their ratios can be defined as follows:

P F U in ch loramphenico l series x IOO

P F U in non- treated series

The percentage ratio observed in six experiments, are shown in Table IV. It can be seen that it is close to IOO at IO rain; the ratio increases significantly at 4o min, and the amount of virus in the chloramphenicol series continues to be comparatively higher at 70 and 13o min. The ratio was significantly lower when the production of virus started in the non-treated series, and during the remaining hours of the observa- tion period; in spite of the fact, that some virus was being produced in the treated cells.

T A B L E I V

PERCENTAGE RATIO OF VIRUS TITERS IN CHLORAMPHENICOL-TREATED OVER NON-TREATED SERIES

Time Number of Probablility rain experiments Percentage ratio* (t test)

IO 5 95 ~Z 8"5 4 ° 5 137 -~ 6.8 P ~ o .o i 7 ° 2 I t i

I 3o 3 lO8 250 3 57 31o 6 29 i .5.3 P ~ o .o i 37 ° 5 26 ~ 9.7 P ~ o .o i 43 ° 6 31 ~_ 8. 5 P ~ o .o i

* The percentage rat io has been calculated as fol lows: P F U ch loramphenico l - t reated × too

P F U non- treated

T A B L E V

RELATIONS BETWEEN INCREMENTS IN SPECIFIC-ACTIVITY OF R N A , ECLIPSED AND PRODUCED VIRUS IN THE CHLORAMPHENICOL TREATED SERIES

E x p t . number

Increment in specific activity A mount o] of R N A " A mount of eclipsed virus produced virus

A i .~i ~ V E V E z V P

22 1.39 i .94 1.6 2.56 3.6 25 1.34 1.79 1.89 3.57 2.4 19 1.39 1.94 2.45 6.o 5.6 27 1.95 3-8o 3.56 12.7 11.9

B i o c h i r n . B i o p h y s . . 4 c t a , 47 ( t 9 6 I ) 1 6 4 - I 7 ~

17o G. CONTRERAS, A. OHLBAUM, J. TOHA

The other effects of chloramphenicol can be briefly stated as follows: (a) The kinetics of multiplication presented in Fig. i seems to be substantially modified; but until more data are available, the changes cannot be satisfactorily analyzed. (b) The relation : VE 2 = VP, between eclipsed and produced virus seems to be also applicable to the chloramphenicol series as shown on Table V. (c) The same is true of the relation : A i 2 = VE, between the increments in specific activity, of RNA and the amount of eclipsed virus, also shown on Table V. (d) The O.D. ratios of the RNA in the chloram- phenicol series did not change significantly within the 2 h studied. (e) There was no differentiation in the dose-effect relation when 15o or 300 t*g/ml of the antibiotic were used.

DISCUSSION

The concept of eclipsed virus arose from the work with bacteriophage, when investi- gators were surprised that no virus could be recovered for several minutes after a bacteria had been infected with virus particles. Such a phenomenon has never been clearly demonstrated within the animal viruses, though it was assumed it would be present in some systems at least.

The data about the kinetics of poliovirus multiplication shown in Fig. I indicates that, in the conditions described, where the usual washings to remove excess virus have been omitted, the amount of virus which is missing at the first io min and which continues to decrease for the next 8o min is comparable to what has been described as eclipsed bacteriophage.

I t seems that the decrease in the total virus amount is not due to inactivation by some non specific factor, (serum, cellular debris, etc.) because it would be rather difficult to understand why there is a direct relation between non-specific virus inactivation and virus produced. I t looks, rather, as if the virus eclipsed in the initial 9 ° min after inoculation goes through some active process of specific degradation necessary for the subsequent replication of its RNA. This process, possibly requires enzymes or other factors that are needed to remove the protein coat from the RNA core of the virus particle, or to produce some unfolding or fragmentation of the viral RNA, (primer, as described by BOLLUMS). The hypothesis that the virus is eclipsed is further substantiated by the two relations described which directly relate the amount of eclipsed virus, VE, at the beginning of the growth cycle with the amount of newly released virus, VP, and with the increments of the specific activity of the RNA during the first 7o min. Perhaps the failure of other workers 1, 2 to find such a relation is due to the fact that they have looked for biochemical changes in the system too late, when the synthetic capacity of the cell has been already seriously damaged by the virus. I t is rather difficult to imagine that the cell has the ability to produce the RNA needed for virus replication in excess of the production of its own RNA, specially in this system, which is known to end with cell destruction as a result of virus multi- plication.

The time sequence observed in the system studied seems to correspond with the assumption that some biochemical changes observed are related to virus multiplication. If the amount of virus begins to increase at 19o min, the process in the RNA concerned with virus multiplication should be looked for earlier. I t seems just right that the increase in specific activity of RNA is observed at 7 ° min, i . e . at a time when the synthetic capacity of the cell is still able to turnover more RNA than the one it needs ;

Biochinl. Biophys..4cta, 47 (196~) 164 I7I

BIOCHEMICAL CHANGES AND POLIOVIRUS MULTIPLICATION 171

tha t enough R N A of a different composi t ion accumula tes in the cells at about 19o min

to give an O.D. ra t io similar to the virus one; and tha t this par t icu lar R N A is later

being lost f rom the cells as shows the significant decrease of R N A after 19o min.

The modificat ions of the growth curve of poliovirus produced by chloram-

phenicol are observed in one cycle of virus mul t ip l ica t ion . In the early l i tera ture no

effect was obta ined when the ant ib iot ic was given to mice inocula ted with Lansing

and Y-SK polioviruses 9. The informat ion about the act ion of this and other anti-

biotics rules out a direct act ion over the polioviruses.

The mechan ism of the modif icat ions observed has to be looked for in the more

subtle a l terat ions of the protein and the nucleic metabol i sm of the infected cell such

as described in the accompanying paper.

ACKNOWLEDGEMENT

The technical assistance of Miss Y. MIRANDA is grateful ly acknowledged.

REFERENCES

1 W. W. ACKERMANN, P. C. LOH AND F. E. PAYNE, Virology, 7 (1959) 17o. 2 N. P. SALZMAN, R. Z. LOCKART, JR. AND E. D. SEBRING, Virology, 9 (1959) 244. 3 G. CONTRERAS, J. TOH/, AND A. OHLBAUM, Biochim. Biophys. Acta, 35 (1959) 268. 4 j. ToHfik, O. CONTRERAS AND A. OHLBAUM, 13iochim. Biophys. Acta, 47 (1961) 158.

G. D. HSlUNG AND J. L. MELNICK, J. Immunol., 78 (1957) 128. 6 F . L . SCAFFER, H. FISCHER AND C. E. SCHWERDT, Virology, IO (196o) 53 o. 7 E. VOLKIN AND W. E. COHN, in D. GLICK, Methods o] Biochemical Analysis, Vol. I, Interscience

Publishers, Inc., N. York, 1954, p. 304 . s F. J. BOLLUM, J. Biol. Chem., 234 (1959) 2733. 9 I. V. MCLEAN, JR., J. L. SCHWAB, A. ]3. ~[{ILLEGAS AND A. S. SCHLINQMAN, J. Clin. Invest.,

28 (I949) 953.

Biochim. Biophys. Acta, 47 (I96I) 164-171