Correlation between loss of enzymatic activity and of protein from cells infected with pseudorabies virus

736

DISCUSSION

ANI)

1’RELIMINAllY

REPORTS

3. K.\I’L.\N, A. d., IJEN-1’01~ \‘I‘. ‘I’., :tt~tl ti \\IIY 1, T., ;l,nn. LY.lT. .lctrt/. sci. 1:30, wri (l!Ni5). A. S., aud HEN-I’oI~.\ I’, T., ./. ,1/o/. 4. K.wL.\N, Hid. 19, 320 (1966). 5. H.\NN.\, C., and \VILKISSON. K. I’., E.r;si”ll. B!/c ffes. 4, 31 (1965). 6. SMIW, K. O., J. Znlnlltnol. 91, 582 (l!)(Y$). Y. I)EINH.\IWI., F., dnn. X.1’. -ICCLII. Sci. 130, 218 (1965). 8. I(.\(-W\R-, II. E., dnn. -V.l-. .2carl. Sci. 130, 167 (1965). 9. 1’~SOFF, TV. .4., B.\KHLE, Y. S., and SEKELY, I,., .-Inn. .V.Y. ilcarz. sci. 130, 135 (1965). 10. K.\PL.\K, A. S., Vi,olog!/ 4, 135 (195T). 11. K.\PI..\x, A. S., and V.~~rsn. A. Ii:., Iriro/og!/ 7, 394 (1959). 12. E.\GLE, H., Science 140, 432 (1959). A. 8., T’i,olog!/ 13. BEN-PoI~.\~~, T., and K.wI,.\N. 20, 310 (1963).

I ~T”KM

I -DENSITY

GRADIENT-

TOP

FIG. 1. Distribution in C&l gradients of 1)NA extracted from cells incubated with various concentrations of IUdR. RK cells were incubated with Eagle’s medium, plus 3% dialyzed bovine serum, containing per milliliter 0.04 MC of TdR-2-X (specific act,ivity, 0.12 mC/mg) for 3 hours. The cultures were washed and further incubated in Eagle’s medium for 18 hours. Different amounts of IUdR, as well as 1 PC of adenine-3I-I (specific act,ivit,y, 3.5 mC/mg) per milliliter, were added 1.0 the cultures. Twelve hours thereafter the c\dt llres were harvested, and the DNA was extract,ed and centrifuged to equilibrium in a C&l solution (average density, 1.780 g/en?). Samples were collected dropwise, and the radioactivity in the DNA was determined. W-labeled DNA, filled circles; 311-labeled DNA, open circles.

(At these concentrations of IUdR, there is a decrease of about 95 % in virus infectivity without any observable decrease in cell multiplication.) The selective antiviral advantage disappears, however, when higher concentrations of the drug are used. REFERENCES

1. EGGEHS, II.

J., and TAMM, I., ‘inn. ZZezl. Pharmacol. 6, 231 (1966). 2. SMITH, K. O., and DUKES, C. B., J. Inlm?mol. 92, 550 (1964).

Department 0J” Jlicrobiology Research Laboratories, Albert Einstein Medical Center Philadelphia, Pennsylvania 19141 Alccepfetl February 7, 1967

Correlation Activity Infected

between and

Loss of Enzymatic

of Protein

from

with Pseudorabies

Cells Virus1

It was shown in a previous publication that between approximately 2 and 6 hours after infection of RI? cells with I+ virus, there is an increase in the level of activity of some of the enzymes involved in the synthesis of DNA (1, 2). The level of activity of the enzymes remains constant thercafter until 10 hours after infection, when it decreases rapidly (3). This decrease seemed 1 This investigation was supported by grants from the Nat,ional Institutes of Health (AI-02432 and AI-033G2), and from the Sational Science Foundation (G&1386), and by a IT. S. Public Health Research Career Program Award (5.K341-19,335) from the National 1nstitut.e of Allergy and Infectious I)iseases. 2 Abbreviations: RK, rabbit kidney; IV, pseudorabies; dAMl’, deoxyadeuosine monophosphate; dGMP, deoxyguanosine monophosphate; TMP, deoxyt,hymidine mon0phosphat.e; dCMP, deoxycytidiue monophosphate.

DISCUSSION TABLE ~)ISTRIBUTION

0F

THE

737

REPORTS

1

Enzyme Fraction

PRELIMINARY

I)E~;YYNuc~E~‘I’IDE

IN CEIJ,UL.\R

KINASES

AND

TMP kinase

;f$;

10.2 79.3 1.5

13.6 81.1 5.0

FRACTIONS”

activity

(s) ____

$yJJz

Protein 2;;:;

co?tjYt

-__

Nuclear Soluble Microsomal and

20.5 08.0 11.5

18.1 20.2 3l.i

37 45 18

mito-

chondrinl a RK cells were infected (adsorbed multiplicity = 10 PFU/cell), as described previously (2). Six hours after infection the cells were washed once with cold phosphate-buffered saline, scraped into 0.25 Al sucrose containing CaClz (0.0018 dI), and fract,ionated according to a modification of the method described by Hogeboom (4). The cell suspension (5 X lo6 cells/ml) was homogenized for 2 minutes at 0” in a Potter-Elvehjem Teflon homogenizer. The homogenate was then layered on 0.34 M sucrose containing CaClz (0.0018 Al) and was centrifuged at’ 700 g for 10 minutes. The pellet (nuclear fraction) contained not more than 5(y0 intact cells. The supernatant was centrifuged at 100,000 q for 1 hour. The supernatant obtained after this centrifugation constituted the soluble fract,ion; and the pellet, the microsomal and mitochondrial fraction. Extracts from the fractions were prepared, and activity of the various enzymes in the extracts was determined as described previously (2).

to be correlated wit’h a leakage of proteins from the cells since both events depend on the presence of a protein(s) which is synthesized by the infected cells between 4 and 7 hours after infection (3). However, we were unable to recover most of t’he enzyme activity

from

the

culture

fluids,

and

the

pos-

sibility remained that a mechanism that regulates enzymatic activity and thereby controls certain phases of the infective process was operative in the infected cells. The experiments presented in this paper are an attempt to clarify this question. The rationale of the experiments to be described is as follows : It is well known that different enzymes are bound to cellular structures to varying degrees and that upon disrupt,ion

solubilized,

of

the

cells,

the

enzymes

each to a different

extent.

are

If

0

4 TIME AFTER

6

12 INFECTION

16 (HOURS)

FIG. 1. ActiviQ of deoxynrlcleotide kinases in RK cells at various t,imes after infection with Pr virus. Ctdtures were infect,ed (adsorbed mult,iplicity = 10 PFU/cell). At various times, cells were harvested and the activity of the enaymes present in the cells was assayed, as described previously (2). The values 011 t,he ordinate are for TMP kinase. To obtain i,he correct, values for the other kinases, multiply the orditlate by the factor given next to each kinase.

the decrease in enzyme act,ivity which occurs late in the infective process were due to the leakage of enzyme proteins from the cells, the more soluble enzymes would be expected to be lost at a more rapid rate than those firmly bound to cellular structures. We examined the distribution of various enzymes among the fractions obtained after disrupt,ion of t’he cells and found that a correlation exists bet’ween t’he degree of loss of activity of an enzyme, during the later stages of infection, and the degree to which this erlzyrw is bound t,o cellular structures. In these experiments, infect’ed and noninfected cells were fractionated, according to a modification of the met’hod described by Hogeboom (/t), into nuclear, microsomal plus mitochondrial, and soluble protein fractions. No at,tempt was made to purify the cellular components ; consequently, there was :I cert#ain degret: of cross-contamination bet’ween the fractions. However, for the purpose of the experiments t’o be described, which was to detcrminc whether different enzymes wcrc bound by

73s

DISCUSSION

AND PRELIMIKARP TABLE

DISTRIBUTION

OF EKZYMES

__-.Time after infection (hours)

Nuclear

G 10

2.2 2.8

14

1.4

IN FRACTIONS

I:F:POI
2

OBTAINED FROM CELLS ;ZT VARIOUS TIMIS~

TMP kinaseb

dAMP kinase”

lMicrosoma1 and mitochondrial

Nuclear

Microsomal and mitochondrial

95.0 86.0 103.0

138.0 96.0 92.0

0.84 0.50 0.06

Soluble 8.6 2.2 0.2

.q IJIXI~ INVIS:(:TIOW

Soluble 34.0 46.0 50.0

Protein total bdsample) G.7 6.0 1.4

a For details of experimental procedure see legends to Table 1 and Fig. 1. b Values stated as millimicromoles phosphorylated per milligram of protein per hour in the stated fraction.

cellular structures to varying degrees, clean separation of the cellular components was unnecessary. Table 1 shows t,he distribution of four deoxyribonucleotide kinases among three fractions obtained from infected cells. Whereas only 20% of the dAMP kinase activity was found in the high speed supernatant of the disrupted cells, 68% of the dGMP kinase activity and approximately 80% of the TMP and dCMP kinase activities were found in this fraction. (Essentially the same distribution of these enzymes among the fractions obtained from noninfected cells was observed.) These result’s do not necessarily imply that dAMP kinase, in contrast to the other enzymes, is localized in the nucleus because, under the experimental conditions used, many of the nuclear enzymes might have leaked out. However, these results do show t’hat dhMP kinase is more tightly bound to the nuclear structures than the ot,hcr enzymes. Since this is the case, one would expect dAMP kinasc to leak out of cells at a slower rate than the others, if the decrease in enzymatic activity observed during the late stages of infection is due to leakage of prot,eins from the ceils. That this is the case is shown in l?ig. 1. Further evidence that leakage is responsible for the loss of enzymatic activity from the cells was obtained from experiment’s in which the specific enzymatic nctivit,ies of the nuclear and extranuclear fractions were compared at various times after infect,ion. Table 2 demonstrates that during the late stages of infection, the nuclear fraction possesses a

higher specific enzymatic activity of TMP kinase than the extranuclear fractions, indicating that the enzyme which was more firmly bound to the nuclear st,ructures was less likely to be lost during the late stages of infection. These experiments confirm t’he conclusion drawn previously (3) that the decrease in enzyme activity during the late stages of infection is due to leakage of enzyme proteins from the cell and is probably correlated with the release of mature virus. REFERENCES 1. NOHARA, H., and KAPLAX, A. S., Riochem. Biophys. Res. Commun. 12, 189 (1963). 2. IIA~~.zIM, C., KAMIYA, T., and KAPLAN, A. S., I’iroZog?/ 28, 271 (19GG). 8. KAMIY.\, T., BEPPORAY~, T., and KAPLAN,

4.

A. S., Virologu 26, 577 (1905). G., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. I, pp. l&19. Arademic Press, New York, 1955.

HOGEROOM,

JAMEL.~V ZEMI,.~~ CELT.1 COTO" hBJS:xT $. K.\PL.w Deparlment

0J Jlicrobiolog~~

Research I,abo,atories, Medical

Philadelphiu, Accepted

Albert Einslein

C’enlcr

Pennsylvania ~ebruur~j

7,

19141

1.967

3 Present address: Institute of Virology, Czechoslovak Academy of Sciences, Bratislava. 4 Fellow of the Consejo National de lnvestigaciones Cientificas y TBcnicas, Argentina. Present address: Facullad de Medicina, CBtedra de Microbiologia, Buenos Aires.