JOURNAL OF BACTERIOLOGY, Feb., 1966Copyright © 1966 American Society for Microbiology

Hydrolytic Enzymes in KB Cells Infected withPoliovirus and Herpes Simplex Virus

JOHN F. FLANAGANDuke University Medical Center, Durham, North Carolina

Received for publication 30 September 1965

ABSTRACTFLANAGAN, JOHN F. (Duke University School of Medicine, Durham. N.C.).

Hydrolytic enzymes in KB cells infected with poliovirus and herpes simplex virus.J. Bacteriol. 91:789-797. 1966.-The effect of poliovirus and herpes simplex virusinfection on the activity of five hydrolytic enzymes was studied in tissue culturecells of KB type. During the course of poliovirus infection, the activity of (3-glucu-ronidase, acid protease, acid ribonuclease, acid deoxyribonuclease, and acid phos-phatase in the cytoplasm rose to levels two- to fourfold greater than the activitypresent in the cytoplasm of uninfected cells. The rise in cytoplasmic activity wasaccompanied by a concomitant decrease in enzymatic activity bound to cell par-ticles. Shift of enzymatic activity from the particulate to soluble state was first de-tected at 6 hr after poliovirus infection, coinciding with the appearance of newinfectious particles and virus cytopathic effect. No net synthesis of these enzymesafter poliovirus infection was found. Hydrocortisone added to the culture mediumfailed to affect either the titer of virus produced in the cells or the release of hydro-lytic enzymes from the particulate state. Herpes simplex infection produced minimalalterations in the state of these enzymes in KB cells. It is hypothesized that thebreakdown of lysosomes and release of hydrolytic enzymes accompanying poliovirusinfection is produced by alterations in cell membrane permeability during thecourse of virus replication and by the consequent change in the ionic content of thecell sap.

Despite the collection of much information onthe biosynthetic events induced by virus infectionof susceptible mammalian cells (11, 14, 15, 26,27), the mechanisms of host injury by viruses areto a large extent unknown. Evidence has recentlybeen presented that poliovirus (16), pseudorabies(4), and adenovirus (Levine and Ginsberg, Fed-eration Proc., 24:597, 1965) infection of tissueculture cells suppresses host messenger ribonu-cleic acid (RNA) synthesis and expression. Thedata suggest that this suppression is produced byaccumulation of virus-specific inhibitory proteinsin the parasitized cell. Diversion of substrate fromthe synthesis of cellular components into virus-directed synthesis has been proposed as a mech-anism of virus injury in T-even bacteriophageinfection (5) and in adenovirus infection (12) ofappropriate host cells. Direct evidence that cellstarvation by such a mechanism is an importantfactor in injury by viruses has not been obtainedyet. The demonstration that infection of Escher-ichia coli by T2, T5, and lambda bacteriophagesis accompanied by severe degradation of deoxy-

ribonucleic acid (DNA) and marked increases innuclease activity within infected cells (17, 29, 30)has raised the possibility that destruction of hostmacromolecules may be an important mechanismof virus damage to host cells.The work described in this report was under-

taken to examine the possible role of hydrolyticenzymes in virus injury to mammalian cells. Asshown by deDuve (7, 8) and others (22, 23) innumerous studies during the past 10 years, thehydrolytic enzymes of mammalian cells are con-centrated in single membrane-enclosed particleswithin the cytoplasm, which deDuve termed"lysosomes." In virtually all of the cells examined,these particles sedimented with a centrifugal forceslightly greater than that necessary to sedimentmitochondria. By cytochemical study, these par-ticles appeared to be exclusively in the cytoplasm(23), whereas cell fractionation procedures con-sistently yielded a portion of the hydrolytic en-zyme activity in the nuclear fraction. Release ofbound enzymes from these structures was shownto be an accompaniment of cell injury from anoxia

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(2), vitamin A toxicity (21), and disturbance inosmotic -or ionic balance (18). Increased fragilityof these particles has been demonstrated in ascitestumor cells exposed to antibody and in certainautoimmune disease states of man (31).

This report describes studies on alterations inactivity of five lysosomal enzymes during virusreplication in KB cells. The enzymes assayed inthese experiments were: acid ribonuclease, aciddeoxyribonuclease, acid phosphatase, acid pro-tease, and /3-glucuronidase. Two viruses wereemployed in these studies for purposes of com-parison: poliovirus, which produces rapid andprofound morphological and chemical changes inthe infected cells, and herpes simplex virus, whichshows less dramatic effect on the host cell as aresult of replication.

MATERIALS AND METHODSTissue culture. These experiments were performed

with monolayer cultures of KB epithelial cells main-tained in Eagle's minimal essential medium supple-mented with 7.5% calf serum. The control and in-fected cells for enzyme assay were grown in 32-ozDuraglas prescription bottles containing at maturity35 X 108 to 45 X 10 cells per bottle. Repetitiveculturing of these cells revealed no pleuropneumonia-like organisms.

Virus and virus infection. The poliovirus was type I,Cox attentuated strain, obtained from Lederle Labora-tories, Pearl River, N.Y. The herpes simplex viruswas a prototype strain isolated by Suydam Osterhout,and was tested for purity against several referenceantisera. Poliovirus pools were prepared by infectingbottle cultures with 2 X 108 plaque-forming units(PFU) per bottle. The cultures were harvested 48 hrlater and clarified by centrifugation for 10 min at10,000 X g. Titers on the supematant virus suspensionwere 107-5 to 108 PFU per ml. Herpesvirus was grownunder similar conditions with the use of 2 X 108'6TCD5o of virus as determined by tube culture assay(13). The cells were harvested 30 to 40 hr after inocu-lation and were centrifuged for 10 min at 1,500 Xg. The infected cells were suspended in one-half thevolume of original medium and homogenized attop speed in a Servall omnimixer. Titers of this virusby tube culture assay were 10"-1 to 107 TcD5o per ml.Infectivity of the herpes preparations was stable onstorage at -70 C.

Cell fractionation. Tissue culture cells were infectedwith either 20 PFU of poliovirus type I or 8 TcDBo ofherpes simplex virus per cell. The virus inoculumwas removed 2 hr later by decanting, the cell sheetswere washed three times with Hanks solution, andEagle's medium supplemented with 7.5% calf serumwas replaced on the cells. The cultures were incubatedat 36 C for the selected time, removed from the glasswith a rubber policeman, and submitted to the frac-tionation procedure described in Fig. 1. Duplicatecultures of control uninfected cells were submitted tothe same procedures with each experiment. Afterpreparation of the pelleted fractions from the cells,

these fractions were suspended in 2 ml of distilledwater, frozen and thawed eight times, diluted with anequal volume of 0.5 M sucrose plus 0.002 M ethylene-diaminetetraacetate (EDTA), and centrifuged at100,000 X g for 1 hr. Enzyme assays were performedon the supematant fractions from this final centrifuga-tion.Enzyme studies. Enzymatic activity of the unin-

fected cells was assayed in citrate, acetate, glycine,and tris(hydroxymethyl)aminomethane (Tris)-male-ate, or Tris-hydrochloride buffers through a widepH range. The ionic composition of the buffer hadno measurable effect on activity of these enzymes;activity of these enzymes was altered by changes inpH, not by change in buffer composition. After opti-mal pH was determined, all enzyme studies were per-formed at 37 C under the standard conditions outlinedbelow.

Acid phosphatase was assayed by incubating amixture of 0.5 ml of enzyme preparation with 0.5 mlof 0.5 M acetate buffer (pH 4) containing 0.1 M P-glycerophosphate. The reaction was stopped after 1 hrat 37 C by the addition of 0.5 ml of 15% trichloro-acetic acid. The inorganic phosphate released by en-zymatic action was measured by the method of Fiskeand SubbaRow (10). Activity was expressed asmicrograms of inorganic phosphate produced perhour per milligram of enzyme protein.

,6-Glucuronidase was assayed in a mixture of 0.5ml of enzyme preparation, 0.4 ml of 0.2 M acetatebuffer (pH 4.5), and 0.1 ml of 0.01 M phenolphthaleinglucuronide. After incubation for 1 hr at 37 C, 1 mlof 10% trichloroacetic acid was added, the precipitatewas removed by centrifugation, and free phenolph-thalein in the supernatant fluid was measured by themethod of Fishman et al. (9). Activity was expressedas the number of micrograms of phenolphthaleinproduced per hour per milligram of enzyme protein.

Acid ribonuclease was determined by incubating0.2 ml of enzyme preparation with 0.7 ml of sodiumcacodylate at pH 6.0 and 0.1 ml of 1% yeast RNA(Worthington Biochemical Corp., Freehold, N.J.).The RNA had previously been precipitated twicefrom phosphate-buffered saline with 2 volumes ofabsolute ethyl alcohol at -20 C. The reaction wasstopped after 30 min of incubation at 37 C with theaddition of 1 ml of 6% HClO4 containing 0.25%uranyl acetate. The precipitate was removed bycentrifugation, and optical density of the supernatantfluid was measured at 260 mu in a B an DUspectrophotometer. The results were expressie asincrements in optical density units in the acid-sdublesupematant fluid per milligram of enzyme protein.

Acid deoxyribonuclease was assayed by mixing 0.5ml of enzyme preparation with 0.4 ml of 0.1 M sodiumcacodylate (pH 5.5) and 0.1 ml of 0.5% thymus DNA(Worthington Biochemical Corp.) suspended in 0.01M sodium chloride. After 1 hr at 37 C, the reactionwas stopped by addition of 1 ml of6% perchloric acidcontaining 0.25% uranyl acetate. The precipitatewas discarded after centrifugation, and optical deintyin the supematant fluid was measured at 260 nmI.Activity was expressed as the increase in optical

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Infected or control cells scraped from monolayer cultures

Cells and medium centrifuged for 10 min at 1,000 X g

Cell pellet washed twice in cold phosphate-buffered saline

Sample removed for cell counting 4

Cell pellet suspended in 2.5 ml of 0.25 M sucrose +0.001 M EDTA (pH 7)

Supernatant fluid

I 1Supernatant fractions pooled

Centrifuged at 15,000 X g for 30 min

Supernatant fraction

Centrifuged 100,000 X g for 1 hr

ISupernatant fluid: cell sap

Cells submitted to gentle homogenization in motor-driven glass grinder for 2 min at 4 C

Centrifuged at 2,500 X g for 10 min

Pellet resuspended in 0.25 M sucrose +0.001 M EDTA at pH 7

Homogenization cycle repeated for total ofthree times

ICentrifuged at 2,500 X g for 10 min

Pellet: nucleic and unbroken cells

- Pellet: lysosomes + mitochondria

- Pell cPellet: microsomes

FIG. 1. Method for preparing lysosome-rich fraction from KB cells.

density in the supematant fluid per milligram of en-

zyme protein.Protease determinations were performed by mixing

0.5 ml of enzyme preparation with 0.5 ml of 6% di-alyzed hemoglobin and 0.5 ml of 0.2 M glycine buffer(pH 3.0). The mixture was incubated at 37 C for 1 hr.The reaction was stopped with 1.5 ml of 10% tri-chloroacetic acid. The amount of acid-soluble tyrosineliberated from hemoglobin into the acid-soluble super-natant per milligram of protein was determined witha tyrosine standard for comparison.

Suitable zero-time controls and incubated blankswere prepared with all the enzyme studies describedabove by precipitating complete mixtures of enzyme,

buffer, and substrate prior to incubation. Enzymaticactivity was computed by measuring the differencesin colormetric or spectrophotometric readings be-tween the zero-time control and the incubated speci-mens.

Substrate concentrations for all enzymatic deter-minations were at a level providing linear enzymeactivity for at least 2 hr of total incubation time underthese experimental conditions.

RESULTS

Determination ofpH optimum for the five hy-drolytic enzymes in uninfected cells. As shown in

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4pH

FIG. 2. Determination ofpH optimum for five lysosomal enzymes. The pH range used for testing ribonucleaseactivity was the same as that used for deoxyribonuclease. The range used for testing activity of acid phosphatasewas identical with that used for testing 3-glucuronidase.

TABLE 1. Distribution ofacid hydrolases in uninfected KB cells

Specific activity of enzymes

FractionPhosphatase Ribonuclease P-Glucuronidase Protease Dxb

Cel sap.8.39 0.41 3.47 2.50 0.124Lysosomes + mitochondria 31.1 3.01 37.0 33.1 0.812Microsomes.......... .... 8.59 0.75 12.3 4.90 0.337Nuclei. 5.24 0.75 6.51 7.75 0.180

* Activity per milligram of protein. Phosphatase activity expressed as micrograms of inorganicphosphate released per hour per milligram of protein; ribonuclease activity as increase in optical den-sity at 260 m,u of acid-soluble supernatant fluid per 0.5 hr per milligram of protein; ,B-glucuronidase ac-tivity as micrograms of phenolphthalein released per hour per milligram of protein; protease activityas micrograms of tyrosine released in acid-soluble supernatant fluid per hour per milligram of protein;deoxyribonuclease activity as increase in optical density at 260 m,u of acid-soluble supernatant fluid perhour per milligram of protein.

Fig. 2, the pH optimum for all enzymes was de-cidedly in the acid range. Despite pH optimaranging from 2.5 to 6, some of these enzymes,notably ribonuclease and deoxyribonuclease,showed appreciable activity at neutral pH. Ribo-nuclease obtained from the various particulatefractions was compared with unbound ribonu-clease of the cell supernatant fraction in respectto heat stability, pH, and temperature optimum.Enzyme preparations from all the particulate andsupernatant fractions showed identical reactions

to these variations in experimental cosuggesting that only one species of thiswas present in KB cells.

Distribution of hydrolytic enzymes in wwfecte4cells. Tables 1 and 2 show the geometric, an results of seven separate studies on the amount ofenzyme activity associated with the varifodt"011fractions. It is evident that for all five eactivity is concentrated in the frction tat 15,000 X g for 30 min (the "lysosona1" r-tion). On the basis of activity per mi am of

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793HYDROLYTIC ENZYMES IN VIRUS-INFECTED KB CELLS

TABLE 2. Total activity of acid hydrolases per million uninfected KB cells

Total activity* per 106 cells

FractionPhosphatase Ribonuclease P-Glucuronidase Protease Deoxyribo-

nuclease

Cell sap ......................... 1.12 5.06 0.475 0.40 0.019Lysosomes + mitochondria. .... 1.62 15.10 1.82 1.87 0.044Microsomes ..................... 0.24 1.68 0.272 0.15 0.009Nuclei ......................... 0.65 7.80 0.695 0.98 0.015

* Units of activity as in Table 2, except activity expressed per 106 cells rather than per milligram ofprotein.

3-0

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4'

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0 8 160 8 160 8 160 8 160 8 16Hours after Infection

FIG. 3. Alterations in the amount of hydrolytic enzyme activity associated with the lysosomal particles andcell sap during poliovirus I replication.

.5-

DN'ase RN'ase CathepsinO Activity/106 Cells Activity/la6 Cells Activity/106 CellsI tv_- , IX '1

-glucuronid aseActivity/106, Cel Is

Acid P'toseActivity/106 Cells

( 8 16

-O 8 16 0 8 16Hours after Infection

FIG. 4. Alterations in total cell content oflysosomal enzyme activities during poliovirus I replication.

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FIG. 5. Multiplication cycle ofpoliovirus in KB cellsand changes in quantity of total cell protein duringpoliovirus I infection.

protein, the enzymatic activity of this fractionwas found to be 4 to 10 times greater than thatpresent in the cell supernatant fluid. Table 2shows the total activity in each fraction per mil-lion uninfected cells. These studies demonstratedthat 10 to 30% of the enzymatic activity was pres-ent in the cell supernatant fraction, whereas theremainder was present in a bound form, prin-cipally in the lysosomal and nuclear fractions.The finding of significant amounts of enzyme ac-tivity in the nuclear fraction was somewhat unex-pected. It is possible that this nuclear enzymeactivity was a result of contamination of the nu-clear fraction with cytoplasmic tags and unbrokencells. Previous cytochemical studies have stronglysuggested that lysosomes are constituents of thecytoplasm only, and it is believed by some workersthat the activity invariably present in the nuclearfraction on physical separation of the cell compo-nents reflects contamination by cytoplasmic ma-terial (Ciba Symposium, Lysosomes, p. 306,1963). At present, this question is not resolved.Attempts were made to separate the mitochon-drial and lysosomal fractions by brief centrifuga-tion at 10,000 X g, followed by more prolongedcentrifugation at 15,000 X g (3). These attemptsproduced no further enrichment of enzymaticactivity in the postmitochondrial fraction oncomparison with the specific activity of the mixedmitochondrial-lysosomal preparation obtained bya single-step centrifugation at 15,000 x g for 30min. In all subsequent experiments, the mito-chondrial and lysosomal fraction was centrifugedwith the one-step procedure.

Hours after Infection

FIG. 6. Changes in total protein content of variouscell fractions during poliovirus infection. The lysosomalfraction also includes the mitochondria fraction asdescribed in the text.

Experimental results with poliovirus infection. Asummary of the results from experiments per-formed at selected time intervals in the replicationcycle of poliovirus is shown in Fig. 3 to 6. Thestudies showed that activity for all enzymes in-

creased in the cytoplasm at 6 hr after infection(Fig. 3). Activity per milligram of protein roserather rapidly in this fraction to reach a maximal

level 12 hr after infection. As protein leaked fromthe cells, these levels declined to a somewhatlower point 16 hr after inoculation. The levels ofactivity at the time of maximal accumulation inthe cytoplasm were two- to fourfold greater thanthe activity of these enzymes in the uninfectedcells. This increase of cytoplasmic activity wasaccompanied by a simultaneous decrease in boundenzyme activity. No net synthesis of any of theseenzymes occurred after infection, and total activ-ity within the cells declined steadily from 6 hrafter infection to the time of last measurement,16 hr past inoculation (Fig. 4). Comparison of therelease of these enzymes with production of in-fectious particles and cytoplasmic effect by mi-

croscopy showed that all of the parameterschanged concomitantly. As shown in Fig. 5, thefirst measurable increase in infectious poliovirusoccurred 6 hr after infection, accompanying theearlier shift in hydrolytic enzymes from the boundto unbound state and the earliest appearance ofcytoplasmic effect. The protein content of all cellfractions decreased rapidly (Fig. 6). This declinebegan 6 hr after infection. The total cell contentof protein at that time was 48% of the contentin control cells.

Since reports have appeared suggesting that

,I i --Intracellular/ * iVirus, pfu.X Extracel lular

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,-Total Protein/106Cel Is

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HYDROLYTIC ENZYMES IN VIRUS-INFECTED KB CELLS

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C.)

0

0.00(/v;3- DN'ase

0

0 1E 1 1 1 1o0. -m Eh

Xj 4 12 24 4 12 24 4 12 24Hours after Infection

FIG. 7. Effect ofherpes simplex virus infection on cellular distribution of three lysosomal enzymes.

U)0=

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FIG. 8. Multiplication cycle of herpes simplex virusin KB cells.

adrenocorticosteroids increased the stability oflysosomes in other cells (32), experiments wereperformed to determine the effect of hydrocorti-sone on the release of bound hydrolytic enzymesaccompanying polio infection. No discernible ef-fect of hydrocortisone hemisuccinate on virus-induced release of these enzymes could be dem-onstrated when the compound was maintained onthe cells at a concentration of 100lOg/ml from 24hr before virus inoculation until the terminationof the experimental period.

Experiments with herpes simplex virus. Studieswith herpes simplex virus infection of KB cellswere performed to compare enzyme alterationsproduced by poliovirus with the effect of a viruswhich initiates less dramatic changes in host cell

function. As shown in Fig. 7 and 8, the enzymealterations during the selected times of observa-tion in the replication cycle of herpes simplexvirus (from the 4 hr after infection until 24 hrafter inoculation) were decidedly less pronouncedthan was the case with poliovirus. During thecourse of infection with this virus, the activity of,B-glucuronidase rose to a maximal level 35%above the enzyme cytoplasmic activity of controlcells, despite the development of frank cytopathiceffect by 12 hr after inoculation. The other hy-drolytic enzymes studied in these experimentsshowed less significant alterations. A 10% loss ofcell protein from the cells occurred during theexperimental period with herpes simplex virusinfection. In addition to measurements of thethree enzymes described in Fig. 7, assay of ribo-nuclease and protease activity in the cytoplasmof herpes-infected cells was performed. Theseenzymes showed approximately 20% increase incytoplasmic enzyme activity of infected cells. Themaximal activity for both enzymes was recorded16 hr after infection.

DiscussIoN

It is apparent from the data reported in thispaper, and from two other studies on virus-in-duced lysosomal enzyme changes reported whilethis work was in progress (1, 33), that hydrolyticenzymes are released from the bound state in cellsduring the course of infection with certain mam-malian viruses. Considerable increases in cyto-plasmic content of these enzymes were found inliver cells of mice infected with mouse hepatitisvirus (1), and during infection of tissue culturecells with poliovirus (33 and our observation).The increase in cytoplasmic activity of lysosomal

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enzymes reported during vaccinia infection ofmonkey kidney cells by Allison and Sandelin (1)was not observed in the experiments reported byWolff and Bubel (33) using vaccinia in KB andguinea pig spleen cells. Our studies with herpessimplex infection of KB cells showed little releaseof hydrolytic enzymes during virus replication.These observations with herpesvirus paralleledthe results of the work by Wolff and Bubel (33)on lysosomal enzymes in vaccinia-infected cells.The precise cause of lysosomal destruction dur-

ing virus replication remains to be determined.The present work suggests that deterioration ofmembrane function may be the initiating factor inhydrolytic enzyme release from these particles.Poliovirus infection, which is accompanied bypronounced release of lysosomal enzymes fromthe bound state, produces obvious alterations incytoplasmic membrane function. Leakage of pro-tein from the polio-infected cell begins 6 hr afterinfection, and by 12 hr after inoculation only one-third of the soluble protein originally present inthe cell remains with the cell body. In herpessimplex infection, loss of protein from the cell isnegligible, and, likewise, alterations in lysosomalenzyme activity within the infected cell are oflimited extent.

Allison and Sandelin (1) pointed out that lyso-somal enzymes may participate in the uncoatingprocess of virus nucleic acid after the intact par-ticle has entered the cell. Myxoviruses have beenshown to enter cells by a process akin to pinocyto-sis (6, 28), and concentration of lysosomes aboutpinocytosis vacuoles has been demonstrated byNovikoff (23). Our failure to demonstrate quanti-tative increases in the activity of unbound hydro-lases within polio-infected cells during the viruslatent period does not rule out such a role forlysosomal enzymes. The local release of enzymesaround a few infecting particles might not bemeasurable by the techniques used in this study.Experiments are presently being performed withpurified virus and lysosomal enzymes to measuredirectly the ability of these hydrolases to rendervirus nucleic acid susceptible to endonuclease ac-tion.

In addition to a possible role in the uncoatingprocess of virus particles, lysosomal enzymes maycontribute significantly to virus injury of the in-fected cell by degrading host macromolecules. Inthe case of bacterial viruses, marked degradationof DNA occurs with T2 and T5 infection of E.coli (30). The importance of virus-induced hydro-lases in parasitism by these agents remains anopen question, since most of the disruption ofhostcell DNA precedes appearance of virus-specificnucleases (20). Infection of L-strain mouse cellswith equine abortion virus has been reported to

be associated with extensive degradation of cellDNA (25). This effect was initially thought to bevirus-induced, but a recent study indicates thatthe host nucleic acid degradation found in thesestudies was produced by enzymes from contam-inating pleuropneumonia-like organisms (24).The data reported in this paper and in two pre-

vious publications (1, 33) indicate that lysosomalenzyme release from the particulate to the freestate occurs with virus infections that produce arapidly cytocidal effect, whereas other viruseswhich show a slower evolution of cellular damageand minimal leakage of constituents from the cellproduce little or no change in the state of theseenzymes. The close time relationship betweenmeasurable release of lysosomal enzymes, matu-ration of virus particles, leakage of virus particlesand protein from the cell, and virus cytopathiceffect in infection of tissue culture cells by polio-virus makes it difficult as yet to ascribe any majorrole in virus pathogenesis to these enzynes. Moreknowledge about the factors producing changes inmembrane permeability and the alterations inionic composition of the cytoplasm of infectedcells at the onset of lysosomal disruption may beuseful in defining the cause of disruption of theseparticles and the role of these hydrolytic enzymesin cellular damage accompanying virus infection.

AcKNowLEDGMENTSThis investigation was supported by Public Health

Service grant AI-05010 and career development awardK3-AI-9858 from the National Institute of Allergyand Infectious Diseases.

LrrERATURE CITED1. ALLISON, A. C., AND K. SANDELIN. 1963. Activa-

tion of lysosomal enzymes in virus-infectedcells and its possible relationship to cytopathiceffects. J. Exptl. Med. 117:879-887.

2. ANDERSON, P. J., S. COHEN, ANm T. BARKA. 1961.Hepatic injury. Arch. Pathol. 71:89-95.

3. APPELMANS, F., R. WATrrIAUX, AND C. DEDUVE.1955. Tissue fraction studies. 5. The associa-tion of acid phosphatase with a special class ofcytoplasmic granules in rat liver. Biochem.J. 59:438-445.

4. BEN PORAT, T., AND A. S. KAPLAN. 1963. MCeh-anism of inhibition of cellular DNA, synthesisby pseudorabies virus. Virology 2:22-29.

5. CoHEN, S. 1961. Virus-induced ofmetabolic function. Federation Proc. 20641-649.

6. DALES, S., AND P. W. CHOPPIN. 1962. At tand penetration of influenza vi Virolog18:489-493.

7. DEDUVE, C., B. C. PREssmAN, R. GLArto, R.WATFIAUX, AND F. APPELMANS. 1955. Tissuefractionation studies. 6. Intracellular dstibu-tion pattems of enzymes in rat liver tissue.Biochem. J. 60:604-617.

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8. DEDUVE, C. 1963. The lysosome concept. CibaFound. Symp. Lysosomes, p. 1-31.

9. FISHMAN, W. H., B. SPRINGER, AND R. BRUNETrI.1948. Application of an improved glucuronidaseassay method to the study of human bloodglucuronidase. J. Biol. Chem. 173:449-456.

10. FISKE, C. H., AND Y. SUBBARow. 1925. Thecolorimetric determination of phosphorus. J.Biol. Chem. 66:375-400.

11. FRANKLIN, R. M., AND D. BALTIMORE. 1962.Patterns of macromolecular synthesis in normaland virus-infected mammalian cells. ColdSpring Harbor Symp. Quant. Biol. 27:175-195.

12. GINSBERG, H. S. 1961. Biological and biochemi-cal basis for cell injury by animal viruses.Federation Proc. 20:651-660.

13. GINSBERG, H. S., G. F. BADGER, J. A. DINGLE,W. J. JORDAN, AND S. KATZ. 1955. Etiologicrelationship of the RI-67 agents to "acuterespiratory disease." J. Clin. Invest. 34:820-831.

14. GINSBERG, H. S., AND M. K. DIXON. 1961. Nu-cleic acid synthesis in type 4 and 5 adenovirus-infected HeLa cells. J. Exptl. Med. 113:283-299.

15. HOLLAND, J. J. 1964. Inhibition of host cellmacromolecular synthesis by high multipli-cities of poliovirus under conditions preventingvirus synthesis. J. Mol. Biol. 8:574-581.

16. HOLLAND, J. J., AND J. J. PETERSON. 1964. Nu-cleic acid and protein synthesis dui-ing polio-virus infection of human cells. J. Mol. Biol.8:556-573.

17. KELLENBERGER, E., 1961. Vegetative bacterio-phage and the maturation of the virus particles.Advan. Virus Res. 8:1-61.

18. KOENIG, H., AND A. JIBRIL. 1962. Acidic gly-colipids and the role of ionic bonds in thestructure-linked latency of lysosomal. Biochim.Biophys. Acta 65:543-545.

19. KoRN, D., AND A. WEISSBACH. 1964. The effectof lysogenic induction on the deoxyribonu-cleases of E. coli K 12 (lambda). II. The kineticsof formation of a new exonuclease and itsrelation to phage development. Virology 22:91-102.

20. LEHMAN, I. R. 1963. The nucleases of E. coli.Progr. Nucleic Acid Res. 2:82-123.

21. Lucy, J. A., J. T. DINGLE, AND H. B. FELL. 1961.

Studies on the mode of action of excess vita-min A. 2. A possible role of intracellular pro-teases in the degradation of cartilage matrix.Biochem. J. 79:500-508.

22. NOVIKOFF, A. B. 1956. Electron microscopy:cytology of cell fractions. Science 124:969-972.

23. NOVIKOFF, A. B. 1963. Lysosomes in the physio-logy and pathology of cells: contributions ofstaining methods. Ciba Found. Symp. Lyso-somes, p. 36-73.

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