JOURNAL OF VIROLOGY, Oct. 1967, p. 843-850Copyright © 1967 American Society for Microbiology

Vol. 1, No. 5Printed in U.S.A.

Inhibition of Host Protein Synthesis in Type 5Adenovirus-infected Cells

LEONARD J. BELLO1 AND HAROLD S. GINSBERG

Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania19104

Received for publication 21 March 1967

The effect of type 5 adenovirus infection on the synthesis of host-cell proteins bysuspension cultures of KB cells was investigated. Although total protein synthesiscontinued at a constant rate for approximately 36 hr, net synthesis of five hostenzymes (lactic dehydrogenase, acid phosphatase, deoxyribonuclease, fumarase,and phosphoglucose isomerase) was found to stop 16 to 20 hr after infection. Thesynthesis of alkaline phosphatase stopped 9 to 12 hr after infection. The inhibitionof host protein synthesis occurred shortly after the synthesis of viral antigens hadbegun, accounting for the continued synthesis of total protein. An investigation ofthe relationship between synthesis of viral antigens and inhibition of host proteinsynthesis yielded results which suggest that the two processes are in some way

coupled.

Adenoviruses comprise a group of deoxyribo-nucleic acid (DNA)-containing animal viruseswhich share a common group-specific antigen.Each particular type of adenovirus possesses, inaddition, a type-specific antigen by which it canbe distinguished from other adenoviruses (18).In recent years, an extensive study has beencarried out in this laboratory on type 5 adeno-virus. It has been demonstrated that this virus in-duces the synthesis of viral DNA about 10 hralter infection (9). Approximately 2 hr after theinitiation of viral DNA replication, the synthesisof three new antigens, now known to be viralstructural proteins, begins (11, 23, 24, 26). Syn-thesis of these antigens not only follows replica-tion of viral DNA but also is dependent upon it;i.e., the addition of 5-fluoro-2-deoxyuridine(FUdR), a well-characterized inhibitor of DNAsynthesis, to cultures of infected cells blocks theproduction of viral antigens (7). The synthesis ofviral antigens, in this respect, is analogous to"late protein" synthesis in phage systems whichalso follows and may require replication of viralDNA (15, 21).Although a 12-hr delay exists between the time

of infection and the onset of antigen synthesis,when exponentially growing cells are infected thislag is not reflected in the kinetics of total proteinsynthesis. Thus, the rate of pt otein synthesis in

1 Present address: Laboratory of Microbiology,School of Veterinary Medicine, University of Penn-sylvania, Philadelphia 19104.

adenovirus-infected cells continues at the prein-fection rate before, as well as after, the initiationof antigen production. The study described in thiscommunication was initiated to determinewhether the protein elaborated before the onsetof antigen production represents the continedsynthesis of host proteins and, if so, whether theproduction of host proteins continues during theperiod of antigen synthesis. To follow the syn-thesis of host proteins selectively, synthesis of sixhost enzymes was studied in infected cells.The results obtained indicate that host protein

synthesis continues in infected cells until viralantigen production begins. Shortly after synthesisof viral antigens commences, host protein syn-thesis is inhibited. The data suggest that the blockin host protein synthesis is linked to productionof viral antigens and is not an independent eventoccurring simultaneously with the synthesis ofviral antigens.

MATERIALS AND METHODS

Tissue culture. All experiments were carried out withsuspension cultures of KB cells (5). Cells were rou-tinely propagated in Eagle's minimal essential mediumsupplemented with 10% calf serum (6). The cell countwas adjusted each day to 150 X 103 cells per ml bythe addition of fresh media. When cells were to be in-fected, a 20-hr culture (2 X 105 to 3 X 105 cells/ml)was diluted with an equal volume of Eagle's minimalessential medium so that the final concentration ofcalf serum was 5%. The concentration of calf serumwas readjusted to 10% 2 hr after infection.

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Virus. Type 5 adenovirus was used in all experi-ments. The strain was identical with that used inprevious studies (24). Stock viral preparations wereprepared as described previously and stored at -20C (24).

Biological determinlationis. Viral infectivity andcomplement-fixation titrations were carried out aspieviously described (8). Cell counts were done intriplicate by use of a hemocytometer.

Preparation of aniliserum. To prepare anti-viralantiserum, rabbits were given two 4.0-ml intramuscu-lar injections of a concentrated homogenate (protein= 1 mg/ml) of type 5 adenovirus-infected cells mixedwith an equal volume of Freund's complete adjuvantand one intraperitoneal infection without adjuvant at10-day intervals. Animals were bled 10 days after thelast injection. Sera were separated from blood clotsby centrifugation, inactivated at 56 C for 30 min, andstored at -20 C. Antiserum to KB cells was pre-pared similarly with extracts of uninfected cells.

Preparation of cell extracts. At various times afterinfection, 50-ml samples of the cell suspension werewithdrawn from the culture vessel and centrifuged for10 min at 160 X g. The cell pellet was washed oncewith 10.0 ml of cold 0.15 M NaCl buffered with 0.01 Msodium phosphate, pH 7.2 (PBS), and stored at -20C until used. Extracts were prepared by dispersing thecell pellets in 5.0 ml of 0.01 M glycyl-glycine buffer(pH 7.4) and treating the suspension for 2 min in anM.S.E. ultrasonic disintegrator (60-w model). Eachsample was then divided into five 1.0-ml fractions, andthose not immediately used were kept at -20 C.

Enizyme assays. Acid phosphatase was always as-sayed on the day that extracts were prepared sincesmall decreases in activity were observed after storageat -20 C. The enzyme was assayed by a modificationof the procedure of Lowry (14). The reaction mixturecontained 300 ,umoles of sodium acetate (pH 4.7),3.0 ,moles of p-nitrophenyl phosphate, and 30 to 90units of enzyme, in a final volume of 3.0 ml. Afterincubation of the mixture at 37 C for 15 min, thereaction was terminated by the addition of 2.0 ml of0.2 N NaOH. Colorimetric determinations were thencarried out by use of a Klett-Summerson photometerwith a 420-m,u filter. One unit of enzyme is defined asthe amount giving a reading of one Klett unit.

Alkaline phosphatase was assayed by the sameprocedure used for acid phosphatase. Glycine buffer(pH 9.5) was substituted for sodium acetate.

Lactic dehydrogenase was assayed in a Zeissspectrophotometer by following the oxidation ofreduced nicotinamide adenine dinucleotide (NADH2).The reaction mixture contained 300 ,umoles of potas-sium phosphate buffer (pH 6.8), 1.0 ,mole of sodiumpyruvate, 0.3 mg of NADH2, and 10 to 30 units ofenzyme, in a final volume of 3.0 ml. After addition ofsubstrate, the reaction mixture was held at 23 C, andthe changes in absorption at 340 mg were recorded at1-min intervals. One unit of enzyme is defined as theamount causing a change of 0.001 in optical densityper minute at 23 C.

Fumarase was assayed in a Zeiss spectrophotom-eter by a modification of the procedure of Racker(19). The reaction mixture contained 300 jumoles of

tris(hydroxymethyl)aminomethane (Tris) buffer (pH8.2), 150 ,umoles of sodium malate, and 10 to 30 unitsof enzyme, in a final volume of 3.0 ml. After additionof the substrate, the changes in absorption at 240 m,>were recorded at 1-min intervals. One unit of enzymeis defined as the amount causing a change of 0.001in optical density per minute at 23 C.

Phosphoglucose isomerase was assayed by a modifi-cation of the procedure of Slein (22), in which theamount of fructose-6-phosphate formed was deter-mined by the method of Roe (20). The reactionmixture contained 50 ,umoles of Tris buffer (pH 8.2),5.0 /Amoles of glucose-6-phosphate, and 30 to 90 unitsof enzyme in a final volume of 1.0 ml. After incuba-tion of the mixture at 37 C for 15 min, the reaction wasterminated by the addition of 3.5 ml of 2 N HCI. Tothis solution, 0.5 ml of a developing reagent contain-ing 0.1 g of resorcinol and 0.25 g of thiourea in 100ml of glacial acetic was added. The solution washeated for 10 min at 80 C, cooled for 5 min at roomtemperature, and read in a Klett-Summerson photom-eter with a 540-mg filter. One unit of enzyme is de-fined as the amount giving a reading of one Klett unit.

Deoxyribonuclease was assayed by measuring theconversion of DNA to acid-soluble material. Thereaction mixture contained 300 ,umoles of sodiumacetate buffer (pH 4.7), 180 jg of salmon sperm DNA,and 10 to 35 units of enzyme, in a final volume of 1.8ml. The mixture was incubated for 2 hr at 37 C, andthe reaction was terminated by the addition of 0.20 mlof 35% perchloric acid. After standing for 15 min at0 C, the mixture was centrifuged for 15 min at 3.000X g. The supernatant solution was removed with apipette and read at 260 m,4 in a Zeiss spectrophotom-eter. A blank obtained by adding acid to an identicalreaction mixture at zero-time was subtacted from thisvalue. Since linearity with enzyme concentration wasobserved only between optical density readings of0.100 and 0.450, a standard curve relating opticaldensity to enzyme concentration was prepared. Areading of 0.100 was defined as 10 units of enzymeactivity. Between optical density readings of 0.100 and0.450, increases of 0.136 equaled 10 additional units.DNA and protein determiniations. DNA and protein

determinations were performed on 1.0-ml fractions ofthe cell extracts. Two ml of cold 10% trichloroaceticacid was added, and after 30 min at 0 C the precipi-tate which formed was sedimented by centrifugation for15 min at 3,000 X g, washed once with 2.0 ml ofcold 5% trichloroacetic acid, and finally resuspendedin 1.0 ml of 5% trichloroacetic acid. The suspensionwas heated for 45 mnin at 90 C, chilled, and centri-fuged. The supernatant solution was used for DNAdeterminations and the precipitate, after being dis-solved in 2.0 ml of 0.2 M Na2CO3-0.1 M NaOH, wasused for protein determination. DNA was determinedby the Burton modification of the diphenylaminereaction (2), and protein, by the method described byLowry (13).

RESULTS

Effect of adenovirus infection on protein syn-thesis. Previous studies carried out with resting

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PROTEIN SYNTHESIS IN ADENOVIRUS-INFECTED CELLS

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FIG. 1. Production of complement-flxing antigens ininfected cells. Two 900-ml cultures ofKB cells contain-ing 100 X 103 cells per ml were incubated at 36 C. Oneof the cultures was infected with an input multiplicity of200 PFUper cell while the other culture was untreated.At 2 hr after infection, calf serum was added to themedium to give a concentration of 10%. At the indi-cated times, samples were withdrawn and complement-fixation assays were done. Infected, X; control, 0.

cells demonstrated that new complement-fixingantigens are produced in cells infected with type 5

adenovirus (11, 24). The kinetics of antigen syn-thesis in exponentially growing cells are shown inFig. 1. Under the conditions employed in theseexperiments, the antigens were first detected 12 to15 hr after infection. Their rate of synthesis in-creased until 20 to 24 hr after infection, afterwhich synthesis proceeded at a linear rate. Netprotein synthesis continued in infected cultures ata linear rate for about 36 hr, despite the fact thatcell division was inhibited about 12 hr after infec-tion. The total amount of protein synthesized by36 hr was essentially equal to the amount made ina control culture of uninfected cells. Similar re-sults have been reported in cells infected with type2 adenovirus (10). These data obtained withexponentially growing cells are in contrast tothose obtained when resting cells are infected.Under conditions of arrested cell division anddepressed cellular biosynthesis, an actual increasein the rate of protein synthesis is observed whenantigen synthesis begins (25).

Selection of enzymes and efficiency of enzyme

845

release. The data described above led to twoquestions. (i) Is host protein synthesized duringthe period prior to viral antigen synthesis? (ii)Does host protein synthesis continue during theperiod of viral antigen synthesis? To answerthese questions, the synthesis of specific hostenzymes was studied in cells infected with type5 adenovirus.The measurement of cellular enzymes, al-

though uncomplicated, contains several pitfalls.The first is the danger that varying inactivationof the enzymes may occur prior to assay. Thisproblem has been obviated by selecting for studyonly stable enzymes. The availability of a rapid,accurate, and convenient assay was the onlyother criterion used to select the enzymes to bemeasured. The enzymes selected were acid andalkaline phosphatase, phosphoglucose isomerase,lactic dehydrogenase, fumerase, and deoxy-ribonuclease. All except acid phosphatase couldbe stored for at least 1 week at -20 C with nodetectable loss in activity.Another possible source of error when enzyme

levels are compared is inefficient and, therefore,variable release of enzymes from the cells understudy. Mechanical breakage of cells, for example,has been shown to release only a fraction of thealkaline phosphatase activity released by de-oxycholate treatment (4). The method for pre-paring cell extracts in the experiment to bedescribed, i.e., sonic treatment of cells in ahypotonic medium, reproducibly released maxi-mal levels of the enzymes studied, provided thecell debris was not removed. Comparable valueswere obtained for alkaline phosphatase whencell extracts were prepared either by sonictreatment or by treatment of cell suspensionswith 0.5% deoxycholate. Since sonic treatmentfor 1 to 4 min gave similar results, there waslittle danger that anomalous values would becaused by minor differences in the efficiency ofsonic disruption.

Synthesis of host enzymes in infected cells.Synthesis of the six host enzymes listed wasstudied in infected and uninfected cell cultures.The results of a representative experiment aresummarized in Fig. 2. In the infected cells, allthe enzymes, with the exception of alkaline phos-phatase, were synthesized at the same rate as inthe control for 16 to 20 hr. By 20 hr after in-fection, however, increases in enzyme activitieshad ceased. Alkaline phosphatase differed onlyin that synthesis stopped about 12 hr after in-fection. The results of these experiments areconsistent with the bulk of the protein synthesizedprior to antigen synthesis being host material.The results also suggest that host protein syn-

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8 16 24 32 40 8 16 24 32 40HOURS AFTER INFECTION

Fio. 2. Effect of type S adenovirus infection on syn-

thesis of host enzymes. Conzditions were as described inFig. 1. At the indicated times, samples were withdrawniand used for enzyme assays as described in Materialsand Methods. The values are expressed as relativeactivity per milliliter of culture with the 2-hr samplefrom the control normalized to a value of 100 for each

enzyme. Inifected, X; control, 0.

thesis was inhibited during the period of viralantigen synthesis. The latter conclusion, however,was not unequivocal, and the following alterna-tives were considered. (i) The release of enzymes

from infected cells may be slower than fromcontrol cells. Thus, the low values observed inextracts of infected cells may reflect the fact thatonly a fraction of the enzyme content of thesecells is actually being assayed. (ii) The enzymesin infected cells may be more sensitive to sonicinactivation than those in control cells. If thiswere true, the lower values could be attributedto a higher fraction of inactivated enzymes. (iii)Infection may result in the production of sub-stances which are enzyme inhibitors. (iv) In-fection may result in the depletion of essentialenzyme activators. (v) Infection may result inan increase in the general rate of enzyme break-down or inactivation. If this were true, the ob-served inhibition of enzyme accumulation wouldnot be equivalent to inhibition of enzyme syn-

thesis.

TABLE 1. Effect of time of soniic treatment onenizyme activities in extracts of infected cells

Relative activity (per cent of maximum) of"

Time ofsonic Ttreat- Alkaline Acid Lactic F Deoxy- Phospho-ment phos- phos- dehy- Fumer- ribonu- glucose

phatase phatase ase clease isomerase

,mi's0 100 100 92 74 95 931 92 94 94 98 100 982 92 88 100 98 98 1004 94 84 94 100 96 100

a Enzyme assays were carried out as describedin Materials and Methods, and the highest valueobtained for each enzyme was standardized at 100.

Release of enzyme activity from infected cells.The first alternative investigated was that en-zymes are released from infected cells at a slowerrate than from uninfected cells, resulting inspuriously low values. The results of experimentsto explore this possibility, summarized in Table1, indicate that enzymes are readily released frominfected cells; in most cases, maximal levels wereobtained even in the absence of sonic treatment.These data also indicate that the reduced levelsin infected cells are not the result of increasedsensitivity to sonic inactivation.

Absence of inhibitors and activators. To de-termine whether the reduced enzyme levels ininfected cells resulted from the formation ofenzyme inhibitors or the absence of enzymeactivators after viral infection, samples of in-fected and control cultures were assayed bothseparately and after mixing. The results (Table2) indicate that enzyme activities measured inthe mixtures were equal to the sums of the in-dividual assays. This evidence implies that neitherenzyme activators nor inhibitors were responsiblefor the phenomenon described.

Evidence for inhibition of enzyme synthesis.The results of these experiments indicate thatnet accumulation of the enzymes studied ceasedby 20 hr after infection. They do not prove,however, that enzyme synthesis was affected,since an increase in the rate of enzyme inactiva-tion to the point where inactivation equaledsynthesis would also prevent a net increase inenzyme activity. The view that the actual syn-thesis of enzymes was inhibited implies that theprotein made after 20 hr is entirely viral. Thispredication was tested by determining at varioustimes after infection, what fraction of the 14C-valine incorporated during a 10-hr pulse couldbe precipitated by antiserum to viral antigens.Four 100-ml cultures of KB cells, containing

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TABLE 2. Additive effect of enizyme activities inmixed extracts of control anid infected cellsa

!Alkaline Acid Lactic Phos- Deoxy-Extrctoos- hos-dehy- Fumer- phoglu-rio-

pfataseb phatase5 raoen aseroases cleasec

(Units)Infected 20 20 8.2 8.4 12.9 23Control 60 58 15.8 17.0 33.8 42Infected 78 90 23.2 24.2 44.0 61+ con- (80) (93) (24.0) (25.4) (46.7) (65)trol

aConditions for preparation of extracts wereidentical to those described in Fig. 2. For eachassay, an equal volume of extract from the con-trol or infected culture was used. The figures inparentheses are the calculated values, assumingno interaction of the extracts.

bBased on a 36-hr sample from infected orcontrol culture.

c Based on a 45-hr sample from infected orcontrol culture.

150 X 103 cells per ml, were infected with aviral input multiplicity of 200. Individual cul-tures were pulsed with 15 ,uc of 14C-valine for10-hr periods. After the pulse, a 50-ml samplewas withdrawn from each culture and centri-fuged for 10 min at 160 X g; the cell pellet waswashed with 10.0 ml of cold PBS. To prepareextracts, the cells were suspended in 5.0 ml ofcold PBS and sonically treated for 2 min. Theextracts were dialyzed for 16 hr at 4C against100 volumes of PBS and were centrifuged for 30min at 3,000 X g. The supematant solutionwhich contained 90% of the incorporated 4C-valine was used to determine total 14C-valineincorporated into soluble proteins. To measurethe fraction of radioactivity incorporated intononviral proteins, 0.24 ml of the supernatantsolution was incubated with 0.24 ml of type 5adenovirus antiserum. After 2 hr at 37C, 0.24ml of PBS was added and the samples werecentrifuged for 20 min at 1,000 X g. A 0.50-mlportion of the supematant solution was placedin a planchet, dried, and counted. The radio-activity remaining in the supematant solutionwas compared with samples treated similarlywith serum from unimmunized rabbits to de-termine the fraction specifically precipitated. Asmall correction (5 %) was necessary to correctfor differences in self-absorption by the twosera.These counts not precipitated by viral antibody

were considered a maximal estimate of hostprotein synthesized during the 10-hr period. Themore direct method of testing the quantity ofprotein precipitated by antiserum to host protein

TABLE 3. Precipitationt of proteinis synithesizedafter type S adenovirus inifection by homologous

adenovirus antiseruma

Total 14C-valine 14C-valineTime 1C-vtaline into incorporated precipitated byTias presalien(hr) soluble proteins type 5 antiserumwas present (hr) (counts per min per

ml of culture) (c)

3-13 1, 258 513-23 1,413 5123-33 1,319 8333-43 1,048 88

a Four cultures of KB cells were infected with aviral input multiplicity of 200. Individual cultureswere pulsed with I4C-valine for 10-hr periods atthe times indicated. After the pulse, extracts wereprepared and incubated with type 5 adenovirusantiserum as described in text.

was precluded by our inability to precipitatesignificant quantities of host protein with specificantibody. The data summarized in Table 3indicate that over 80% of the "4C-valine incorpo-rated into protein later than 23 hr after infectionwas precipitated by virus-specific antibody.Thus, even if all of the radioactivity that was notprecipitated represented continued synthesis ofhost proteins, the rate of synthesis was less than20% of that in uninfected cells.

Coordination of antigen synthesis with inhibitionof host protein synthesis. The fact that hostprotein synthesis was inhibited after synthesisof viral antigens began suggested the possi-bility that the two processes might be related.To investigate this possibility, the synthesis ofhost proteins was studied in cells infected underconditions in which the synthesis of viral antigenswas blocked. This was accomplished by theaddition of FUdR to an infected culture. FUdRinhibits the replication of viral DNA and inso doing blocks the synthesis of viral antigens(7). The addition of thymidine prevents theblock in antigen synthesis, indicating that FUdRacts by virtue of its inhibitory effect on thymidy-late synthesis rather than by some less specificmechanism.Although antigen synthesis is not initiated

in the presence of FUdR, net protein synthesiscontinues. The data presented in Fig. 3 indicatethat the addition of a 2 X 10-6 M FUdR atthe time of infection caused only a slight de-pression in the rate of protein synthesis whencompared with the control which receivedFUdR and thymidine. Apparently, the toxiceffects of unbalanced growth (3) were somehowminimized in cells infected with type 5 adeno-virus, since uninfected cells treated with FUdR

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FIG. 3. Effect ofFUdR on protein synthesis in cellsinfected with type 5 adenovirus. Two 450-mi cultures ofKB cells containing 120 X 103 cells per ml were incu-bated at 36 C. Both cultures were infected with a viralmultiplicity of200 PFUper cell. After inJection, FUdRwas added to both cultures at a final concentration of2 X 10-6 M. One culture also received thymidine at a

final concentration of 10-5 MI. At the indicated times,samples were withdrawn and concentrations of proteinwere determined. FUdR, 0; FUdR + thymidine, X.

synthesized only 70% as much protein in 36 hras infected cells (unpublished data). Since viralantigens, which normally constitute over 80%of the protein made later than 23 hr after in-fection, were not made in FUdR-treated cells,the continued synthesis of total protein indicatesthat host protein synthesis continued in thetreated, infected cells past the time when in-hibition normally occurs. Otherwise, inhibitionof host synthesis would be reflected in an in-hibition in total protein synthesis.

Experiments to test the above interpretationwere carried out. The data summarized in Fig.4 indicate that acid phosphatase, fumarase, anddeoxyribonuclease continued to be synthesizedpast the time when inhibition occurred in theinfected culture which received thymidine inaddition to FUdR. Although phosphoglucoseisomerase was synthesized at the uninhibitedrate for only 16 hr, synthesis continued longerin the FUdR-treated cells than in the cells thatreceived FUdR and thymidine. Synthesis ofthis enzyme was apparently sensitive to the toxic

T-100

ACID PHOSPHOGLUCOSE>200

PHOSPHATASE ISOIMERASE

200

8 16 24 32 40 48 8 16 24 32 40 48

HOURS AFTER INFECTION

FIG. 4. Effect of FUdR on enzyme synthesis in type5 adenovirus-infected cells. Conditions were as de-scribed in Fig. 3. At the indicated times, samples were

withdrawn and used for enzyme assays as described inMaterials and Methods. Values are expressed as rela-tive activity with the zero-hour sample from the FUdR+ thymidine culture normalized to a value of 100 foreach enzyme. FUdR, 0; FUdR + thymidine, X.

effects of unbalanced growth (3). The synthesis oflactic dehydrogenase was even more sensitiveto the toxic effects of FUdR and for that reasoncould not be included in the study. It shouldbe pointed out, however, that infected cellstreated with FUdR synthesize more lactic de-hydrogenase than uninfected cells treated simi-larly. Alkaline phosphatase synthesis was in-

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FIG. 5. Effect of FUdR on alkaline phosphatasesynthesis in cells infiected with type S adenovirus.Conditions were as described in Fig. 3. At the indicatedtime, samples were withdrawn and assayed for alkalinephosphatase activity as described in Materials andMethods. FUdR, 0; FUdR + thymidinie, X.

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hibited in both the FUdR-treated culture and inthe control, i.e., FUdR plus thymidine (Fig. 5).This inhibition cannot be attributed to the toxiceffect of FUdR, since synthesis of the enzyme inuninfected cells treated with FUdR proceededfor longer periods of time than in infected cells.It appears that the early inhibition of alkalinephosphatase synthesis is effected by a mechanismdistinct from that which inhibits the synthesisof the other five enzymes studied. Since severalother examples of a specific alteration in the rateof alkaline phosphatase synthesis in culturedcells are known to be caused by such diversefactors as hormones and alterations in the ionicstrength of the culture medium, the atypicalbehaviour of this enzyme in infected cells maynot be representative of host protein synthesisin general (4, 17).

DIscussIoN

The results described indicate that infectionof KB cells with type 5 adenovirus has littleeffect on host protein synthesis until after viralantigen synthesis begins. Host protein synthesisis then markedly inhibited. Whereas synthesis ofhost protein is probably blocked completely by20 hr after infection, synthesis of total proteinin the infected cells is not impaired until about36 hr after infection. The phenomenon, there-fore, is one of selective rather than general in-hibition of protein synthesis. The protein madefrom 20 to 36 hr after infection consists largely,if not entirely, of viral antigens. This has beendemonstrated by the specific precipitation of4C-valine incorporated during this period byantiserum directed against viral antigens. How-ever, the absence of material precipitable byvirus-specific antibody early in infection is not,in itself, sufficient evidence that host protein ismade during this period. New proteins that arecoded for by the virus but which are poor im-munogens, or which are not readily precipitatedby the antiserum used, would be undetectedby this method. The failure to precipitate a

significant amount of host protein by antiserumdirected against uninfected cells serves to illustratethis point. However, if one accepts the assump-tion that the producion of the host enzymesstudied reflects the synthesis of all host proteins,the demonstration that during the initial 16 hrafter infection specific host enzymes were syn-thesized at the same rate as in uninfected cellsindicates that host, rather than viral, proteinsmake up the bulk of the material made duringthis period.The experiments in which FUdR was used

indicate that inhibition of host protein synthesis

in infected cells requires synthesis of viral DNA.Hence, it is unlikely that an "early protein"synthesized only before production of viral DNAis responsible for the inhibition. Since DNAsynthesis is required both for initiating the pro-duction of viral antigens and inhibiting the syn-thesis of host proteins, a "built-in" control ispresent for eliciting the inhibition at the timethat synthesis of viral proteins is taking place.This effect is manifested in the lack of a quanti-tative alteration in the rate of total proteinsynthesis despite the extensive qualitative altera-tion in the kinds of proteins made. It is con-ceivable that inhibition of host protein synthesisincreases the efficiency of production of viralantigens. This seems especially likely in ex-ponentially growing cells in which protein syn-thesis is probably proceeding at a maximalrate before infection. The precise way that in-hibition is coordinated with synthesis suggeststhat some component of the reactions involvedin synthesizing viral antigens, or alternatively,one of the antigens synthesized, may be involvedin the inhibition. Indeed, one of the viral antigenscan inhibit protein synthesis when added directlyto cultured cells (9). It is not yet certain, however,that the latter phenomenon is affected by thesame mechanism which brings about inhibitionin infected cells. The selective inhibition of pro-tein synthesis in cells infected by an adenovirusappears analogous to the inhibition of hostprotein synthesis in cells infected with somecoliphages (1, 12, 16) and poliovirus (27). Incontrast to adenovirus-infected cells, however,these viruses block protein synthesis immedi-ately after infection rather than during the syn-thesis of viral subunits.

Conceivably, the inhibition of host proteinsynthesis could occur at any of the followingthree steps: (i) transcription of host messengerRNA (mRNA), (ii) transport of host mRNAinto the cytoplasm, and (iii) translation of hostmRNA. Recent evidence (9) indicates thatsynthesis of host protein is inhibited at somestep after transcription of host mRNA, indicatingthat the block involves interference with eitherthe transport or translation of host mRNA.

ACKNOWLEDGMENTS

This investigation was supported by Public HealthService grants AI-03620 and 2-TI-AI-203 from theNational Institute of Allergy and Infectious Diseases.

LrERATuRE CIrr

1. BENZER, S. 1953. Induced synthesis of enzymes inbacteria. Biochim. Biophys. Acta 11:383-393.

2. BURTON, K. 1956. A study of the conditions andmechanism of the diphenylamine reaction for

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the colorimetric estimation of deoxyribonucleicacid. Biochem. J. 62:315-323.

3. COHEN, S. S., AND H. D. BARNER. 1954. Studies onunbalanced growth in E. coli. Proc. Natl.Acad. Sci. U.S. 40:885-893.

4. Cox, R. P., AND C. M. MAcLEON. 1962. Alkalinephosphatase content and the effects of predniso-lone on mammalian cells in culture. J. Gen.Physiol. 45:439-485.

5. EAGLE, H. 1955. Propagation in a fluid mediumof a human epidermoid carcinoma strain KB.Proc. Soc. Exptl. Biol. Med. 89:362-364.

6. EAGLE, H. 1959. Amino acid metabolism in mam-malian cell cultures. Science 130:432-437.

7. FLANAGAN, J. F., AND H. S. GINSBERG. 1962.Synthesis of virus-specific polymers in adeno-virus-infected cells: effect of 5-fluorodeoxy-uridine. J. Exptl. Med. 116:141-157.

8. GILEAD, Z., AND H. S. GINSBERG. 1965. Char-acterization of a tumorlike antigen in type 12and type 18 adenovirus-infected cells. J.Bacteriol. 90: 120-125.

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