Mechanism of the Arginine Requirement for ... - Journal of Virology

JOURNAL OF VIROLOGY, Nov. 1971, p. 742-753

Vol. 8, No. 5 Printed in U.S.A.

Copyright © 1971 American Society for Microbiology

Mechanism of the Arginine Requirement for Adenovirus Synthesis I.

Synthesis of Structural Proteins

EINAR EVERITT, BO SUNDQUIST, AND LENNART PHILIPSON Departmenit of Microbiology, The Walleniberg Laboratory, Uppsala University, Uppsala, Sweden

Received for publication 9 August 1971

The mechanism of the arginine requirement for adenovirus was studied in cultures of KB cells infected with adenovirus type 2. Macromolecular synthesis was found to be severely impaired in uninfected cells under complete arginine deprivation, whereas an arginine concentration of 50 AM yielded a moderate and reversible inhibition of growth and nucleic acid synthesis. At this concentration, viral structural proteins were accumulated in excess although the virus yield was reduced more than 1,000-fold. The arginine-sensitive step appeared to occur early during the first 15 hr postinfection in the virus growth cycle. Virus-infected cells deprived of arginine to 50 ,M showed, when reversed, a 4- to 5-hr lag period before the increase in virus growth was observed. Analysis of the radioactive pattern of labeled virions synthesized after reversion showed that all polypeptides were synthesized after addition of arginine to the medium, and none of the virion-polypeptides which are revealed by gel electrophoresis appeared to be preferentially synthesized after arginine reversion. The excess pool of structural proteins formed during depletion appeared to a large extent to be unavailable for virus assembly.

Synthesis of adenovirus has been shown to be strongly dependent on the concentration of arginine in the growth medium (23; V. H. Bonifas and R. W. Schlesinger, Fed. Proc. 18:560, 1959). Synthesis of herpes simplex virus (1, 7), simian virus 40 (4), polyoma virus (35), and reovirus (10) also appears to require arginine for production of infectious progeny. Adenoviruses contain an internal protein component associated with the viral deoxyribonucleic acid (DNA), which is exceptionally rich in arginine (23 mole per cent) (8, 21, 28). None of the outer capsid proteins or the remaining core proteins contains arginine to higher concentrations than 12 mole per cent (8, 17). It has therefore been suggested that the arginine requirement reflects a maturation process in which the synthesis of the arginine-rich protein is ratelimiting for assembly of mature progeny (26, 30). This hypothesis was based on kinetic studies of viral DNA synthesis and the presence of structural proteins at arginine depletion. Both viral DNA and structural proteins can be identified at depletion (23, 26), but it is not clear whether the preformed proteins are utilized for virus progeny at reversion. The present study reports quantitative data on synthesis of virus and structural proteins at

different levels of arginine depletion and after reversion. MATERIALS AND METHODS Cells. KB cells were grown either in Spinner cultures in Eagle's Spinner medium (3) with 7% calf serum, penicillin, streptomycin, and kanamycin or in 32-oz (ca. 900 ml) prescription bottles with 30 ml of Eagle's minimal essential medium (MEM) and 15%,o calf serum. Virus. The prototype strain of adenovirus type 2, originally obtained from R. J. Huebner, National Institutes of Health, Bethesda, Md., was used. Unlabeled purified pools were used for infection, and purification was performed by a modification of the procedure of Green and Pifia (5, 19). Radioactively labeled virus was purified further by sedimenting partially purified preparations on 15 to 30% sucrose gradients formed on a cushion of CsCl (p = 1.40 g/cc) for 45 min at 150,000 X g. Virus materials from the sucrose gradients were then banded on self-generating CsCI gradients. Purified virus was dialyzed against 0.25 M sucrose containing 0.02 M

tris(hydroxymethyl)aminomethane (Tris)-hydrochloride (pH 7.4), 1 mm MgCl2, and 0.5% n-butanol (18). Virus infectivity. Adenovirus infectivity was assayed by fluorescent focus formation in KB cells grown in 35-mm plastic petri dishes with Eagle's MEM with 15% calf serum (19). The results are expressed in fluorescent focus units (FFU). 742

VOL. 8,1971

ARGININE REQUIREMENT FOR ADENOVIRUS SYNTHESIS

Virus infection of Spinner cultures. KB cells in Spinner cultures at cell densities of 105 to 4 X 105 cells/ml were harvested by centrifugation at room temperature for 10 min at 300 X g. The cells were washed twice in 10% of the original volume in Eagle's Spinner medium with 2% calf serum at 37 C. The cells were resuspended in Spinner medium without calf serum, and virus was added to a multiplicity of 100 FFU/cell at a cell density of 107 to 3 X 107 cells/ml. Adsorption was carried out in a water bath at 37 C for 30 min. After attachment, the cells were centrifuged and resuspended in Spinner medium with 7%calf serum at a density of about 3 X 105 cells/ ml. In all arginine depletion experiments, washing of cells and virus adsorption were carried out in arginine-free media and dialyzed serum was used. Virus infection of monolayer cultures. After washing twice with Eagle's MEM without calf serum, bottles were infected at a multiplicity of 30 FFU/cell in 2 ml of phosphate-buffered saline (PBS) for 60 min at 37 C with gentle shaking every 15 min. After adsorption, the cells were washed once with MEM containing 10% calf serum, and then 30 ml of the same medium was added. The 37 C environment was maintained during all steps. Immunoassays. Quantitative determinations of hexon, penton base, and fiber antigens were carried out by the Mancini technique (16). Standard curves with pure preparations of antigens and monovalent antisera showed a linear relationship between the square of the precipitate ring diameter and antigen concentration. The penton base antigen was assayed with an anti-penton serum adsorbed with purified fiber antigen until no further fiber antibodies were detectable by immunodiffusion. The amount of penton base is given in terms of penton equivalents, since the assay was standardized with purified intact penton. Interference of penton antigen on the assay of fiber antigen was studied separately, and it was found that the fiber content could be accurately estimated at a twofold or higher molar excess of fiber over penton antigen. This condition is fullfilled in the experiments reported. Antisera were prepared by methods described previously (17). Complement fixation was carried out with the microassay described by Sever (32). Antigen titers were determined in a total volume of 0.125 ml with 4 units of antibody and 2 units of complement. All titers given represent the dilution before the addition of antiserum, complement, and hemolytic system. Radioimmunoprecipitation. The procedure described by Scharff et al. (29) was followed. Hyperimmune antisera against hexon and heavy chains from immunoglobulin E (anti-epsilon) were used. Equivalent concentrations of the two rabbit antisera and goat anti-rabbit immunoglobulin G (IgG) (Microbiological Associates, Bethesda, Md.) were determined by immunodiffusion. Unspecific precipitation was estimated by using uninfected '4C-amino acid-labeled KB cells and an antihexon serum. Sufficient amount of anti-epsilon was included in each experiment in a separate tube to

743

give the same unspecific precipitation as anti-hexon serum.

Samples were incubated at 37 C for 1.5 hr with antihexon and anti-epsilon sera followed by 1.5 hr with anti-IgG, and the precipitates were washed three times in 0.14 M NaCl, 0.01 M Tris-hydrochloride (pH 7.4) at 4 C. The precipitates were dissolved in 0.2 M NaOH before counting. Arginase assay. Arginase was assayed by determining labeled urea from guanido-labeled arginine by thin-layer chromatography (TLC; reference 24) according to the reaction:

AC-arginine

arginase

ornithine + '4C-urea Uninfected or infected KB cells were withdrawn, washed twice in cold PBS, resuspended in 0.01 M maleate-NaOH (pH 7.0) with 0.01 M MnCl2, sonically treated for 30 sec in the cold, and incubated at 45 C for 2 hr to activate the enzyme. The extract was then diluted with an equal volume of 0.1 M glycine-NaOH (pH 9.5) and assayed for enzyme activity. Reaction products were separated by cellulose TLC (Whatman CC 41) with the solvent system ethylacetate-pyridineacetic acid-water (5:5:1:3). Radioactivity was eluted from the TLC plates with ethanol. Arginase activity is expressed as micromoles of urea per microgram of protein per hour. Assay of cellular pools of amino acids. Cells (107 to 2 X 107) were sedimented by centrifugation for 3 min at 1, 500 X g at 37 C, washed twice with 0.15 M NaCl, 0.01 M Tris-hydrochloride (pH 7.5), and resuspended in distilled water. Cold 10% trichloroacetic acid was added, and the supernatant fluid was extracted repeatedly with diethyl ether in the cold, lyophilized, hydrolyzed with 6 N HCl, and analyzed on a "Biochrom" automatic amino acid analyzer. To detect small amounts of arginine during arginine depletion, an isotope dilution technique based on the following reaction was used: -

tRNA + amino acids + adenosine triphosphate

aminoacyl-tRNA synthetase

amino acids-tRNA + adenosine monophosphate +

inorganic pyraphosphate Cells were washed as above, sonically treated for 15 sec in the cold, and centrifuged at 3,250 X g for 15 min. The supernatant fluids were tested for free L-arginine. The assay conditions were as described in detail by Rubin and Goldstein (25), with the following exceptions. Instead of Escherichia coli "soluble" ribonucleic acid (RNA), a preparation of Saccharomyces sp. "soluble" RNA (Schwarz BioResearch) was used, and the aminoacyl-transfer RNA (tRNA) synthetase mixture was prepared from Saccharomyces sp. Sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polypeptides were dissociated essentially as described by Maizel et al. (15). Newly made virus suspensions at a concentration of 200 to 2,000 ,ug of protein per ml were dialyzed against 0.05 M Tris-

744744EVERITT, SUNDQUIST, AND PHILIPSON hydrochloride (pH 8.0) and added in final concentrations of sodium dodecyl sulfate (SDS) to 2%, dithiothreitol to 0.2 M, and ethylenediaminetetraacetic acid (EDTA) to 0.005 M. The samples were then incubated at 95 C for 3 min and subsequently made 10% with respect to glycerol. Acrylamide gels contained 10% acrylamide and 0.25% N'N'-methylene bisacrylamide both of which were recrystallized before use (9). The buffer contained 0.1% SDS (recrystallized from ethanol) and 0.005 M EDTA in 0.1 M Tris-hydrochloride (pH 8.0). Polymerization was catalyzed by 0.1% (v/v) tetraethylmethylenediamine and 0.04% (v/v) ammonium persulfate. The gels were made in 13-cm glass tubes with an internal diameter of 6 mm. After the gels had solidified, they were prerun at 8 v/cm for 1 hr (6 to 7 ma/cm). The samples were layered on the gel surfaces and overlaid with the electrophoresis buffer 0.1 M Trishydrochloride (pH 8.0) with 0.1% SDS and 0.005 M EDTA. Electrophoresis was carried out for 15 min at 3 v/cm and then for 2.5 hr at 8 v/cm. The gels were stained for 18 hr in Coomassie Brilliant Blue R250 dissolved in 10% acetic acid-30% methanol. Destaining was performed in 10% acetic acid-30% methanol. Gels were cut in 1-mm slices, and radioactivity was extracted with 0.4 ml of 1 M NaOH at 37 C for 18 hr and counted. Assay of trichioroacetic acid-insoluble radioactivity and protein. Cells were withdrawn from Spinner cultures, sedimented by centiifugation (for 10 min at 300 X g) at 4 C, and washed once in 2 ml of ice-cold PBS. Tpe cell pellet was resuspended in 0.5 ml of ice-cold PBS and an equal volume of ice-cold 10% trichloroacetic acid was added. After thorough mixing, the material was incubated overnight at 4 C. The precipitate was sedimented, washed twice with 1 ml of ice-cold 5% trichloroacetic acid, and then extracted once with a mixture of ethanol and diethyl ether (3:1). After complete drying at 45 C, the precipitate was dissolved in 0.1 M NaOH and counted. Protein was measured by the method of Lowry et al. (12) with bovine serum albumin as standard. Isotopes. L-Arginine-monohydrochloride-guanido14C (4.6 mCi/mmole) and 14C-L-arginine-monohydrochloride (uniformly labeled) (254 mCi/mmole) were obtained from New England Nuclear Corp., Boston, Mass. Adenosine-T-G (500 mCi/mmole) and L-arginine-monohydrochloride-5-T (500 mCi/mmole) were obtained from Radiochemical Centre, Amersham. 14C-L-reconstituted protein hydrolysates (1 mCi/mg) were obtained from International Chemical and Nuclear Corp., Irvine, Calif. Radioactive isotopes were counted in a Packard liquid scintillation spectrometer with a dioxane-based scintillation fluid (6) containing an equal dry volume of Cab-O-Sil (Cabot Corp., Boston, Mass.).

RESULTS Synthesis of virus and structural proteins under normal growth conditions. To establish the kinetics of synthesis of both virus and structural proteins during one-step growth conditions, virus and antigens were quantitated both during the loga-

J. VIROL.

rithmic and the linear phase of virus production. Monolayers and Spinner cultures of KB cells were infected and cultivated at the normal arginine concentration of 1.2 mm. At different time intervals, approximately 1.5 X 107 cells were withdrawn, washed, and, after sonic treatment, assayed for virus infectivity and virus antigens by the complement fixation and radial immunodiffusion techniques. The time course for virus synthesis was the same in monolayers and Spinner cultures. The logarithmic increase in structural proteins was followed with the complement fixation test since it is about 10 times more sensitive than radial immunodiffusion. Late in infection during the linear increase, quantitative estimates were made of the excess antigens. As shown in Fig. 1, the four antigens hexon, fiber, penton base, and the major core protein (the alanine and arginine rich core protein, AAP; 21) appeared one after another at 1-hr intervals. The antigen first detectable was the hexon, followed by the fiber and penton base. The AAP was detected only 1 hr before the rise in virus infectivity. The quantitative data show that the fiber antigen was in excess over hexon and penton base up to 22 hr after infection, but at this time there was a shift and hexon antigen increased more rapidly than the other capsid antigens (Fig. 2). A correlation in time between the linear increase in the antigens and the infectivity at 20 to 35 hr after infection also appeared to exist. f 41 u

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FIG. 1. Logarithmic synthesis of infectious virus and structural proteins in Spinner cultures of KB cells under normal one-step growth conditions. Top: Cell-associated fluorescent focus units (FFU) at 0 to 64 hr postinfection. Bottom: Antigen synthesis measured by complement fixation at 5 to 24 hr postinfection. No antigens were detected between 5 and 9 hr postinfection when samples were analyzed at hourly intervals.

the rate exceeded by 20% the rate in control cells; afterwards there was a continued decrease. The net increases in protein content of virusinfected and uninfected cells were similar, sug-

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gesting that the rates of protein synthesis are the same (not shown). Cellular pool of arginine in normal and argininedepleted KB cells. The pools of free arginine and other amino acids in normal and arginine-depleted cells were analyzed. The arginine concentration was slightly lower than that for all other amino acids analyzed and was sensitive to com-

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ARGININE REQUIREMENT FOR ADENOVIRUS SYNTHESIS

VOL. 8, 1971

45

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plete arginine depletion (Table 1). The concentration of free arginine in the cell was determined at depletion and upon reversion. KB cells were maintained in normal medium (1.2 mm arginine) for 2 hr and then divided into

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FIG. 2. Linear synthesis of infectious virus and structural proteins in Spinner cultures of KB cells under normal one-step growth conditions. Top: Cellassociated fluorescent focus units (FFU) in per cent of maximum yield at 10 to 64 hr postinfection. Bottom: Production of viral antigens measured as micrograms of protein per 106 cells. The antigens were measured with radial immunodiffusion by using specific antisera.

Incorporation of arginine into protein in control and virus-infected cells. To reveal differences in arginine incorporation into protein between infected and control cells, Spinner cultures of KB cells were infected at a multiplicity of 100 FFU/ cell in normal medium. Control cultures were examined in parallel. At intervals, 106 cells were withdrawn and pulse-labeled for 30 min at 37 C with 3H-arginine at 1 ,uCi/ml. Cells were assayed for total protein, trichloroacetic acid-insoluble radioactivity, and virus infectivity. Figure 3 shows that the rate of arginine incorporation in control cells was constant over the period examined. On the other hand, there appeared to be a slight increase in the rate of arginine incorporation in infected cells up to 12 to 14 hr after infection, coinciding with the logarithmic rise in structural proteins. At this time of maximum arginine incorporation

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FIG. 3. H-arginine incorporation in protein at intervals in a one-step growth curve of infected cells. Uninfected cells were included as a control. The percentages of the maximum rate of arginine incorporation in infected cells calculated as trichloroacetic acid-insoluble radioactivity per milligram of protein per 30min pulse is plotted versus the time of incubation. Time zero is the time of infection. TABLE 1. Pools offree amino acids in KB cells after incubation in complete medium (1.2 mM) and arginine-deprived medium (0 uM)a Amin Normalmediu Nomlmeim Amino acid d cells) (uamoles/108

Arginine.. Lysine .......... Proline ..........

Glycine.. Alanine ......... Leucine ......... a

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746

EVERITT, SUNDQUIST, AND PHILIPSON

two equal parts. One was transferred to argininedepleted medium, and the other was kept in normal medium as a control. After 2 hr of additional incubation, the depleted culture was reversed to 1.2 mm arginine. Samples were analyzed by the isotope dilution method described above. By 15 min after removal of arginine, the pool was decreased by 80%;o, and at 15 min after reversion to normal arginine concentration the cellular arginine pool was completely restored

(Fig. 4).

Growth of uninfected KB cells at different concentrations of arginine. Elimination of single amino acids from the medium of mammalian cells drastically influences the synthesis of macromolecules (14). Therefore, it was necessary to study the effect of arginine depletion on uninfected KB cells. Monolayers of KB cells were trypsinized, washed, and incubated on plates in medium with final concentrations of arginine ranging from 0 to 1,200 Mm. At zero time and at 24 and 48 hr after plating, the cells were gently scraped off in their growth medium, washed in PBS, and resuspended in 0.1 M NaOH, and the total cell protein was determined. The amount of protein synthesized decreased when arginine was eliminated, but in the presence of 50 UM arginine cell protein remained constant (Fig. 5). Higher concentrations of arginine allowed an increase in

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TIME (HOURS) FIG. 4. Cellular pool of free arginine at arginine depletion (O A,M) and upon reversion to normal concentration (1.2 mm) expressed as per cent of the maximum concentration in micromoles per microgram of protein ini control cells. The arrow A indicates the time point of depletion, antd B indicates the time point of reversion. Symbols: *, cells exposed to depletioni anid reversion; 0,

control cells.

J. VIROL.

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