Degradation of adenovirus by isolated KB cell membranes

EXPERIMENTAL

AND

MOLECULAR

PATHOLOGY

Degradation

326-333 (1972)

17,

of Adenovirus KB Cell

by

Isolated

Membranes

P. A. BOULANGER AND R. WAROCQUIER Unit&

de Rechetches

SW la Virologie Received

de I’I. April

N. S. E. R. M.,

Lille,

France

%,1972

Adenovirus type 5 labeled with ‘H-thymidine was adsorbed onto isolated KB cell plasma membranes. After incubation with them, 45% of the label became deoxyribonuclease sensitive and was converted to a trichloroacetic acid-soluble form. After further incubation with trypsin or with KB cell nuclear membranes, 65-70% of the label was rendered deoxyribonuclease accessible. When ‘H-valinelabeled adenovirus was incubated with KB cell plasma membranes, a negligible amount of protein label was converted to an acid-soluble form, and after a further incubation with nuclear membranes, only 4% of the protein was released as free peptides and amino acids. Trypsin released 6% of the protein label as soluble material in control virus preparation, whereas it converted 25% of the label to a soluble form in adenovirus preparation previously incubated with plasma membranes. These results suggest that adenovirus uncoating is a two-step process whose first step, occurring at the plasma membrane, would be a labilization of the virus capsid, and the second one, occurring at the nuclear membrane, would be a proteolytic degradation of the labilized capsid.

The electron microscopic and biochemical aspects of adenovirus adsorption, penetration, and uncoating have been well documented in several studies (Lawrence and Ginsberg, 1967; Philipson, 1967; Sussenbach, 1967; Schlesinger, 1969). First, it, had been thought that adenovirus enters the cell by phagocytosis and is uncoated within phagosomes (Dales, 1962), but further investigations showed that adenovirus can penetrate directly through the cell membrane (Morgan et ai., 1969) and that viral uncoating follows penetration closely (Lawrence and Ginsberg, 1967; Morgan et al., 1969). If uncoating is defined as-to quote Lawrence and Ginsberg (1967)-(‘the development, of sensitivity of the parental viral nucleic acid to deoxyribonuclease,” evidence has been recently provided (Lonberg-Holm and Philipson, 1969) that adenovirions are uncoated in close vicinity of the plasma membrane, as they gain entry into the cytoplasm. Moreover, in vitro experiments using purified lysosomal fractions have shown that lysosomal enzymes are incapable of uncoating adenovirus particles and further supported the assumption that an “uncoating factor” is located at the cell surface (Boulanger et al., 1970 a). It was the aim of the present work to obtain new data on the localization and mechanism of the adenovirus uncoating process by in vitro study of the action of KB cell plasma and nuclear membrane preparations on adenovirus particles. 326 Copyright All rights

Q 1973 by Academic Press. of reproduction in any form

Inc. reserved.

ADENOVIRUS

DEGRADATION

MATERIALS

BY KB CELL

AND

MEMBRANES

327

METHODS

Virus Growth and Purification

Adenovirus type 5 was propagated in KB cell spinner cultures in Eagle’s medium supplemented with 5% horse serum. Virus was extracted with fluorocarbon and purified by two cycles of centrifugation in CsCl gradient (Green and PiEa, 1964). Isotopically

Labeled Virus

Labeling adenovirus DNA with 3H-thymidine, 1 &X/ml (sp act 20 Ci/ mmole), was carried out from 630 hr postinfection. The proteins of adenovirus particles were labeled with 1 &i/ml 3H-L-valine (sp act 1 Ci/mmole), and incorporation of isotope was enhanced by reducing 10 times the concentration of cold valine in the nutrient medium. Isolation and Purification

of

KB Cell Plasma Membranes

The “Tris-method” of Warren et al. (1966) was employed for the isolation of the surface membranes from KB cells. The membranes were harvested from the band at the top of the 45% sucrose layer of the final centrifugation stepwise sucrose gradient. The yields of this procedure were 0.8-1.0 ml of plasma membrane per 20 ml of packed whole cells (corresponding to 3-4 x 10e cells). The membrane pellet was washed three times in MST buffer (10e3 M MgC12, 0.25 M sucrose, 0.02 M tris-hydroxymethyl-aminomethane, pH 7.4)) and resuspended in the same buffer. Purity of the membrane fraction was controlled by electron microscopy. It appeared inutile to test for eventual lysosomal contaminations by enzyme assays, since lysosomal enzymes have been proved inefficacious against adenovirions and their soluble antigens (Boulanger et al., 1970 a). Isolation and Purification of KB Cell Nuclear Membranes

The procedure of Franke et al. (1970) was used. The approximate nuclear membranes was 0.2-0.3 ml/10 ml packed nuclei.

yield of

Biochemical Determinations

Protein was measured by the method of Lowry et al. (1951), with bovine serum albumin (BSA) used as a standard. Radioactive acid-precipitable material was extracted three times with cold trichloroacetic acid (TCA) 5%, then collected on membrane filters, dried, and counted in a liquid scintillation spectrometer. The 3H-TCA-soluble radioactivity corresponding to released amino acids and peptides from labeled adenovirus proteins was determined after filtration on Millipore membrane (0.45-pm pore size) by counting a l-ml aliquot from the filtrate in Bray’s solution. Adsorption of Adenovirus to Plasma Membranes

The plasma membrane pellet was resuspended in a modified MST buffer used for adsorption, containing 0.05 in lieu of 0.01 M MgC12 and labeled adenovirus

328

BOULANGER

AND WAROCQUIER

particle suspension was added to the membrane preparation at a multiplicity of 10810’ infecting cell units (ICU, Warocquier et al., 1966) per mg of plasma membrane protein. Adsorption was allowed to proceed for 2 hr at OC with stirring. The mixture was then centrifuged at 5360g for 20 min, and the membrane pellet washed twice with 0.05 Mg 2+ MST buffer. The two washes were combined with the original supernatant fraction. The labeled adenovirions contained in the washed membrane pellet fraction was designated as plasma membrane-adsorbed virus (PmAV) , the pooled supernatant fraction as unadsorbed virus (UAV), and adenovirus not exposed to plasma membrane as control virus (CV). Under these conditions lO-15% of adenovirus label was adsorbed onto plasma membrane. RESULTS Tentative Localization of the Uncoating Processin Situ

KB cells maintained in suspension in culture medium (l-2 X lo6 per ml) were treated with deoxyribonuclease I (DNase EC, Sigma Co, 100 rg per ml) during the period of adenovirus adsorption, and the production of infectious adenovirions was studied by titrating the virus progeny yielded at the end of the multiplication cycle (Warocquier et al., 1966). Relatively low input multiplicities were employed (two to five particles per cell) to sensitize the assay. The activity of deoxyribonuclease employed during the period of virus adsorption was controlled by determining its activity against calf thymus DNA as substrate under the same conditions, i.e., in the adsorption medium containing the same KB cell concentration: no inhibition of the deoxyribonuclease activity was found. A slightly but constantly lower virus particle production was observed in deoxyribonuclease-treated KB cells: 46% under untreated cells (average of five separate experiments). Effects of Incubating Adenovirus with KB Cell Membranes on DeoxyribonucleaseSensitivity of the Viral Nucleic Acid

PmAV, UAV, and CV were incubated in parallel in MST buffer for 2 hr at 37°C. Aliquots were removed at intervals, further incubated with deoxyribonuclease (100 pg of enzyme per ml) for 1 hr at 37”C, and tested for TCA-precipitable radioactivity. The DNA label of CV and UAV remained deoxyribonuclease resistant: 95-85s of the DNA remained in a macromolecular form. In the Pm AV preparation, 4045% of the label was converted to a deoxyribonuclease-accessible form during incubation (Fig. 1A). An extensive alteration of the virus particles was produced by incubating the preparations with trypsin and with KB cell nuclear membrane preparation. CV, UAV, and PmAV preparations were incubated in parallel 2 hr at 37”C, aliquots were withdrawn at intervals and incubated with trypsin (100 pg per ml) for an additional 1 hr at 37°C. At the end of this incubation period, trypsin was inhibited with 1% diisopropylfluorophosphate (10 ~1 per ml), and each aliquot was then further incubated with deoxyribonuclease for 1 hr at 37°C and finally

ADENOVIRUS

DEGRADATION

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329

FIG. 1. Kinetics of degradation of aH-thymidine-labeled adenovirus by KB cell membranes. Virus was adsorbed for 2 hr at OC on plasma membrane (Pm) and then centrifuged. Virus adsorbed to the Pm (PmAV), virus that remained in the supernatant fraction (UAV) and control virus (CV) unexposed to Pm were then incubated in parallel for 2 hr at 37°C. Aliquots were withdrawn at intervals as indicated on the graph. One series of samples (A) were incubated with 100 rg of deoxyribonuclease per ml for 1 hr at 37°C and processed for TCA-precipitable counts. Another set of samples (B) was incubated with 100 pg of t.rypsin per ml for 1 hr at 37”C, trypsin inhibited by diisopropylfluorophosphate and samples further incubated with 100 pg of deoxyribonuclease for 1 hr at 37°C and then assayed for TCA-precipitable counts. The third set (C) was incubated consecutively first with nuclear membrane for 1 hr at 37”C, then with deoxyribonuclease, and finally processed for TCA-precipitable radioactivity. Counts are plotted against time of incubation as percentage of the TCA-precipitable counts at zero time. In A, the total number of counts (100%) involved were 1800,3300, and 2250 cpm for CV, UAV, and PmAV. respectively. In B, 1700, 3600, and 1200 cpm. In C, 1500, 3100, and 1050 cpm. The percentage of TCA-precipitable counts in PmAV preparation without subsequent deoxyribonuclease treatment remained at over 98%, indicating a negligible level (if any) of deoxyribonuclease activity at pH 7 in the membrane preparation. Symbols: q , CV; 0, UAV; A, PmAV.

tested for TCA-precipitable radioactivity. In CV, UAV, and PmAV preparations consecutively treated with trypsin and deoxyribonuclease, 65% of the label became acid-soluble at the samerate of conversion (Fig. 1B). In the same way, aliquots from CV, UAV, and PmAV preparations were removed at intervals during the 2-hr incubation and further incubated with KB cell nuclear membrane preparation (0.5 ml of a nuclear membrane suspension obtained by resuspending 0.5 ml of nuclear membrane pellet in 10 ml MST buffer, per ml incubat’ion mixture) for 1 hr at 37°C. The aliquots were then treated with deoxyribonuclease for 1 hr at 37°C and tested for TCA-precipitable counts. The DNA label of CV and UAV treated with nuclear membranes remained deoxyribonuclease resistant, and only a low percentage became acid soluble, 20 and 40%, respectively. When adenovirus was consecutively incubated with KB cell plasma membrane (PmAV) and with nuclear membrane, 70% of the label became deoxyribonculease accessibleand acid soluble (Fig. 1C). Effect of Incubating Proteins

Adenovirus with KB

Cell Membranes on Viral

Capsid

In order to determine which alteration occurred in virus capsid, “H-valine-labeled adenovirus was used. CV, TJAV, and PmAV preparations were incubated in parallel and three aliquots of each preparation were removed at intervals during

BOULANGER

330

0

30

AND WAROCQUIER

60

90

120

MINUTE5

Release of TCAsoluble amino acids and peptides from aH-valine-labeled adenovirus after incubation with KB cell membranes. Control (CV), unadsorbed (UAV), and plasma membrane-adsorbed (PmAV) adenovirus preparations were incubated in parallel for 2 hr at 37°C. Aliquots were removed at intervals as indicated. One set of samples (dark symbols) was processed for TCA-soluble counts; another set of samples (open symbols) was incubated with 100 N of trypsin per ml for 1 hr at 37°C and then assayed for TCA-soluble radioactivity; the third set (half-tilled symbols) was incubated with nuclear membrane for 1 hr at 37°C and then counted for TCA-soluble radioactivity. Counts are plotted against time of incubation as percentage of the total TCA- precipitable counts at zero time. The total numbers of counts (100%) involved were, respectively 445 X 108,460 X lOa, and 682 X 10’ cpm for untreated, trypsintreated, and nuclear membrane-treated CV; 355 X lo”, 362 X lo* and 126 X l@ cpm for UAV treated in the same way; 31.5 X 108,27.6 X IO*, and 16.5 X 10’ cpm for PmAV. Symbols: 0, CV; 0, UAV; A, PmAV. FIG.

2.

the 2-hr incubation period. One aliquot was precipitated with TCA, the second one was further incubated with trypsin (100 pg per ml) for 1 hr at 37°C and then TCA precipitated, and the third one was incubated with nuclear membrane

preparation for 1 hr at 37°C and TCA precipitated. were tested for TCA-soluble radioactivity. Figure

2 shows

that only

about 0.2% of total

The three series of aliquots initial

protein

label was con-

verted spontaneously to a soluble form in CV and UAV incubated for 2 hr in MST, and 0.5% in PmAV preparation. When CV, UAV, and PmAV were treated with

nuclear membranes

for a further

1 hr at 37”C, the peptides

and free amino

acids released from the viral capsid account for 24% of the initial protein label. About 6% of this protein label was converted to a soluble form in CV and UAV preparations incubated with trypsin. In PmAV preparation, trypsin released 25% of the protein label as free amino acids and peptides. Trypsin treatment on aH-valine-labeled adenovirus sequentially exposed to both plasma and nuclear membranes released a maximum of 25% of the protein label.

ADENOVIRUS

DEGRADATION

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MEMBRANES

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DISCUSSION

From the results of these in vitro experiments it appears that KB cell plasma membrane is active on adenovirus capsid: nearly half of the viral DNA became deoxyribonuclease accessible in 60- to 90-min incubation at 37°C. After incubating CV, UAV, and PmAV 1530 min in isotonic buffer and subsequently with trypsin, two thirds of adenovirus DNA became deoxyribonuclease sensitive. KB cell nuclear membrane alone does not affect the integrity of adenovirus particles since 80% of the viral DNA remains deoxyribonuclease resistant. However, adenovirions consecutively incubated, first with plasma membrane, then with nuclear membrane, were extensively modified and 70% of the viral DNA was converted to a deoxyribonuclease sensitive form. The KB cell plasma membrane does not seem to possess a proteolytic action on virus particles, as shown in Fig. 2: the same extent of proteolysis was observed in control and membrane-adsorbed adenovirus preparations. In the contrary, a proteolytic effect occurs with nuclear membrane, with a degree of proteolysis similar in control, unadsorbed, and plasma membrane-adsorbed adenovirus preparations. In spite of this apparent similar degree of alteration, the DNA of PmAV was deoxyribonuclease sensitive to a much higher extent than DNA of control and unadsorbed virus. The protein bulk released from the capsid by trypsin in CV and UAV preparations (6% of protein label) is in good agreement with previous experiments of chemical and enzymatic degradation of adenovirus capsid (Valentine and Pereira, 1965; Russel et al., 1967; Prage et al., 1970; Pereira and Skehel, 1971) : the penton base (the point of weakness of the adenovirus capsid) is sensitive to tryptic digestion and the whole penton accounts for about 5% of the capsid protein (Sussenbach, 1967) and 3-4% of the total viral protein (Schlesinger, 1969). The small discrepancy between the percentage of soluble label released from the virion in our experiments (6%) and the theoretical relative amount of penton in adenovirus (4%) might be interpreted in two manners: either valine (hot valine is used for labeling) is not evenly distributed in the adenovirus capsid and core proteins (Petterson and Hoglund, 1969; Boulanger et al., 1969, Boulanger et al., 1970 b, Laver, 1970), or trypsin releases not only the penton but some other protein(s) from the viral capsid. The effect of trypsin on adenovirus incubated on plasma membrane is particularly net: after 30-min incubation on plasma membrane followed by trypsin digestion, 25% of protein label was released in soluble form. Since it is known that internal core accounts for 1820% of the adenovirus protein moiety (Laver et al., 1967), the released part of protein label could represent the penton and most of the core proteins digested by trypsin after plasma membrane action. The results herein presented are more easily understandable if it is postulated that adenovirus uncoating is a two-step process: the first step occurring at the plasma membrane would be a virus labilization (the “labilizer” in the plasma membrane could be the same substance as the cell membrane receptor). That would render the virus sensitive to further alteration and permit a more extensive degradation of the virus capsid and core by proteases such as trypsin. Such a labilization of virus particle by susceptible cell membrane has been described in

332

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AND WAROCQUIER

the case of poliovirus (Chan and Black, 1970). The second step occurring at the nuclear membrane could be a proteolytic action achieving the degradation of labilized capsid and opening it to passage of DNA-core complex. That an energetic process should be involved in this transfer has been recently shown by Chardonnet and Dales (1972). The data of our in vitro biochemical experiments confirm the morphologic observations of Morgan et al. (1969) and the results of in viva investigations by Sussenbach (1967) and Philipson (1967) on adenovirus uptake and eclipse, and seem to exclude the role of lysosomes as the main uncoating pathway (Boulanger et al., 1970a). The fact that adsorption of adenovirus processed at low multiplicity in the presence of deoxyribonuclease affects slightly the viral progeny yield suggests that penetration of adenovirus through phagocytotic vacuoles is presumably a means-though not the predominant one-of uptake and uncoating. This result also supports the assumption that a certain degree of alteration of adenovirus capsid does occur in vivo at the plasma membrane. It remains to decide whether uncoating must be defined as any intracellular alteration of the capsid rendering the viral genome deoxyribonuclease accessible (e.g., labilization at the plasma membrane) or as the complete removal of the viral coat, as it occurs at the nuclear membrane (Morgan et al., 1969, Chardonnet and Dales, 1970), or as a two-step process including these two sequential phenomena. ACKNOWLEDGMENTS This investigation was supported by Contract CRL 71 50533 from the Inst,itut National de la Sante et de la Recherche Medicale. The excellent technical assistance of Mr. J. C. D’Halluin is gratefully acknowledged. REFERENCES P. A., FLAMENCOURT, P., and BISERTE, G. (1969). Isolation and comparative chemical study of structural proteins of the adenoviruses 2 and 5: hexon and fiber antigens. Eur. J. Biochem. 10,1X-131. BOULANGER, P. A., BREYNAERT, M. D., and BISERTE, G. (1970 a). Lysoeomes and the problem of adenovirus uncoating. Exp. Mol. Pathol. 12, 235-242. BOULANGER, P. A., JAIJME, F., FLAMENCOURT, P., and BISERTE, G. (1970 b). Acid soluble material of adenovirus. J. Viral. 5,109-113. CHAN, V. F., and BLACK, F. L. (1970). Uncoating of poliovirus by isolated plasma membranes. J. Virol. 5,309-312. CHARD~NNET, Y., and DALES, S. (1970). Early events in the interaction of adenovirus with HeLa cells. I. Penetration of type 5 and intracellular release of the DNA genome. Virology 40,46!2-477. CHARDONNET, Y., and DALES, S. (1970). Early events in the interaction of adenoviruses with HeLa cells. II. Comparative observations on the penetration of types 1,5, 7 and 12. Virology 40,478-485. CHARDONNET, Y., and DALES, S. (1972). Early events in the interaction of adenovirus with HeLa cells. III. Relationship between an ATPase activity in nuclear envelopes and transfer of core material : a hypothesis. Virology 48,342-359. DALES, S. (1962). An electron microscope study of the early association between two mammalian viruses and their hosts. J. Cell. Biol. 13,303-322. FRANKE, W. W., DEUMLING, B., ERMEN, B., JARASCH, E. D., and KLEINIG, H. (1970). Nuclear membranes from mammalian liver. I. Isolation procedure and general characterization. J. Cell. Bid. 46, 379-395. BOULANGER,

ADENOVIRUS

DEGRADATION

BY

KB

CELL

MEMBRANES

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Virology 42,341-358. C., VALENTINE, R. C., and PEREIRA, H. G. (1967). The effect of heat on the of the adenovirus. J. Gen. Viral. 1,50%522. SCHLESINGER, R. W. (1969). Adenoviruses : The nature of the virion and of controlling factors in productive or abortive infection and tumorigenesis. Adwan. Virus Res. 14, 1-61. SUSSENBACH, J. S. (1967). Early events in the infection process of adenovirus type 5 in HeLa cells. Virology 33,567-574. VALENTINE, R. C., and PEREIRA, H. G. (1965). Antigens and structure of the adenovirus. J. RUSSEL,

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WAROCQUIER,

particules

de

titrage

des

infectieuses

cells.

Methods

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Ass%. Vice

President