ph-dependent exposure of endoproteolytic cleavage sites of the adenovirus 2 hexon protein

FEMS Microbiology Letters 49 (1988) 229-233 229 Published by Elsevier

FEM 03039

pH-Dependent exposure of endoproteolytic cleavage sites of the adenovirus 2 hexon protein

Einar Everitt, Mikael J. Persson and Claes Wohlfart

Department of Microbiology, University of Lund, Sweden

Received 23 March 1987 Revision received 18 September 1987

Accepted 22 September 1987

Key words: Adenovirus 2; Hexon; Low pH; Conformational change; Dispase

1. SUMMARY

Physiological ionic strength conditions prevented low pH-mediated destabilization of the adenovirion. A conformational change of the virion was induced at low pH as demonstrated by endoproteolytic cleavage of virions with dispase at pH 5.0. Hidden cleavage sites of the hexons were exposed and upon enzymatic digestion, virions still were intact as physical entities. Enzymatic cleavage of the hexon protein increased its hydrophobicity.

2. INTRODUCTION

somal membrane. Treatment of virus-infected cells with lysosomotropic agents increase the endo- somal pH and subsequently interferes with the viral release from these vesicles [6,7]. We have also shown that chemical cross-linking of the virus, anti-hexon antibody-mediated neutralization and treatment of cells with reagents affecting initial virion destabilization, all provoke virion accumu- lation inside cytoplasmic vesicles [8]. In this report we directly demonstrate that the hexon proteins of the virion at low pH expose novel sites for an endoproteolytic metalloprotease with specificities for sites harbouring hydrophobic amino acid residues.

Adenoviruses are internalized via a receptor- mediated endocytotic pathway [1-3], and possibly also by an alternative mechanism of 'direct' penetration [4,5]. To escape from endocytic vesicles, endosomes, the virions hypothetically expose hydrophobic sites of the capsid in response to the low pH conditions of this compartment, and thereby interact with and penetrate the endo-

Correspondence to: E. Everitt, Department of Microbiology, University of Lund, S61vegatan 21, S-223 62 Lurid, Sweden.

3. MATERIALS AND METHODS

3.1. Cells and virus

Human adenovirus 2 (Ad2) was propagated in HeLa cells, metabolically labelled with [3H]thymidine (Amersham) and purified as described previously [9-11]. The virus was extensively dialysed against autoclaved 10% glycerol, I mM MgC12 in 50 mM Tris-HC1 buffer, pH 8.1 (freeze buffer), passed through 0.22-/xm filters (Millex GV, Milli-

0378-1097/88/$03.50 © 1988 Federation of European Microbiological Societies

230

pore) and stored unfrozen at 8 °C until used. The specific radioactivity of the 3H-labelled virions was 7.05 × 104 cpm/101° virions.

3.2. Analysis if virion destabilization and aggregation

Destabilization of [3H]thymidine-labelled virions was monitored as viral genome sensitivity to exogenously added DNase I as previously described [3]. Spontaneous and antifiber-mediated virion aggregation was quantified by analytical rate sedimentation centrifugation in sucrose gradients as recently described [12].

3.3. Purification of the Ad2 hexon protein Preparat ive anionic ch roma tog raphy on

DEAE-cellulose (Whatman) of the excess pool of structural proteins accumulating late in infection was performed as described previously [13]. Fur- ther purification of the hexon protein was obtained by gel exclusion chromatography on a Sep- harose CL-6B column (1.5 × 85 cm) at a rate of 4.3 m l / h / c m z with 0.15 M NaC1 in 50 mM Tris-HC1, pH 7.5, as eluting buffer. The hexon was extensively dialysed against autoclaved 0.15 M NaC1, passed through a 0.22-/~m Millex GV- filter and stored at 8°C until used. The specific radioactivity of the [35 S]methionine-labelled hexon was 5.40 × 104 cpm/ t tg protein.

3. 4. Hydrophobicity analysis of the hexon protein The assay according to Seth et al. [14] was

followed with 50 mM MES buffers for the indicated pH-values.

3.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

Linear gradient (6-20%) polyacrylamide slab gels were run and stained as described earlier [13]; after drying under vacuum the gels were directly autoradiographeu on Hyperfilm-/3max (Amers- ham).

3.6 Proteolytic digestion of cirions and hexon protein

Unfrozen virions were diluted 40 times in 0.15 M NaC1 buffered with 0.01 M Na-acetate/acet ic acid, pH 5.0 or 0.01 M Tris-HC1, pH 7.5. The

samples were preincubated at 37°C for 30 min. The proteolytic enzymes, frozen as stock solutions of 10 m g / m l in 0.015 M NaC1 (thermolysin, dispase I, chymotrypsin A 4 from Boehringer Man- nheim, and subtilisin and trypsin from Sigma), were prior to use diluted 25 75 times in 0.15 M NaC1 containing 50 mM CaC12- From such freshly made working solutions appropriate volumes were removed and added to virions to give calculated ratios of virion protein over enzyme protein. The final concentration of CaC12 was always 2.5 mM~ and incubations were at 37 °C for indicated periods of time. Preparations of hexon protein in 0.15 M NaC1 were added 1 /10 volumes of 0.1 M stock solutions of the pH 5.0 and 7.5 buffers above, and subsequently the procedure for virions was followed as described above.

4. RESULTS AND DISCUSSION

Under low ionic strength conditions and upon lowering of the pH, the adenovirion undergoes destabilization, with a concomitant loss of infectivity [15]. As shown in Fig. 1, such a partial degradation could efficiently be prevented under physiological ionic strength conditions. Already one cycle of freeze-thawing in the freeze buffer, which fully retains infectivity, rendered the virions below pH 6 twice as sensitive to the DNAse-treatment as unfrozen control virions at pH 7.5. Stor- age of virions at 8°C for 11 days caused no detectable change in DNAse sensitivity; longer periods up to 105 days increased the degree of destabilization at low pH to the level of virions frozen and thawed once. These observations em- phasize the importance of avoiding freeze-thawing and non-physiological ionic strength conditions when looking for possible pH effects on the stabil- ity and structure of adenovirions. Since the pH of endocytic vesicles is about 5 [16], and this pH was the highest pH causing a maximum degree of destabilization under hypotonic conditions, pH 5 was chosen to somewhat mimic the endocytic ves- icle as the acidic counterpart of the control series of pH 7.5. An increase in the intra-vesicular pH obstructs the adenovirus-mediated release of an EGF-conjugated Pseudomonas exotoxin adminis-

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Fig. 1. pH-dependent destabilization of Ad2 virions. Five /~1 samples of [3H]thymidine-labelled Ad2 ( = 1.8 × 10 l° virions) were incubated at 37 ° C for 30 min in 200 ~tl of the following buffers: 0.01 M Na-aceta te /acet ic acid, pH 4.0, 4.5, 5.0, 5.3 and 5.6; 0.01 M Tris-maleate , pH 6.0 and 6.5, and 0.01 M Tr is -HCl , pH 7.0, 7.5 and 8.0. Freshly prepared unfrozen virus in buffer (O) and in buffer with 0.15 M NaCI (O) was assayed for DNAse sensitivity, and 100% corresponds to a degradation of the entire viral genome. Virus once frozen and thawed and assayed in buffer with 0.15 M NaCI (I). The filled circles indicated by 1, 2 and 3 show DNAse sensitivity in 0.15 M NaCI of unfrozen virus stored at 8 ° C for 11, 105 and 150 days, respectively. Destabilization of such virus at pH 7.5 was as for control virus.

tered to cells together with adenoviruses [6]. Like- wise, pH-raising lysosomotropic agents apparently delay the escape of adenovirions from endocytic vesicles as directly observed in the electron mi- croscopy [7]. Thus, an exposure of hydrophobic sites of the virion has been suggested as an ex- planation for the low pH-induced and adenovirus-mediated 51Cr-release of virus-treated cells [17]. Taken together we believe that a hypothetical pH-induced conformational change of the adenovirion at physiological ionic strength was directly demonstrated by comparing the proteolytic cleavage patterns obtained at pH 5.0 and 7.5 (Fig. 2).

231

At both pH-values and for the two bacterial endoproteolytic metalloproteases recognizing hydrophobic amino acid residues at the cleavage site (thermolysin and dispase; for both EC 3.4.24.4), the penton base disappeared from its position in the SDS-gels, indicating that all available cleavage sites were maximally exposed irrespective of the pH. On the other hand, the hexon protein revealed different cleavage patterns at pH 6.0 and 7.5 for thermolysin and dispase, and also one fast migrat- ing polypeptide of an estimated M r of 15 kDa specifically appeared upon digestion at pH 5.0. The differences displayed at pH 5.0 and 7.5 were demonstrated not to be due to drastic changes in enzyme activity, since in an assay system with

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Fig. 2. Stained electropherogram of enzymatically cleaved Ad2. Electrophoresis on a 6-20% linear gradient gel was done on samples containing unlabelled virions ( = 4.2 ttg) and 1.3 fig enzyme, previously incubated for 15 min in the buffer systems of pH 5.0 (lanes 1, 3 and 5) and pH 7.5 (lanes 2, 4 and 6). The pairs of sample wells ter, dis and chy show virions treated with thermolysin, dispase and chymotrypsin, respectively. Lanes 7 and 8 are Ad2 markers treated as controls without enzymes at pH 5.0 and 7.5, respectively. On each side of the small letters a, b and c the polypeptides of thermolysin, dispase and chymotrypsin are located, respectively. The polypeptides of the hexon (II), penton base (III), fiber (IV), proteins V, VII, VIII and IX are indicated by their corresponding molecular masses of 120 kDa, 85 kDa, 62 kDa, 48.5 kDa, 18.5 kDa, 13 kDa and 12 kDa, respectively [20]. The 15kDa polypeptide is marked by a double arrow head in the left margin.

232

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Fig. 3. Sucrose gradient sedimentation of Ad2. The left and right hand columns show the sedimentation of 1 .8x101° [3H]thymidine-labelled virions ( = 5 /~g protein) after various treatments at pH 5.0 and 7.5, respectively. Panels a and b demonstrate virus sedimentation of control series; c and d after anti-fiber antibody precipitation; e and f after a 30-min dispase digestion (virus pro te in /enzyme ratio = 3.5/1); g and h antifiber precipitation after dispase treatment. The low recovery of radioactivity after immunoprecipitat ion is solely due to quenching.

azoalbumin as the substrate in a 10-fold molar excess over the enzyme [18], dispase exerted 90% of its activity at pH 5.0 as compared with the control situation at pH 7.5. Dispase was chosen for the further studies and the physical status of enzyme-digested [3H]thymidine-labelled virions was examined by rate sedimentation analysis in sucrose gradients in the absence and presence of antifiber antibodies. As shown in Fig. 3, the virions were not spontaneously aggregated due to the low pH treatment alone, and after enzyme digestion the virions also retained the penton base- anchored fiber antigen in proper position available for quantitative antifiber-mediated immunoag- gregation. Destabilization analyses revealed that

the virions were fully intact. Since the hexon protein showed different cleavage patterns at pH 5.0 and 7.5, this protein was digested separately with dispase. The number of pH-dependent cleavage products for this enzyme was limited and maximal within 30 min, and one polypeptide fragment of 15 kDa migrated in the position of a tryptic fragment (Fig. 4). Control led cleavage of [3H]amino acid-labelled hexon protein and subse- quent recovery of radioactivity from SDS gels demonstrated that the ratio obtained for the number of created 15 kDa fragments over the number of initial hexon polypeptides was 0.88; quantitative determination of the specific radioactivity of incorporated [35S]methionine residues of the intact hexon polypeptide and the fragment revealed that the latter contained 3.3 + 0.3 (SD), (n = 2) methionine residues. Based on this information we suggest that the fragment of this size and methionine content should tentatively be cleaved off from the N-terminal end of the 967 amino acid residues long hexon polypeptide - possibly close to one of the established tryptic cleavage sites [19].

7 8 I I

I I I

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Fig. 4. S D S - P A G E of enzymatically cleaved hexon protein. [35S]hexons were digested at pH 5.0 (lanes 1, 3 and 5) and at pH 7.5 (lanes 2, 4 and 6) for 15 min at a hexon /enzyme ratio of 5 /1 . Twenty #1 samples ( = 1 . 3 ~g) were withdrawn and electrophoresed on a linear gradient gel; sample-well pairs dis, try and sub show hexons treated with dispase, trypsin and subtilisin, respectively. Lane 7 contains an [35S]Ad2 marker and lane 8 hexons incubated at pH 5.0; some Ad2 marker polypeptides are indicated.

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Fig. 5. IAydrophobicity of the hexon protein. The partitioning for 0.33-t~g samples of 35S-labelled hexon protein between various buffers and a Triton X-114 phase was determined. The relative figures for hexon association with the hydrophobic detergent phase were normalized to the untreated control series of pH 7.5. Hexons treated with buffer at pH 5.0 (©), digested with dispase for 15 min at a hexon protein/enzyme ratio of 5/1 at pH 5.0 (O) and at pH 7.5 (m).

The exposure of hypo the t i c a l l y h y d r o p h o b i c re- g ions of the h e x o n p ro t e i n u p o n ac id i f i ca t ion of the e n v i r o n m e n t a n d a poss ib le ' f r eez ing ' of a n e w

c o n f o r m a t i o n a l s tage u p o n d ispase c leavage was s tud ied b y the re la t ive d i s t r i b u t i o n of the h e x o n in a T r i t o n X-114 two-phase sys tem. As s h o w n in Fig. 5, the h y d r o p h o b i c p rope r t i e s o b t a i n e d at ac idic p H va lues were d ras t i ca l ly e n h a n c e d u p o n d i spase d iges t ion - a n effect b e i n g less p ro - n o u n c e d u p o n d iges t ion at p H 7.5 or af ter mere i n c u b a t i o n in low p H buffer . In s u m m a r y , the p r e sen t ed d a t a s u p p o r t a suggested n o t i o n of a

p H - d e p e n d e n t c o n f o r m a t i o n a l change of s t ruc t - u ra l p ro t e ins w i th in the a d e n o v i r i o n capsid, a n d

fu r the r s tudies o n the poss ib le o b s t r u c t i o n of b io - logical f unc t i ons af ter d i spase d iges t ion of v i r ions w o u l d be reward ing .

233

Th i s s t udy was s u p p o r t e d b y the Swedisch N a t - u ra l Sc ience R e s e a r c h Counc i l , C r a f o o r d s k a Stif- te lsen a n d K u n g l . Fys iog ra f i ska S~illskapet, b o t h in L u n d .

R E F E R E N C E S

[1] Dales, S. (1973) Bact. Rev. 37, 103-135. [2] FitzGerald, D.J.P., Padmanabhan, R., Pastan, I. and Wil-

lingham, M.C. (1983) Cell 32, 607-617. [3] Svensson, U. and Persson, R. (1984) J. Virol. 51,687-694. [4] Morgan, C., Rosenkranz, H.S. and Mednis, B. (1969) J.

Virol. 4, 777-796. [5] Brown, D.T. and Burlingham, B.T. (1973) J. Virol. 12,

386-396. [6] Seth, P., FitzGerald, D.J.P., Willingham, M.C. and Pastan,

I. (1984) J. Virol. 51,650-655. [7] Svensson, U. (1985) J. Viriol. 55,442-449. [8] Everitt, E., Svensson, U., Wohlfart, C. and Persson, R.

(1986). In: Virus Attachment and Entry into Cells (Crowell, R.L. and Lonberg-Holm, K., Eds), pp. 196-204. American Society for Microbiology, Washington, D.C.

[9] Edvardsson, B., Everitt, E., J6rnvall, H., Prage, L. and Philipson, L. (1976) J, Virol. 19, 533-547.

[10] Svensson, U., Persson, R. and Everitt, E. (1981) J. Virol. 38, 70-81.

[11] Everitt, E., Meador, S.A. and Levine, A.S. (1977) J. Virol. 21, 199-214.

[12] Wohlfart, C.E.G., Svensson, U.K. and Everitt, E. (1985) J. Virol. 56, 896-903.

[13] Wohlfart, C. and Everitt, E. (1985) Virus Res. 3, 77-85. [14] Seth, P., Willingham, M.C. and Pastan, I. (1985) J. Biol.

Chem. 260, 14431-14434. [15] Prage, L., Pettersson, U., HOglund, S., Lonberg-Holm, K.

and Philipson, L. (1970) Virology 42, 341-358. [16] Mellman, I., Fuchs, R. and Helenius, A. (1986) Ann. Rev.

Biochem. 55,663 700. [17] Seth, P., Willingham, M.C. and Pastan, I. (1984) J. Biol.

Chem. 259, 14350-14353. [18] Kroll, S. and Klostermeyer, H. (1984) Lebensm. Unters.

Forsch. 178, 179-196. [19] JiSrnvall, H., Akusj~irvi, G., Alestr6m, P., von Bahr-Lin-

dstr6m, H., Pettersson, U., Appella, E., Fowles, A.V. and Philipson, L. (1981) J. Biol. Chem. 256, 6181-6186.

[20] Anderson, C.W., Baum, P.R. and Gesteland, R.F. (1973) J. Virol. 12, 241-252.

A C K N O W L E D G E M E N T S

T h e technica l ass i s tance b y B l a n k a Boberg is gra tefu l ly apprec ia t ed .

ph-dependent exposure of endoproteolytic cleavage sites of the adenovirus 2 hexon protein

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