J. Mol. Biol. (1991) 217; 477-486

Functional and Structural Effects of an Ala to Val Mutation in the Adenovirus Serotype 2 Fibre Marie-Laure

Caillet-Boudin’, Pierre Lemay’“f and Pierre Boulanger2$

‘Unite’ de Virologie Mole’culaire de 1’INSERM Place de Verdun, 59045 Lille, France ‘Laboratoire Faculti

(U-233)

de Virologie et Pathoge’nBse Mole’culaires de Me’decine, Boulevard Hem-i IV 34060 Montpellier, France

(Received 6 April

1990; accepted 8 October 1990)

H2ts125 is a fibre-defective, temperature-sensitive mutant of adenovirus serotype 2. H2ts125 fibre is unstable at the non-permissive temperature (ts phenotype), and does not migrate in the same way as the wild-type fibre in an SDS/polyacrylamide gel (elm phenotype). Sequence analysis has shown that H2ts125 carries two mutations on the fibre gene: Leu105 to Phe, and Ala434 to Val. Analysis of the structural modifications occurring in H2ts125 fibre was performed using peptide finger-printing and antipeptide sera as immunological probes. We found that all the detectable structural alterations in the mutant fibre were due to the substitution on codon 434. In addition, the ts phenotype was rescued by a wild-type DNA fragment containing the 3’ moiety of the fibre gene and overlapping the 434th codon. Morphological analysis of fibre molecules observed under the electron microscope showed minor but statistically significant differences in the fibre length between mutant and wild-type. The mutant fibre was found to be slightly longer (3088f 1.9 A) than the wild-type fibre (3001 f2.1 A). Thus both ts and elm phenotypes were carried by the same Ala434 to Val mutation which probably resulted from a change in the threedimensional structure of the fibre protein, and not from some proteolytic cleavage.

1. Introduction

fibre (60 x lo3 compared to 62 x 103). The same apparent Mr of 60 x lo3 is exhibited by H2ts125 fibre synthesized by infected cells at the permissive temperature (33 “C), and by cell-free extracts in vitro (Boudin et al., 1983). Moreover, the mutant fibre polypeptides seem to undergo a normal glycosylation, N-acetylation, and assembly into 6 S oligomerit fibre capsomers and into 11 S penton capsomers (Boudin et al., 1983). Pulse-chase labelling experiments indicate that the 60 x lo3 Mr species is not derived from a normal 62 x lo3 M, polypeptide precursor, and mRNA mapping analysis has excluded the possibility of a spliced sequence within the mutant fibre messenger RNA (Boudin et al., 1983). Sequence analysis has shown two point mutations at nucleotide numbers 31,342 and 32,330, respectively (Boudin et al., 1983). These mutations generate two amino acid substitutions, Leu to Phe at codon 105, and Ala to Val at codon 434 on the fibre sequence. Although the oligomeric nature of the fibre is still a matter of controversy (Devaux et al., 1984, 1990;

A temperature-sensitive, fibre-defective mutant of adenovirus serotype 2 (Ad2§), H2ts125, has been isolated in our laboratory, phenotypically characet al., 1980; Martin et al., 1978) terized (D’Halluin and genetically mapped within the fibre region of the adenovirus genome (D’Halluin et al., 1982). Previous biochemical characterization of the mutant fibre has shown that H2tsl25-infected cells synthesize a fibre polypeptide that is unstable at 39.5”C (ts phenotype), and has an altered electrophoretic mobility (elm phenotype). H2ts125 fibre migrates in an SDS/polyacrylamide gel with a lower apparent relative molecular mass (JZ,) than the WT i Present address: Centre de Transfert en Biotechnologie et Mierobiologie, INSA, Avenue de Rangueil, 31077 Toulouse, France. $ Author to whom all correspondence should be addressed. 0 Abbreviations used: Ad2, adenovirus serotype 2; WT, wild-type; TP, terminal protein.

477 0022-2836/91/030477-10

$03.00/O

0 1991 Academic Press Limited

478

!?I.-L.

Caillet-Boudin

Lemay & Boulanger, 1980; Pettersson, 1984; va,n Oostrum & Burnett, 1985), the polarit,y of the a.denovirus fibre has been confirmed recently by experimental data, and unambiguously determined (Devaux et al., 1987; Caillet-Boudin, 1989; Weber et al., 1989). The N-terminal tail of the fibre binds to the penton base to form the penton capsomer, and the distal knob containing the C terminus is the site of recognition and attachment to the cell membrane receptor (Boulanger & Lonberg-Holm, 1981; Boulanger & Philipson, 1981; Hennache & Boulanger, 1977; Hennache et al., 1979; Nermut 1984; Pettersson, 1984; Svensson et al., 1982). In addition, it has been suggested that, the Pi-terminal tail of the fibre binds to complementary peptide sequences(s) on the penton base (Caillet-Boudin, 1989). In adenovirus serotype 2, the N and C termini are separated by 22 repeats of a consensus sequence that forms the shaft (Green et al., 1983). Amino acid substitutions in putative critical domains have been widely used to probe the stability and/or folding of proteins (for a review, see Alber, 1989; Shortle, 1989). In the light of the accumulated data, and the distinctive roles assigned to the ?I’ and C ends of the fibre molecule, it seemed interesting to re-examine the effect of the HBts125 mutations on the structure and physiology of the adenovirus fibre. In the present, study we show that the altered mobility of the mutant fibre in an SDS/polyacrylamide gel does not result from a proteolytic cleavage at the N or C terminus, but rather from a change in the secondary structure of the H2ts125 fibre, as suggested by the discrete morphological alterations observed in electron microscopy. In addition, we show that this structural ehange and the temperature-sensitive character are both due to the Ala434 to Val substitution.

2. Materials

and Methods

(a) Cell cultures

and viruses

Suspension cultures of KB cells were grown in Eagle’s spinner medium supplemented with 5% (w/v) horse serum. HeLa and 293 cells were cultured as monolayers in Dulbecco’s modified Eagle’s medium supplemented with 10% (w/v) newborn calf serum. Stocks of ts mutants were grown on HeLa call monolayers at 33°C. Ad2 WT titers were determined by the fluorescent focus assay on HeLa cell monolayers at 37”C, and titers expressed as f.f.u. (fluorescent focus units; Philipson et al., 1968). Adenovirus ts mutant titers were determined by the plaque assay on HeLa cell monolayers at 33°C and 395°C and expressed as plaque-forming units (p.f.u.). (b) Puri$cation viral

DNA

of virus and isolation of and of DNA

fragments

Ad2 particles produced at 37°C by WT or at 33°C by HZts125 were extracted from infected cells and purified in self-generating gradients as described (D’Halluin et al., 1978). Viral DNA-terminal protein (TP) complex was prepared as described (D’Halluin et al., 1984). Free viral DZLA was obtained by treatment of the virus with Sarkosyl-Pronase according to conventional techniques

et al.

(Pettersson & Sambrook, 1973). Ad2 DNA restriction fragments were purified in i y/, (w/v) agarose gels electro90 mM-Tris-borate buffer (pH 8.3); phoresed in 2.5 mM-Pu’a,EDTA. visualized with ethidium bromide and recovered by eledro-elution (Maniatis et aZ., 1982). re.scue of temperature-sensitive

(c) Xarker

mutation

Electrophoretically purified fragments from Ad2 WT DNA overlapping one or other mutation of the H2ts125 were co-precipitated onto 293 cells at 39°C along with H2ts125 DNA-TP complex, using the calcium phosphate technique (Arrand, 1978). Plaques were counted after 10 days at 395°C. (d) Radioactive labelling and purijieation adenovirus serotype 2 $bre

of

Fibre was uniformly labelled in viva by addition of “C-labelled sodium formate (50 to 60 mCi/mmol; Amersham; UK) at a final concentration of 1 PCi per ml of infected cell culture medium. Labellings were performed for 24 h at 37°C for the WT (at 16 h after infection), or for 72 h at 33°C for the H2ts125 mutant (at 48 h after infection). Native, soluble fibre was purified from t,he cellular pool of adenovirus-coded proteins by a 4-step procedure described by Boulanger & Puvion (1973). (e) Chemical

cleazage of Ad2 fibre

Cleavage at Asp-Pro bonds was performed using diluted HCl (10 to 50 rnM) at 56°C overnight, and Iiydrolysates were analysed in SDS/polyacrylamide gel as described (Caillet,-Boudin et nl., 1988). (i) Electrophoresis

(i) Standard

in gels

anaEytica1 SDSlpolya.crylamide

electrophoresis

gel

(SDSIPAOE)

Polyacrylamide (15 %) slab gels were electrophoresed under the conditions described by Laemmli (1970). Unless otherwise stated, samples were dissociated by heating at 100°C for 2 min in 0.0625 x-Tris.HCl (pH 6.8), 4% (w/v) SDS, 10 o/0 (v/v) 2-mercaptoethanol. 0002 o/0 (v/v) bromophenol blue. (ii) Urea-gradient XLM/polyacrylamide gels These gels contained a 0 to 8 M urea gradient perpendicularly oriented to the direction of migrat,ion. The sample was loaded as a layer all along the top of the gel (CaillebBoudin & Lemay, 1986; Goldenberg & Creighton, 1984). (iii) Aron-dissociating gels Protein samples in their native state were elecerophoresed in polyacrylamide (acrylamide : 5% bisacrylamide ratio of 100 : 2.66), cast in 0375 M-Tris. HCl buffer (pH 8.9) overlaid w&h a 3% stacking gel buffered with 0125 M-Tris.HCl (pH 68). The tank buffer was 0435 M-Tris-O.4 M-glycine, (pH 8.3). (g) Synthetic

peptides,

anti-peptide

antibodies

and immunobrol analysis The E-terminal tridecapeptide Met-Lys-Arg-Ala-ArgPro-Ser-Glu-Asp-Thr-Phe-Asn-Pro, and the C-terminal dodecapeptide Thr-Asn-Ser-Tyr-Thr-Phe-Ser-Tyr-Ile-AlaGin-Glu were synthesized by a solid-phase method (Merrifield et al., 1982). Purification of the peptides, conjugation to tetanus toxoid as the carrier and immunizaton of rabbits have been described in detail (Caillet-Houdin et

Ala434 to Val Mutation al., 1988). Polypeptides electrophoresed in SDS/polyacrylamide gels were analysed with the rabbit anti-peptide sera, using a peroxidase-immunoblotting technique (Burnett’e, 1981).

in Ad2 Fibre

479

II---

m(h) Electron

microscopy

Samples were stained with 1% (w/v) uranyl formate, buffered with 91 M-sodium cacodylate (pH 7.0), and examined in a Hitachi HU12 electron microscope. The magnification of the microscope was calibrated using the lattice spacing of negatively stained catalase crystals (Wrigley, 1968). Length measurements of fibre were performed on penton capsomers, using a MOP/AM 01 Quantitative Image Analyzer (Kontron Messgerate). More than 200 molecules were taken at random on at least 4 prints (final magnification: 168,000) of each WT and H2ts125 penton preparation. The accuracy of our length measurements was estimated to be *@I mm. Experimental values were statistically analysed and the popula,tion of lengths was plotted with the aid of a computer (Hewlett-Packard PC-AT), using the SigmaPlot-Version 4.0 program.

IUO--IF-P-

P[---

Xlt-

XmIIX-

ut

I,

(b)

(i) Hydrophobicity analysis and prediction of secondary structure Hydropathy plots were generated using a program derived from the one described by Kyte & Doolittle (1982), with a scanning window of 9 amino acid residues. Amino acid conformational parameters were determined using the predictive method described by Chou & Fasman (1974).

Figure 1. Comparative analysis of the P; and C termini of WT (wt) and HZts125 (ts) fibres. 14C-labelled fibres were analysed by electrophoresis in an SDS/polyacrylamide gel. Gels were dried and autoradiographed (a), or electrically transferred onto nitrocellulose membranes and probed with anti-N-terminal peptide serum (b), or anti-C-terminal peptide serum (c). Lane 1: adenovirus polypeptide markers. (f) fibre. Immunoperoxidase labelling.

3. Results (a) &ructural

integrity of the N and C term&i HZts125 jibre polypeptide

of

A tridecapeptide and a dodecapeptide corresponding to the N and C termini of the Ad2 fibre polypeptide, respectively, were synthesized and anti-peptide antibodies were raised in rabbits. Anti-N and anti-C terminus antibodies reacted on immunoblots with both WT and H2ts125 fibres (Fig. 1). This suggested that both polypeptide chain extremities were structurally identical in the WT and mutant fibres. Therefore, no proteolytic cleavage, occurring at one end or the other end, can explain the difference in electrophoretic mobility between the WT and H2ts125 fibres, observed in SDS/polyacrylamide gels. (b) The H2ts125 mutation(s) organized

domain(s)

involve(s) highly in the j&e polypeptide

The difference in apparent relative molecular mass (n/r,) between the WT and H2ts125 fibre polypeptides in SDS/polyacrylamide gels might be due to some conformational change(s) occurring in the mutant fibre protein, which is not completely abolished by SDS denaturation and migration in a.n SDS-containing gel in the standard conditions (Laemmli, 1970). To address this question, H2ts125 fibre was mixed with a WT Ad2 virion sample, denatured by heating in the SDS sample buffer without urea, and electrophoresed in an SDS/lS%

polyacrylamide gel containing a gradient of urea (0 to 8 M) perpendicular to the direction of migration. As shown in Figure 2(a), the differences in apparent M, between the mutant and WT fibre polypeptides (60 x lo3 compared to 62 x 103) disappeared in the presence of urea. In high concentrations of urea (6 to 8 RI), both fibre polypeptides co-migrated with the IIIa (68 x 103). This confirmed our previous finding that some structural domain(s) of the fibre polvpeptide resist(s) the standard SDS denaturation procedure (Caillet-Boudin & Lemay, 1986).. This alsn suggested that the difference in electrophoretie mobility between WT and H2ts125 fibres in an SDS/polyacrylamide gel resulted from a change in the conformatonal structure of the fibre poly\peptide. This was confirmed by the next experiment in which WT and H2ts125 fibres were subjected to heat-SDS denaturation in the presence of urea. The final ooncentrations of denaturing agents were 3 M-urea, 2% SDS and 5% 2-mercaptoethanol. Fibre samples were incubated at 100°C for different lengths of time, and electrophoresed in a standard SDS-containing 15% polyacrylamide gel. After 30 minutes of denaturation, no difference in electrophoretic mobility between the two fibre polypeptides could be detected, and both fibre polypeptides co-migrated with an apparent M, of 75 x lo3 (Fig. 2(b)). The fuzzy aspect of the fibre polypeptide bands after 10 minutes denaturation

M.-L.

480

Caillet-Boudin

et al.

(a)

--II --m mo t( ar(f)

f-

--P

lone

:

I

2

3

4

567891011 ibl

Figure 2. Kinetics of denaturation of Bd2 fibre poiypeptide. (a) Urea denaturation. H2ts125 fibre was mixed with a sample of Ad2 WT virion, denatured in SDS sample buffer in the a.bsence of urea, and loaded on top of a SDS/lS% polyacrylamide gel, containing a urea gradient (O-8 M) perpendicular to the direction of migration. Polypeptide bands are: l> hexon; 2; penton base; 3, IIIa; 4; WT fibre; 5: HZts125 fibre; 6, IVal; 7, IVa2; 8, core protein V. WT and mutant fibres co-migrate in the presence of a high concentration of urea (arrowhead), whereas other polypeptides of similar Mr (e.g. IVal, IVa2 and V) still migrate as separate bands. Coomassie blue staining. (b) Heatdenaturation. Samples of WT (lanes 1, 3, 5, 7 and 9) and H2ts125 (lanes 2, 4, 6, 8 and 10) fibres (f) were heated at 100°C in SDS-urea sample buffer for different’ lengths of time and loaded on a standard SDSjl5oi, polyacrylamide gel. La.nes: 1 and 2, 2 min; 3 and 4, 5 mm; 5 and 6, 10 min; 7 and 8, 20 min; 9 and 10, 30 min; 11. Ad2 WT polypeptide markers (M,): II; hexon (120 x 103); III, penton base (85 x 10s); IIIa (68 x 103); IV, fibre (62 x 103): V; core protein (45 x 103). Note that a contaminating polypeptide of 98 x lo3 Nr, present in H2ts125 fibre samples was not modified in its apparent MC by heat-denaturation. Autoradiography.

(lanes 7 and 8) suggested the existence of a heterogeneous population of fibre polypept’ides in both samples. This critical time of denaturation could thus correspond to a transition state in which several classes of intermediate molecules co-existed at different stages of denaturation (Boulanger & Loucheux, 1972), or an equilibrium between two forms of fibre molecules in a structured (0 to 5 min, lanes 1 to 4) or totally destructured state (30 min, lanes 9 and 10). The possible influence of the HZtsl25 mutation on fibre conformation was also studied by gel electrophoresis under non-denaturing conditions. The mutant fibre was found to migrate more slowly than the WT fibre (Fig. 3). Since aone of the mutations

loi

Figure 3. Comparative mobility of H2tal25 (ts) fibres electrophoresed in SDS-containing polyacrylamide gel (a) dissociating gel (b). v, adenovirus type used as ma,rkers; p. adenovirus penton.

WT (wt) and a denaturing and in a non2 pofgpeptides

at position 106 or 434 modifies the net electric charge of the protein molecule, this strongly suggested that one or both mutations introduced a significant change in its conformat,ional structure. Alternatively, a conformational change might unmask polar groups of charged amino acids at the prot,ein surface. Whichever is the mechanism, such a change might be significant, enough to become detectable by secondary structure analysis and by electron microscopy. (c) ISlectron microscopic analysis vf N2txd25 and penton capsomers

jibre

Possible changes in the morphology of the mutant fibre were therefore examined under the electron microscope. WT and H2ts125 fibres were morphoindistinguishable logically from each othr. However, since the t,ail of the fibre could be buried in the stain, comparative measurements were performed on WT and H2ts125 penton ca,psomer. i.e. on fibre bound t’o penton base. The length between the distal knob of the fibre and its anchorage point on the penton base was measured for more than 200 molecules of each WT and mutant penton. As shown on the corresponding histograms (Fig. 4), the fibre length distribution appeared t,o be monomodal for both types of fibre molecules. After mathematical transfarmation into the closest fitting

Ala434

0.10

in Ad2 Fibre

0.10

I

1

I

,

I

to Val Mutation

I

481

I

,

I

I

F wt

0.08 s 2 006 5 5 e 5 0.04 0

0 .oo

150

200

250 Flbre

length

Fibre

(8)

length

(8)

(b)

(a)

wt

te 125

040 -

Fwt

0.08 T .3 0.06 .-s ‘I e f 6

0.02

0.02

o.oc

‘.., ‘.

/ .I I

:5cI

I

1

325

300

275 Fibre

length

350

(8)

(c)

Id

1

Figure 4. Electron microscopic analysis of WT and H2ts125 fibres. (a) and (b) Histograms of length measurements for WT (a) and HZts125 mutant fibres (b); 219 molecules of WT and 234 molecules of mutant fibres were measured on several prints of electron micrographs at a final magnification of 168,000. The x-axis represents the fibre lengths expressed as a (1 ii per class interval), and the y-axis represents the number of molecules of fibres in each class interval, expressed as arbitrary units (100 molecules = 0.1 a.u.). The maximum length values were 430 and 420 A, and the minimum values were 189 and 236 d for the WT and mutant fibres, respectively. (c) Enlargement of the closest-fitting Gaussian curves representing the size distribution of fibre WT (continuous curve) and mutant fibres (dotted curve) in the 250 to 350 B interval. The mean value is at 3001 A for the WT and 308.8 ,& for the mutant. (d) Gallery of 20 molecules of each wildtype (wt) and mutant (ts125) fibre taken at random from electron micrographs, like the ones used for drawing the histograms depicted in (a) and (b). Bar represents 300 8.

Gaussian curve, the values obtained were 300.1+2.1 a (1 d = 61 nm; n = 219) for the WT fibre (meanIfrs.E.M.), and 308.8t 1.9 a (n =234) for the H2ts125 fibre. The standard deviation of the population of lengths was 32.0 a and 29.3 A for WT and H2ts125 fibres respectively. The mutant fibre therefore appeared to be slightly longer than the WT fibre. Student’s t-test was applied to determine whether the difference of

8.8 A ( -3%) in mean values was significant. values of t= -3.0196 and P = Respective 2.675 x 10e3 were obtained, implying that the two means were significantly different at the P = 0.05 level. The discrete change in morphology of the capsomeric fibre seemed therefore to reflect the modification of structure observed in gel electrophoresis analysis of native fibre and fibre polypeptide subunit (Figs 2 and 3).

482

M-L.

Caillet-&m&n

et ai. Asp-Pro 406-407 I

Asp-Pm 194-195 I

*

* coo

NH2

44

K

34

K

15 K

I

2

3

5

4

6

7

6

9

IO

!I

!2

13

62 60-

34 32 -

20 -

(bi

Figure 5. (a) Sketch illustrating the Asp-Pro bonds on the fbre amino acid sequence, and t.he peptide resulting from complete or partial hydrolysis. *: the point, mutations, PhelO5 to Leu, and Ala434 to Val. K, x lo3 Mr. (b) Peptide fingerprint analysis in an SDS/polyacrylamide gel of fibres from Ad2 WT (lanes 1j 3. 5 to 8) and H2ts125 (lanes 2.4, IO to 13). Fibre uniformly labelled with [‘%]formate was cleaved at aspartyl-propyl bonds with HCl under miid conditions (56”C, overnight), and hydrolysates were analysed in an SDS-containing, 150,& p 01y acrylamide gel. Gels were dried and autoradiographed (lanes 1 and 2) or electrically transferred onto nitrocellulose membranes (lanes 3 to 13). The blots were probed with anti-N terminus (lanes 3 and 4) or anti-C terminus serum (lanes 5 to 13). SDS-denatured WT virion is il: lane 9. Lanes 1 to 4, hydrolysis with 25 mu-HCl; lanes 5,9 and 10,O mu-HCI; lanes 6 and 11. 10 mu-HCI; lanes i and 12, 25 m&I-HCl; lanes 8 and 13, 50 mM-HCl. Arrows indicate the 34/32 x lo3 Xr C terminus-containing peptide. Mr ( x i03) values are indicated on the left. Immunoperoxidase labelling.

The value found here for the length of the WT fibre (300.1 12.1 8) was significantly higher than the value of 250 to 280 a previously reported in the literature for negatively stained serotype 2 and 5 fibres (h’ermut, 1984; Pettersson, 1984). A higher

value for fibre Length has aiso been recently reptrrted from a series of measurements of penton capsomers observed after various negative stainings (Ruigrok et al., 1990). The mean value obtained was 331 &5 A (n = 35).

Ala434

to Val Mutation

(d) Localization of the mutation responsible for the altered electrophoretic mobility of H2ts125 jibre polypeptide In order to determine which of the two mutations in the H2ts125 fibre gene caused the change in apparent Mr of the fibre polypeptide, WT and H2ts125 fibres were cleaved at their Asp-Pro bonds using HCl hydrolysis under mild conditions (CailletBoudin et al., 1988; Rittenhouse & Marcus, 1984). The sequence Asp-Pro occurs twice in the Ad2 fibre at positions 194-195 and 406-407 sequence, (H&is& et al., 1981; H&is& & Galibert, 1981). Asp-Pro bond hydrolysis would therefore generate three peptides of theoretical n/r, of 20, 22 and 19( x 103), corresponding to the N-terminal, internal and C-terminal fragments, respectively (Fig. 5(a)). Single HCl cleavage at position 406-407 or at position 194-195 would give rise to two partial cleavage products of theoretical n/l, 43 x lo3 and 42 x lo3 containing the N terminus and the C terminus, respectively. Hydrolysates of [‘4C]formate-labelled fibres were analysed by electrophoresis in an SDS/polyacrylamide gel and autoradiographed (Fig. 5(b), lanes 1 and 2), or transferred to nitrocellulose membranes and immunologically probed using anti-N and anti-C terminus sera (Fig. 5(b), lanes 3 to 13). An autoradiogram of WT fibre HCl hydrolysate showed an uncleaved fibre polypeptide unit (62 x lo3 M,), three major discrete peptide bands of apparent niir, 44, 34, 20( x 103) and a broad band at 16 to 15x lo3 (Fig. 5(b), lane 1). In a previous study, we have identified the 44 x lo3 and 34 x lo3 M, species as the partial cleavage products resulting from a single cut at position 406-407 or 194-195, and containing the N terminus and the C terminus, respectively. The blurred band at 16 to 15 x lo3 Mr was found to contain the C-terminal fragment (Caillet-Boudin et al., 1988). The only visible differences in M, between WT and mutant fibre patterns resided in the uncleaved fibre polypeptide (60 x lo3 instead of 62 x 103), and in a peptide band of 32 x lo3 (Fig. 5(b), lane 2) instead of 34 x 103. Both 34 and 32( x 103) M, fragments reacted with anti-Cterminal peptide antbody in immunoblotting (Fig. 5(b), lanes 5-13). The chemical fingerprint analysis of the WT and H2ts125 fibres therefore suggests that the structural change responsible for the altered mobility in an SDS/polyacrylamide gel is located in the C-terminal domain of the fibre polypeptide chain, and is due to the Ala to Val substitution at position 434 on the amino acid sequence.

(e) Hydropathy structures

projiles and predicted secondary of WT and H2ts125$bres

Hydropathy plots (Kyte & Doolittle, 1982) of WT and H2ts125 fibre polypeptides were compared in the regions corresponding to each mutation. No significant difference was seen in the N-terminal portion of the fibre sequence, around the Leu105 to

in Ad2 Fibre

483

Table 1 Marker

of HZts125 temperature-sensitive mutation by WT DNA fragments rescue

Ad2 WT

Map co-ordinates of rescuing fragment

Number of plaques per dish at 39.5”C

0 DNA-TP EcoRI E EeoRI C XhoI D

O-100 83.6-898 89.8-100 82.9-100

2 30 0 10 50

Type of DNA H2ts125 DNA-TP 0 DNAPTP DNA-TP DNA-TP

+ + + + +

Monolayers of 293 cells were co-transfected with H2ts125 DNA-terminal protein complex (DNA-TP) and restriction fragments from Ad2 WT DNA; 3 pg of each DNA was coprecipitated per Petri dish, with 3 pg of herring sperm DNA as carrier.

Phe mutation. On the contrary, a slightly more pronounced peak of hydrophobicity was visible in a hydrophobic domain around the residue Va1434 of the mutant fibre (data not shown). The predictive method of Chou & Fasman (1974) suggested that the Leu to Phe mutation at position 105 did not significantly modify the values of the conformational parameters: l-21 to 1.13 for an a-helix, and 1.30 to 1.38 for a b-sheet, respectively. On the contrary, Ala to Val substitution at codon 434 has a high probability of modifying an cc-helix stretch (as in the WT predicted structure) into a b-sheet conformaton: a-helix parameters are 1.42 and 1.06, respectively, whereas /?-sheet parameters are 0.83 and 1.70, respectively. Therefore the Va1434 mutation introduces into the sequences a hydrophobic amino acid known to be one of the best p-sheet formers, whereas alanine is an indifferent former. This again suggests that the mutation responsible for the observed phenotype is at codon 434. (f) Marker rescue of the H2ts125 temperature-sensitive mutation The functional role of each mutation on H2ts125 physiology and ts phenotype was analysed by marker rescue experiments. H2ts125 DNA-terminal protein complex was co-precipitated onto 293 cells with WT Ad2 DNA restriction fragments overlapping one of the two H2ts125 mutations. As shown in Table 1, plaques of ts+ recombinants were obt’ained with EcoRI fragment C from WT DNA (map coordinates 89.8-100) and not with EcoRI fragment E (83.6-89.8 map units). These data suggest that the mutation of Ala to Val at position 434, rescued by the WT sequence, causes the temperature-sen.sitive phenotype of the H2ts125 mutant. 4.

Discussion

The fibre gene of the H2ts125 mutant has been found to contain two mutations (Boudin et al.,

484

M.-l;.

Caillet-Boudin

1983). One of the mutations, Leu105 to Phe, usually considered as conservative (Dayhoff, 1969), has been localized in the N-terminal portion of the shaft, whereas the other mutation, a non-conservative substitution of Ala434 to Val, is situated within the terminal knob of the molecule, near the knob-shaft junction. Cells infected with HZts125 mutant at either permissive or non-permissive temperature synthesize a fibre polypeptide that migrates with an apparent M, of 60 x lo3 in contrast to the WT of 62 x lo3 in an SDS/polyacrylamide gel (Boudin et al., 1983). Since H2ts125 is a double mutant, with two phenotypic characters, temperature sensitivity (ts) and a,n altered electrophoretic mobility of the fibre polypeptide (elm), it was essential to determine which character(s) might be assigned to which of the two mutant codons, and hence the domain(s) of the fibre molecule that is (are) critical for its structural integrity and biological function. The temperature-sensitive mutation was rescued by a fragment, of WT DNA corresponding to the C-terminal moiety of the fibre sequence (Table l), suggesting that the ts phenotype is carried by the Ala to Val substitution at, position 434. Using antipeptide sera specific for the N and C termini to probe the structural integrity of the fibre polypeptide ends, we found that the higher mobility of the mutant fibre in an SDS-containing gel was not due to a proteolytic cleavage at the N or C terminus of the fibre polypeptide (Fig. 1). Results of SDS-urea denaturation experiments (Fig. 2) suggest that the apparent nil, of 60 x lo3 is due to a change in the secondary structure of t,he mutant detectable in standard fibre polypeptide, SDS-containing gels, as well as in non-denaturing conditions (Figs 1 and 3). The peptide patterns of chemical cleavage at Asp-Pro bonds indicate that the Ala to Val mutation at position 434 is responsible for the elm phenotype (Fig. 5). The altered mobility of the fibre polypeptide in an SDS/polyacrylamide gel and its temperature sensitivity seemed therefore to be at the carried by the same Ala to Val mutation 434th codon. The Phe to Leu substitution is therefore a silent mutation. The H2ts125 mutation seemed also to provoke discrete morphological changes in native fibre molecules, visible under electron microscopy as a slight but significant increase (3%) in the length of the mutant fibre molecules: 308.8 A for the mutant instead of 300.1 A for the wild-type fibres (Fig. 4). Several examples of point mutations that involve the replacement of a small residue such as Gly or Ala by the sterically hindered Val have been reported in the literature (Alber, 1989). Most of them ha,ve been shown to have dramatic functional and structural effects. Modification of the electrophoretic mobility in an SDS/polyacrylamide gel as a result of a point mutation has been reported for the proto-oncogene ras in human bladder carcinoma cells (Reddy et al., 1982; Tabin et al., 1982), the NS protein of vesicular stomatitis virus (Rae & Elliott,

et al.

1986), and the 14 x lo3 111,envelope oligomeric protein of vaccinia virus (Gong et al., 1989). Extensive although subtle conformational changes have also been described in bacteriophage T4 lysozyme after a similar Ala to Val mutation (Alber, 1989). An Ala to Vs,l substitution in the N-terminal domain of the phage i repressor has been found to reduce the helical propensity of the Val-containing to the Ala-containing WT doma,in, relative sequence, decreasing the thermal stability favouring the aggregation of the protein (Hecht et al., 1984). This was also observed for H2ts125 fibre, which has a tendency to self-aggregate spontaneously at high concentrations (unpublished results). This behaviour of the mmant fibre made it impossible to perform comparative physical st,udies (e.g. circula,r dichroism or differential ultraviolet spectroscopy) with the WT fibre. Temperature sensitivity, conformational stability and in vitro spontaneous aggregation might be three related phenomena in the H2ts125 fibre. Aggregation might also be mediated by DNA fragments to which fibre could bind. Adenovirus type 5 fibre has been shown to have some affinity towards DNA (Levine & Ginsberg, 1967, 1968). Indeed, DNA was detected in the fibre fraction eluted from DEAE-Sephadex columns (unpublished results). H2ts125 fibre might have a, stronger affinity for DNA than WT fibre. From the model of structural organization of the adenovirus type 2 fibre polypeptide subunit (Green et al., 1983), 22 stretches of a 15 amino acid repeat motif form the shaft of the fibre, whereas the first 45 amino acids of the sequence constitute the t’a.il and the C-terminal portion of the polypeptide chain (amino acids 402 to 582) forms the distal knob. According to the predictive method of Chou $ Fasman (1974); the conservative substitution of LeulO5 by Phe, another hydrophobic residue with a similar p-sheet parameter, had very little probability of changing the putative b-sheet structure present in the fourth repetitive motif of %he fibre. Indeed, we found that all of the conformational and physiological changes observed in the HXtsl25 mutant were due to the substitution of Ala434 by Va,l. This mutation introduces an additional strong g-sheet inducer in a hydrophobic stretch of amino acids with a probable a-helix conformation near the It seemed likely that the knob-shaft junction. mutation had weakened the hydrophobic core of t,he knob-shaft junction, with a resulting expansion of the protein tertiary packing caused by the misfit of Val into a site adapted to Ala. Disruption of the knob-shaft junction structure and the subsequent exposure of a more highly hydrophobic patch might explain the tendency of HZts125 fibre to self-aggregate. Taken altogether, our results strongly suggest that a particula,r domain of the fibre molecule, located near the junction of the shaft, and the knob? has a critical function in a,denovirus physiology, It is imeresting to note that this junction contains the sequence TLWTTPDPSPN (amino acids 400 to 410), which has been found to be conserved with

Ala434

to Val Mutation

more than 80% homology in all adenovirus serotypes sequenced to date (Chroboczek & Jacrot, 1987; HBriss6 et al., 1981; Hong et al., 1987; Kid & Erasmus, 1989; Pieniazek et aZ., 1989, 1990; Sign& et al., 1985). It remains to be determined how a rather distal substitution, such as Ala by Val at codon 434, might alter virion morphogenesis and provoke an accumulation of empty capsids at the non-permissive temperature (D’Halluin et al., 1980). The role of the adenovirus fibre in virion assembly (Chee-Sheung & Ginsberg, 1982; D’Halluin et al., 1980), and of the fibre protein domain(s) involved in this process need to be elucidated. The authors thank Didier Petite for providing cell cultures, Jean-Claude GesquiBre, Andre Tartar and Armelle Novelli for their help in peptide synthesis and production of anti-peptide sera, Gerard Topier and Bernard Gay for electron microscopy, and Douglas Barker and SawSee Hong for critical reading of the manuscript. Work in Lille was supported by the Institut National de la Sante et de la Recherche MBdicale (INSERM-U 233) and the Universitk du Droit et de la was Sante de Lille (UER III). Work in Montpellier supported by grants from the Agence Nationale de Lutte contre le SIDA, the Fondation pour la Recherche MBdicale, the Association pour la Recherche contre le Cancer, the F&d&ration des Groupements des Entreprises Franqaises pour la Lutte contre le Cancer and le Conseil RBgionsl du Languedoc-Roussillon.

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Edited by R. Huber