J. Mol. Biol. (1990) 215, 589-596

Structure of Adenovirus Fibre II. Morphology of Single Fibres Rob W. H. Ruigrokt, Annie Barge, Corinne Albiges-Rizo and Sonia Dayan EMBL

Grenoble Outstation, c/o I L L , 1 5 6 X 38042 Grenoble Cedex, France

(Received 13 M a r c h 1990; accepted 20 J u n e 1990) Adenovirus type 2 fibres in crystals appear to be significantly longer than found previously (accompanying paper). We therefore examined isolated fibre by electron microscopy and measured a length of 370 A, consistent with the length found in the crystals. The specific N-terminal structure of the fibre caused a heterogeneity in the length that may at least partially explain the values of 280 to 310 A published previously. Green et al. described a 15 amino acid repeat in the primary structure of the shaft of the fibre thought to be associated with the specific three-dimensional fblding of the shaft. We compared the adenovirus type 2 (with 22 repeats) and type 3 (with 6 repeats) fibre lengths and derived a contribution of 13"2 A to the length of the shaft per 15 amino acid repeat. Specific morphological features of the fibre are discussed in relation to its amino acid sequence.

Hoglund, 1969, Norrby, 1969). To clarify this discrepancy, we reinvestigated the adenovirus fibre using various electron microscope techniques and found a length of 370A. The behaviour of the N-terminal end of the fibre appeared to be the cause of an apparent variability in length and of specific interactions between fibres in solution. We further compared the fibre lengths of adenovirus type 2 (with 22 repeats of 15 residues) and type 3 (with only 6 repeats) to calculate the shaft length per repeat.

1. Introduction The pentons of adenoviruses protrude from the 5-fold vertices of the icosahedral capsid (Valentine & Pereira, 1965). The penton consists of a penton base with 5-fold symmetry that is locked in the capsid and a fibre that extends from the centre of the base outwards (Pettersson & Hoglund, 1969; Laver et al., 1969; Boudin et al., 1979). The fibre is responsible for the haemagglutination activity of the virus, attaches to a cell receptor to initiate infection (Pettersson, 1984) and consists of a head on a long shaft. The N terminus is located at the end of the shaft opposite the head and interacts in a non-covalent manner with the penton base (Devaux et al., 1987; Boudin & Boulanger, 1982), whereas the C terminus is probably located somewhere in the head. The fibre is most likely a trimer of identical subunits (Sundquist et at., 1973; Van Oostrum & Burnett, 1985; Van Oostrum et al., 1987) and shows an interesting 15 amino acid pseudo-repeat in the shaft region (Green et al., 1983). Crystals of the adenovirus type 2 and type 5 fibres were grown (Devaux et al., 1987; Devaux et al., 1990) and the crystal lattice in some types of crystals could be explained only by assuming a length ['or the fibre of 370A (1 A = 0.1 nm), whereas previous estimates from electron microscopy gave lengths between 270 and 310 A (Valentine & Pereira, 1965; Pettersson &

2. M a t e r i a l s a n d M e t h o d s

(a) Proteins Adenovirus type 2 was grown in KB cells (D'Halluin et al., 1978). Free fibre and intact penton were isolated from the pool of cellular, non-assembled viral proteins as described (Devaux et al., 1987; Boudin & Boulanger, 1982) and stored in 40 mM-Tris-HCI (pH 7-8). Cleaved fibre of 60,000 Mr was made from intact fibre by digestion with chymotrypsin (1 : 10 enzyme to substrate ratio, at 37°C for 2 h in 40 mM-Tris-HCI (pH 7"8)). Chymotrypsin was removed from fibre by chromatography over DEAE-Sepharose (Devaux et al., 1990). Type 3 virus was grown in KB or HeLa cells (infected HeLa cells were kindly provided by Dr C. Tibbetts). The penton was again isolated from the pool of free non-assembled viral proteins and purified via a 3-step procedure of polyethylene glycol precipitation and DEAE- and Blue-Sepharose .chromatography. The detailed purification procedure will be

t Author to whom all correspondence should be addressed. 0022-28361901200589-08 $03.00/0

589

(c') 1990 AcademicPress Limited

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