Annie Barge

Structure of adenovirus fibre. II. Morphology of single fibres.

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 folding 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.

Characterization of rabies virus nucleocapsids and recombinant nucleocapsid-like structures.

Rabies virus nucleoprotein (N) was produced in insect cells using the baculovirus expression system described by Préhaud et al. (Virology 178, 486-497, 1990). The protein was either purified on a CsCl gradient, resulting in a mixture of nucleocapsid-like structures and beaded rings, as observed by electron microscopy, or on a glycerol gradient that resulted in a preparation of the rings only. The rings and nucleocapsid-like structures had the same morphological characteristics as viral nucleocapsids. N in these structures is an 84 A long and thin molecule that is spaced at around 34 A along the length of the nucleocapsid, identical in shape and spacing as the nucleoprotein in nucleocapsids of rabies virus and very similar to those of vesicular stomatitis virus. The recombinant nucleocapsids contained RNA with a stoichiometry similar to that found in viral nucleocapsids. The RNA bound in the beaded rings was a subset of the insect cellular RNA. One of the RNA species was partially sequenced and, although a positive identification could not be made, could correspond to a tRNA. With respect to sensitivity to trypsin and RNase digestion, the recombinant and viral nucleocapsids behaved similar. Trypsin cleaved a 17 kDa fragment from the carboxy terminus of N with only a very small effect on the morphology of the nucleocapsids. RNase A completely digested the resident RNA in both viral and recombinant nucleocapsids into fragments of 4-5 nt long, again with no effect on the morphology of the nucleocapsids. Thus, when the RNA is cleaved, the structure must be maintained by protein-protein contacts. Experiments to remove the resident RNA from viral and recombinant rabies virus nucleocapsids failed, whereas the same methods used to eliminate the RNA from vesicular stomatitis virus nucleocapsids was successful.

Human adenovirus serotype 3 (Ad3) and the AD fiber protein bind to a 130-kDa membrane protein on HeLa cells

The fiber protein of adenovirus mediates the interaction of adenovirus with cell membrane receptors. We have produced the Ad3 fiber protein in the baculovirus expression system. Biochemical, morphological and functional analyses showed that the recombinant fiber was properly folded and functionally competent. The specific binding of Ad3 virus to two HeLa membrane proteins of 130 and 100 kDa was demonstrated with an overlay protein binding assay. In the same assay, Ad3 fiber only recognized the 130-kDa protein. Divalent cations seemed to be important for the interaction of both virus and fiber with these proteins.

The fibre of bovine adenovirus type 3 is very long but bent

The sequence of the fibre of bovine adenovirus type 3 (BAd3) predicts an extremely long structure due to a large number of 15-residue repeats in the fibre shaft, the tail and head domains being similar in size to the human adenovirus fibres. The length of the fibre was confirmed using negative-strain electron microscopy of BAd3 pentons (fibre plus penton base). The fibre was found to be bent in several discrete places and the bending sites appear to correspond with irregular repeats in the shaft. We suggest that bending of the fibre is needed for the interaction of the penton base with the secondary receptors on the cell surface.