SUMMARYHuman embryonic kidney cells have been transformed by exposing cells to sheared fragments of adenovirus type 5 DNA. The transformed cells (designated 293 cells) exhibited many of the characteristics of transformation including the elaboration of a virus-specific tumour antigen. Analysis of the polypeptides synthesized in the 293 cells by labelling with 35S-methionine and SDS PAGE showed a variable pattern of synthesis, different in a number of respects from that seen in other human cells. On labelling the surface of cells by lactoperoxidase catalysed radio-iodination, the absence of a labelled polypeptide analogous to the 25o K (LETS) glycoprotein was noted. Hybridization of labelled cellular RNA with restriction fragments of adenovirus type 5 DNA indicated transcription of a portion of the adenovirus genome at the conventional left hand end.

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Adenovirus protein V is associated with the DNAcontaining virus core and functions as a bridge between the capsid and the core. A yeast two-hybrid analysis performed with a human cDNA library using protein V as ' bait ' selected a cellular protein, p32 -described previously as associated with the splicing factor ASF/SF2. By expression and purification of p32 and preparation of an antibody we confirmed the binding of p32 to V by a variety of methods including immune precipitation. We demonstrated

The cellular protein p32 was isolated originally as a protein tightly associated with the essential splicing factor ASF/SF2 during its purification from HeLa cells. ASF/SF2 is a member of the SR family of splicing factors, which stimulate constitutive splicing and regulate alternative RNA splicing in a positive or negative fashion, depending on where on the pre-mRNA they bind. Here we present evidence that p32 interacts with ASF/SF2 and SRp30c, another member of the SR protein family. We further show that p32 inhibits ASF/ SF2 function as both a splicing enhancer and splicing repressor protein by preventing stable ASF/SF2 interaction with RNA, but p32 does not block SRp30c function. ASF/SF2 is highly phosphorylated in vivo, a modification required for stable RNA binding and protein-protein interaction during spliceosome formation, and this phosphorylation, either through HeLa nuclear extracts or through specific SR protein kinases, is inhibited by p32. Our results suggest that p32 functions as an ASF/SF2 inhibitory factor, regulating ASF/SF2 RNA binding and phosphorylation. These findings place p32 into a new group of proteins that control RNA splicing by sequestering an essential RNA splicing factor into an inhibitory complex.

A ' west-Western ' blotting procedure indicates that adenovirus core protein V is linked to the capsid via protein VI. Double-labelling techniques employing confocal microscopy and immunofluorescence suggest that this linkage is disrupted following infection and penetration of the host cell. Protein V enters the nucleus presumably still in association with the other core proteins attached to the virus genome. Later in infection protein V rapidly accumulates in the nucleus in close association with nucleoli.Virus capsid proteins play a key role in the recognition of cell receptors and consequential adsorption and penetration of the virus. In the case of adenoviruses there is a complex capsid structure comprising seven different proteins. The major capsomere is the hexon of which there are 240, forming an icosahedral shell with the 12 apices of the icosahedron accommodating pentons which are themselves bipartite, consisting of the penton base and fibre. A number of other proteins, viz. VI, VIII, IIIa and IX, interact to stabilize this structure and to contact the nucleoprotein core of the virus. The latter consists of a linear double-stranded DNA genome, with a covalently linked terminal protein (TP) at each 5h terminus, in close association with the highly basic protein VII and a more loosely associated protein V. The locations of the major components of the capsid have been well-defined and tentative assignments have been made for the other capsid proteins based on cryoelectron microscopy and knowledge of the structure of the hexon from X-ray analysis (Stewart et al., 1993). The disposition of the core proteins is not so wellestablished but there have been indications that the genome is held in a tightly coiled configuration. A number of these

The amino acid sequence of the adenovirus fibre protein reveals an approximately repeating motif of 15 residues. A diagonal comparison matrix established that these repeats extended from residue 43 to residue 400 of the 581 residue sequence. Assignment of secondary structure combined with model building showed that each 15‐residue segment contained two short beta‐strands and two beta‐bends, one of which incorporated an extra residue in a beta‐bulge of the Gx type. The 44 strands together gave a long (210 A) narrow, amphipathic beta‐sheet, which could be stabilised by dimer formation to give the shaft of the fibre. The knob could arise from a dimer of the C‐terminal 180 residue segment, predicted to be an 8‐10 stranded beta‐sandwich. This model is consistent with the electron micrographs of the fibre and it was supported by measurements of c.d. and of electron diffraction from microcrystals. The latter gave a pair of wide angle arcs, corresponding to a repeat of 4.7 A, oriented appropriately for a cross‐beta structure. The relation of this structure to globular structures is discussed and a folding pathway is proposed. In its general features the structure resembles that proposed for the tail fibre of bacteriophage T4.

SUMMARYPeptides were synthesized based on the cleavage sites in the adenovirus type 2 proteins pVI and pVII. The synthetic peptides were incubated with disrupted, purified adenovirus as a source of proteinase and specific cleavages were monitored by fast protein liquid chromatography and amino acid analysis. Using this approach it was established that all the peptides cleaved were of the form M(L)XGX~G or M(L)XGG~X. Thus we have shown that the adenoviral proteinase recognizes a specific secondary structure formed by a sequence of at least five amino acids, the main determinants of specificity being two and four residues to the N-terminal side of the bond cleaved. We were able to examine the relevant structural features of the peptide substrates by utilizing the CHEM-X molecular modelling package. Using our consensus sequence we were able to predict the cleavage sites in the viral proteins pVIII, pre-terminal protein (pTP), IlK and IIIa. Octapeptides containing the predicted sites in pVIII and the pTP were synthesized and shown to be cleaved by the proteinase.

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