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In common with many other viruses, adenoviruses code for a protease essential for the development of infectivity. Recombinant adenovirus protease was active in crude in vitro complementation assays but was inactive with peptide or purified protein substrates. Activity was reconstituted by a component of adenovirus virions, which was identified as GVQSLKRRRCF, a peptide derived from the virus protein pVI. Synthetic peptides were used to demonstrate that the cysteine is essential and that the disulphide-linked dimer is required for activity. It is proposed that the adenovirus protease is a cysteine protease and that its activation by the peptide involves thiol-disulphide interchange, which serves to expose the active site cysteine. This represents a novel strategy for controlling the activity of a protease that is required for virus maturation.

Mutant dl 309 is a viable Ad5 deletion mutant. Whereas wild-type Ad5-infected HeLa cells contain two VAI RNA species [VAI(A) and VAI(G)] which differ by three nucleotides at their 5' ends, dl 309-infected HeLa cells contain VAI(G) but no VAI(A) RNA. Nucleotide sequence analysis indicates that dl 309 lacks two base pairs which precede the 5' end of VAI(A) by 22 nucleotides. Since the 5' ends of VAI RNAs are not processed, the 309 deletion serves to identify a portion of the sequence required for RNA polymerase III initiation. Since dl 309 grows as well as wild-type Ad5 in HeLa cells, the VAI(A) species is not essential for viral growth in these cells.

Temperature-sensitive mutants in the N complementation group of human adenovirus type 5 are defective at the nonpermissive temperature for replication of virus DNA and for transformation of rat embryo cells. We show that nuclear extracts prepared from Ad5ts 149-infected cells grown at the nonpermissive temperature fail to replicate DNA in vitro. The defect lies in the first step in the initiation of viral DNA synthesis, the formation of a covalent linkage between the terminal protein precursor (pTP) and dCMP. A 140 kilodalton (140 kd) protein which complements these defective extracts and contains DNA polymerase activity has been purified from HeLa cells infected with wild-type Ad2. It is tightly associated with the 80 kd pTP in a replication complex. Both of these proteins are products of the E2B region of the adenovirus genome, and the 140 kd protein coding sequences lie immediately downstream from those encoding the 80 kd protein. These results demonstrate that adenovirus encodes a novel DNA polymerase that is required for priming of DNA synthesis at the origin of replication. This protein may also function in the initiation of transformation of cultured cells.