Analysis of 100K-defective temperature-sensitive adenovirus mutants confirmed the multifunctional character of the nonstructural, virus-coded 100K protein. In addition to its function in hexon trimerization (altered in H5ts1), and its possible direct or indirect role in hexon transport to nucleus (mutated in H2ts118), genetic and biochemical evidence was presented that 100K play some critical role in the scaffolding process of adenovirus capsid. This function appeared to be defective in H2ts107 and to map between coordinates 69.0 and 69.9, leftward from the H5ts1 lesion (70-73 map units; Arrand, 1978). This corresponded to the central domain of the 100K protein, between amino acid 300 and 400 from the N end. DNA sequencing of cloned fragments of H2ts107 DNA overlapping the mutation revealed two point mutations on the same codon at nucleotide 25,082 and 25,083 (GAC----GCA), corresponding to a nonconservative amino acid change (aspartic acid----alanine) at position 324 in the 100K sequence. 100K of adenovirus 2 wild type (WT) was found to bind in significant amounts to novobiocin-affinity column, and to be coeluted with hexon, penton, IIIa, and cellular topoisomerase II activity, by novobiocin- or ATP-Mg2+-containing buffers. H2ts107 100K also bound to novobiocin column, but the elution pattern differed from that of WT, suggesting some alteration in the affinity of the mutated 100K for novobiocin. The same behavior on affinity column as H2ts107 100K was observed for 90K, a cleavage product of the 100K, found in great abundance in H2ts107 at 39.5 degrees and corresponding to the C-terminal moiety of the 100K molecule. This implied that the "novobiocin-binding" domain of the 100K was not confined at its N terminus, and was altered in the H2ts107 mutant.