Minichromosomes of wild-type polyomavirus were previously shown to be highly acetylated on histones H3 and H4 compared either to bulk cell chromatin or to viral chromatin of nontransforming hr-t mutants, which are defective in both the small T and middle T antigens. A series of site-directed virus mutants have been used along with antibodies to sites of histone modifications to further investigate the state of viral chromatin and its dependence on the T antigens. Small T but not middle T was important in hyperacetylation at major sites in H3 and H4. Mutants blocked in middle T signaling pathways but encoding normal small T showed a hyperacetylated pattern similar to that of wild-type virus. The hyperacetylation defect of hr-t mutant NG59 was partially complemented by growth of the mutant in cells expressing wild-type small T. In contrast to the hypoacetylated state of NG59, NG59 minichromosomes were hypermethylated at specific lysines in H3 and also showed a higher level of phosphorylation at H3ser10, a modification associated with the late G 2 and M phases of the cell cycle. Comparisons of virus growth kinetics and cell cycle progression in wild-type-and NG59-infected cells showed a correlation between the phase of the cell cycle at which virus assembly occurred and histone modifications in the progeny virus. Replication and assembly of wild-type virus were completed largely during S phase. Growth of NG59 was delayed by about 12 h with assembly occurring predominately in G 2 . These results suggest that small T affects modifications of viral chromatin by altering the temporal coordination of virus growth and the cell cycle.Histone modifications play important roles in regulating cellular DNA transcription, replication, and repair. Histones undergo a variety of reversible covalent modifications including acetylation, methylation, and phosphorylation which contribute to the control of these processes (37,52,57). While much has been learned about the chemistry and enzymology of histone modifications, knowledge of their functional importance and how they are regulated is lagging. The mouse polyomavirus and other members of the polyomavirus group package their ϳ5-kb circular DNA as a minichromosome utilizing cellular histones ordered into roughly 24 nucleosomes (42). We have used the minichromosomes of wild-type (WT) and mutant polyomaviruses as a model system to study the regulation of histone modifications by the viral T antigens.Minichromosomes of polyomavirus and simian virus 40 (SV40) show an unusually high degree of acetylation of histones H3 and H4 compared to normal cell chromatin (43). Interestingly, minichromosomes of polyomavirus hr-t mutants (43), a class of host range mutants that are defective in cell transformation, were not hyperacetylated (3). These mutants are altered in sequences shared by the small T (sT) and middle T (mT) antigens but encode a normal large T (6, 22). Large T mutants of the ts-A class of both viruses were highly acetylated (43). Recent studies with SV40 have revealed a dynamic st...