THE REGULATION OF ADENOVIRUS DNA SYNTHESIS IN VITRO BY VIRAL ENCODED PROTEINS

Abstract

Three viral proteins are needed for adenovirus (Ad) DNA replication in vitro. They include a 72 kDA DNA binding protein, a 80 kDa preterminal protein (pTP) and a 140 kDa Ad DNA polymerase (Ad pol). The open reading frame for the Ad2 pol gene has been cloned behind the bacteriophage SP6 promoter, transcribed in vitro (Ad pol ORF mRNA) using SP6 RNA polymerase and translated in rabbit reticulocyte cell lysates (RCL). In addition to the synthesis of a 120 kDa protein corresponding to the size of the ORF, a major peptide of 62 kDa was also produced. Data presented here showed that the synthesis of the 62 kDa peptide arose as a result of an internal initiation during translation of the Ad pol ORF mRNA at 11 or 12 AUGs (1638 or 1655 base pairs) from the 5' end of the mRNA. If the internal initiation site is utilized in vitro, it could have profound effects on the synthesis of the Ad pol during a viral infection.;The Ad2 pTP ORF was also similarly cloned and expressed in RCLs. The size of the pTP made in vitro was shown to be similar to the pTP from infected cells. Therefore the contribution of additional coding sequences by upstream leaders found on the pTP mRNA in infected cells seemed unlikely. In addition, the pTP ORF mRNA was translated more efficiently than the Ad pol ORF mRNA and had no significant sites of internal initiation.;In addition, the effects of the viral core proteins on Ad DNA replication in vitro were examined. It was demonstrated that the core proteins polypeptide V and VII could inhibit Ad DNA replication in vitro by binding to the templates and rendering them inaccessible to the replication proteins. These results suggest that the core proteins must be removed from the template before viral DNA replication can occur in vivo and that the presence of core proteins during viral assembly could terminate viral DNA replication.