Herpes simplex virus is a useful model for studying the mechanisms involved in DNA replication in eukaryotic cells. Our current efforts are directed toward studying this process with purified proteins. We have previously found seven viral genes which are both necessary and sufficient to carry out authentic origin- dependent DNA replication. The sequence of each one of these seven genes has been determined. Using the predicted amino acid sequence as a starting point, we have raised rabbit antisera against the product of each of the seven genes. The antisera have been used in immunoassay experiments to identify the proteins in HSV-infected cells. All seven proteins localize to the proteins in HSV-infected cells. All seven proteins localize to the nucleus and are expressed in a manner consistent with the idea that they are the products of early genes. Various immunological assays suggest that four of these proteins (UL5, UL8, UL9, and UL52) are made in infected cells in very low abundance relative to the other three. In UL5, UL8, UL9, and UL52 in insect cells using the baculovirus expression system. The HSV proteins made in insect cells are immunoprecipitable with the appropriate antisera and the size of each protein is indistinguishable from the corresponding protein made in HSV infected Vero cells. We have purified one of these proteins (UL). Preliminary characterization of purified UL9 has shown that it binds specifically to the HSV origins of DNA replication.
| Vashee, S; Simancek, P; Challberg, M D et al. (2001) Assembly of the human origin recognition complex. J Biol Chem 276:26666-73 |
