AN ACTIVITY SPECIFIED BY THE OSTEOSARCOMA LINE U2OS CAN SUBSTITUTE FUNCTIONALLY FOR ICP0, A MAJOR REGULATORY PROTEIN OF HERPES-SIMPLEX VIRUS TYPE-1

Among the five immediate-early regulatory proteins of herpes simplex v irus (HSV) type 1, only ICP0 is capable of activating all kinetic clas ses of viral genes. Consistent with its broad transactivating activity , ICP0 plays a major role in enhancing the reactivation of HSV from la tency both in vivo and in vitro. Although not essential for viral repl ication, ICP0 confers a significant growth advantage on the virus, esp ecially at low multiplicities of infection. In this report we describe the expression of a novel activity by the osteosarcoma cell line U2OS that can substitute functionally for ICP0. Compared with Vero cells, both U2OS cells and cells of the ICP0-expressing line 0-28 significant ly enhanced the plating efficiency of an ICP0 null mutant, 7134. In co ntrast, the plating efficiencies of the wild-type virus in all three c ell types were similar. Single-step growth experiments demonstrated th at the yield of 7134 in U2OS cells was severalfold higher than that in 0-28 cells and about 100-fold higher than that in Vero cells, In orde r to identify the viral genes whose expression is enhanced by the acti vity in U2OS cells, levels of expression of selected viral proteins in extracts of Vero and U2OS cells were compared by Western blot (immuno blot) analysis following low-multiplicity infection. At a multiplicity of 0.1 PFU per cell, the levels of expression of the immediate-early protein ICP4 and the early protein go in 7134-infected U2OS cells were significantly higher than those in 7134-infected Vero cells. When inf ections were carried out at a multiplicity of 1 PFU per cell, however, no major differences in the levels of expression of these proteins in U2OS and Vero cells were observed. Cycloheximide reversal experiments demonstrated that the cellular activity expressed in U2OS cells that promotes high-level expression of ICP4 is not synthesized de novo but appears to exist as a preformed protein(s). To confirm this observatio n and to determine whether, like immediate-early genes, early, delayed -early, and late viral genes are also responsive to the cellular activ ity, transient-expression assays were performed. The results of these tests demonstrated that basal levels of expression from immediate-earl y, early, and delayed-early promoters, but not that from a late promot er, were significantly higher in U2OS cells than in Vero cells and tha t this enhancement occurred in the absence of viral proteins. Mutation al analysis of the ICP4 promoter, the most responsive of the HSV type 1 promoters tested, has shown that the 139-bp sequence between the Sph I and SacII sites is required for the high basal level of expression o f ICP4 in U2OS cells.