Abstract
As a first step in identifying the functions and intramolecular functional domains of herpes simplex virus type 1 infected cell protein 0 (ICP0) in productive infection and latency, a series of mutant plasmids specifying varying amounts of the ICP0 primary amino acid sequence were constructed. In transient expression assays with mutant and wild-type plasmids, the N-terminal half of the ICP0 molecule was found to be sufficient to transactivate a variety of viral promoters. Although promoters representing the immediate-early, early, and late kinetic classes were transactivated by wild-type ICP0, individual promoters responded to mutant forms of ICP0 in a manner consistent with the possibility that ICP0 transactivates different promoters by different mechanisms. Unlike infection with virus particles, which contain the 65-kilodalton transcriptional transactiovator, the initiation of viral replication after transfection of cells with purified viral DNA requires de novo protein synthesis. In order to assess the role of ICP0 in the de novo synthesis of infectious virus, Vero cells were transfected with purified DNA of wild-type virus or an ICP0 null mutant and the production of infectious virus was monitored. In cells transfected with mutant DNA, virus production was delayed by 2 days and the level of virus was reduced by several orders of magnitude relative to Vero cells transfected with wild-type viral DNA, suggesting an important role for ICP0 in the de novo synthesis of infectious particles. In cotransfection experiments with infectious DNA of the ICP0 null mutant and a plasmid specifying wild-type ICP0 titers of infectious virus were significantly enhanced relative to transfection with mutant DNA alone, confirming the role of ICP0 in de novo synthesis. These findings are consistent with the proposed role of ICP0 in reactivation of herpes simplex virus from latency (D. A. Leib, D. M. Coen, C. L. Bogard, K. A. Hicks, D. R. Yager, D. M. Knipe, K. L. Tyler, and P. A. Schaffer, J. Virol. 63:759-768, 1989), a process also thought to require de novo protein synthesis. The complementing activities of ICP0 mutant plasmids for ICP0 null mutant DNA in cotransfection assays correlated well with their transactivating activities for viral promoters in transient assays, indicating that the transactivating function of ICP0 is a critical factor in the de novo synthesis of infectious particles.(ABSTRACT TRUNCATED AT 400 WORDS)
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