Substitution of a TATA box from a herpes simplex virus late gene in the viral thymidine kinase promoter alters ICP4 inducibility but not temporal expression

A N Imbalzano, N A DeLuca
1992 Journal of Virology  
The role of cis-acting promoter elements associated with herpes simplex virus type 1 (HSV-1) early and late genes was evaluated during productive infection with regard to activation of gene expression by the HSV-1 transactivator ICP4 and control of temporal regulation. A set of recombinant viruses was constructed such that expression of an HSV-1 early gene, thymidine kinase (tk), was placed under the control of either the tk TATA box or the TATA box from the late gene, glycoprotein C (gC), in
more » ... rotein C (gC), in the presence or absence of the upstream Spl and CCAAT sites normally found in the tk promoter. The presence of Spl sites in the promoter or replacement of the tk TATA box with the gC TATA box resulted in a decreased activation of tk mRNA expression by ICP4. Substitution of the A+T-rich region from the gC TATA box in the context of the remainder of the surrounding tk sequences resulted in a promoter that bound recombinant TATA-binding protein (TBP) better at lower concentrations than the wild-type tk promoter did. These results indicate that tk promoters that are better able to utilize TBP are less responsive to ICP4 activation and suggest that activation by ICP4 involves the general transcription factors that interact with TBP or TBP itself. Additionally, all of the viruses expressed tk at early times postinfection, indicating that cis-acting promoter elements that control the level of expression of HSV-1 early and late genes do not determine temporal regulation. Herpes simplex virus type 1 (HSV-1) genes can be roughly categorized into three classes on the basis of their time of expression during productive infection (18). Immediate-early (IE) genes are expressed shortly after infection, and their transcription does not require prior protein synthesis (18, 19) . Early genes are the next class expressed; their synthesis requires the activity of at least one IE protein (19). Among the products of early genes are the DNA replication enzymes. Finally, late genes are expressed to maximum levels following viral DNA synthesis (18, 19) . Late genes encode predominantly the structural proteins of the virion. Expression of the early and late genes is absolutely dependent on the presence of functional IE proteins and, in particular, the IE protein ICP4 (11, 42). ICP4 is a 170-kDa phosphoprotein that is required for activating transcription from most HSV-1 genes during viral infection (11, 42, 58) . Mutational analyses have identified domains of the ICP4 molecule that are associated with its transcription-activating function (9, 10, 38, 39, 48) ; however, the mechanism by which ICP4 transactivates HSV-1 early and late gene promoters remains unclear. ICP4 binds specifically to diverse sequences present in many HSV-1 early and late genes (14, 22, 27, 34, 36, 54) ; however, the ability of ICP4 to bind to DNA has not been clearly shown to contribute to transactivation (2, 22, 40, 46, 47, 49, 54, 55) . The viral thymidine kinase (tk) promoter is transactivated by ICP4 (6, 8, 35 ). It is transcribed by RNA polymerase II and contains a CCAAT box and two Spl sites upstream of a * Corresponding author. TATA box (24, 33). Studies of this promoter have found that only the cis sequences that interact with cellular transcription factors are required for expression of tk during viral infection; no induction-specific sequences have been identified (3, 5, 12) . A more recent study directly evaluated the role of these cis sites in ICP4 induction of the tk promoter and found that induction by ICP4 was not dependent on the integrity of these cis sequences (21). Efficient induction by ICP4 could be observed in the presence of only the tk TATA box. Therefore, the induction of HSV-1 genes by ICP4 may be mediated through TFIID, the other general cellular transcription factors (TFIIA, TFIIB, and TFIIE/F), or RNA polymerase II. TFIID is composed of TATA-binding protein (TBP) and a number of TBP-associated factors that often mediate interactions with activating proteins (lla). Biochemical studies on the IE transactivator of pseudorabies virus, which shows sequence conservation with ICP4 (4, 57), have shown that IE stabilizes the interaction of TFIID with an inducible promoter (1). Therefore, promoters that possess TATA boxes with a greater affinity for TFIID or TBP may not be affected by the activity or presence of ICP4 as much as are TATA boxes that have a lower affinity for these factors. In this study, the effects of ICP4 mediated induction of two different TATA box sequences with different affinities for TBP were evaluated to test this hypothesis. HSV-1 late gene promoters differ from early gene promoters in that the cis-acting sequences 5' to the TATA box, such as Spl or CCAAT sites, that are prevalent in early promoters (53) are usually not present in late promoters. In general, prior analyses of late gene promoters have indicated that DNA sequences 5' to the TATA box are not required for induction or for regulation as a late gene (14, 16, 17, 23, 45) . Late gene promoters also differ from early promoters by their inability to be maximally expressed until viral DNA replication occurs (18). However, a tk promoter lacking the 5453 Vol. 66, No. 9 on May 8, 2020 by guest
doi:10.1128/jvi.66.9.5453-5463.1992 fatcat:ff32wadllzfgzdgelr2zqjywb4