The start site regions of late genes of herpes simplex virus type 1 are similar to the eukaryotic initiator sequence (Inr), have been shown to affect the levels of expression, and may also play a role in transcription activation by the viral activator ICP4. A series of linker-scanning mutations spanning the start site of transcription and several downstream mutations in the true late gC promoter were analyzed in reconstituted in vitro transcription reactions with and without ICP4, as well as in the context of the viral genome during infection. The nucleotide contacts previously found to be important for Inr function were also found to be important for optimal induction by ICP4. While the Inr had a substantial effect on the accumulation of gC RNA during infection, no other sequence downstream of the TATA box to ؉124 had a significant effect on levels of expression during infection. Therefore, these studies suggest that TATA box and the Inr are the only cis-acting elements required to achieve optimal expression of gC, and that the high levels of late-gene transcription may be largely due to the induction by ICP4, functioning through the Inr element.During productive infection by herpes simplex virus type 1 (HSV-1), approximately 80 genes are sequentially expressed in three major kinetic groups: immediate-early (IE, ␣), early (E, ␤), and late (L, ␥) genes (36,37,46,60,61). Each HSV-1 gene has its own promoter with an obvious TATA box homology that is recognized by the host RNA polymerase II transcription machinery (1,11,89). A viral protein VP16, carried in with the virion, activates transcription of the five IE genes in a complex with two cellular proteins, Oct-1 and HCF (5,55,66). Once expressed, the IE gene products regulate expression of the early and late genes by a variety of mechanisms in the course of infection (75).One of the IE proteins, ICP4 (infected-cell polypeptide 4), is essential for viral growth as it is required for optimal expression of most early and late genes (15,19,22,25,67,68). ICP4 is a nuclear phosphoprotein that acts as a homodimer to activate or repress transcription depending upon the promoter (12,15,26,54,63,67,71). Genetic and biochemical studies have shown that ICP4 binds to DNA and interacts with a component(s) of general RNA polymerase II transcription machinery to activate or repress transcription (2,8,17,29,42,64,65,84). ICP4 forms a tripartite complex with TFIIB and TATA binding protein (TBP) on DNA, interacts with TBP-associated factor 250 (8, 84), and promotes the formation of transcription preinitiation complexes on promoters (28).The transcriptional regulation of HSV genes during infection is mostly determined by the structure of a given promoter. The promoters of IE and E genes contain a number of cisacting elements upstream of the TATA box (89, 90). Viral and cellular proteins (Oct-1/VP16, Sp1, CTF or NF1, etc.) bind to these cis-acting elements to activate transcription (21,24,25,45,89). An important characteristic of late genes is that their expression is significantly i...

The expression of most herpes simplex virus type 1 (HSV-1) immediate-early (IE) and early (E) genes decreases late in productive infection. IE and E promoters contain various binding sites for cellular activators, including sites for Sp1, upstream of the TATA box, while late gene promoters generally lack such sites. To address the possibility that Sp1 function may be altered during the course of infection, the modification state and activity of Sp1 were investigated as a function of infection. Sp1 was quantitatively phosphorylated in HSV-1-infected cells without a significant change in abundance. The kinetics of accumulation of phosphorylated Sp1 immediately preceded the decline in E gene (thymidine kinase gene [tk]) mRNA abundance. Phosphorylation of Sp1 required ICP4; however, the proportion of phosphorylated Sp1 was reduced during infection in the presence of phosphonoacetic acid or in the absence of ICP27. While the DNA binding activity of Sp1 was not greatly affected by phosphorylation, the ability of phosphorylated Sp1 isolated from HSVinfected cells to activate transcription in vitro was decreased. These studies suggest that modification of Sp1 may contribute to the decrease of IE and E gene expression late in infection.During productive infection by herpes simplex virus type 1 (HSV-1), viral genes from three major kinetic classes are sequentially transcribed by host RNA polymerase II machinery (1,9,23,28). The activated expression of immediate-early (IE) genes, which is required for the subsequent cascade of gene expression, is achieved immediately after infection by the function of the virion transcription activator VP16 (3, 5). IE gene promoters also contain multiple binding sites for the transcription factor Sp1. Following the expression of IE proteins, the expression of early (E) genes begins, and it is maximally induced by the IE transcription activator ICP4 (25,40,55). Expression of most IE and E genes is subsequently attenuated, and with viral DNA replication, late (L) genes are expressed (14,24,55). Even though both E and L genes are highly activated by ICP4 and transcribed by the same transcription machinery, only the E genes are efficiently shut off later in infection. The mechanisms involved in attenuating IE and E gene expression late in infection remain unclear.