The downstream activation sequence of the strict late Herpes Simplex Virus Type 1 U(L)38 promoter interacts with hTAF(II)70, a component of TFIID.

A class of strict late Herpes Simplex Virus Type 1 (HSV-1) promoters contains a conserved sequence element (termed the downstream activation sequence, DAS) located downstream of the transcription start site. These DAS-containing promoters also require both a TATA box and an initiator element for maximal levels of transcription. In this communication, we demonstrate that the downstream promoter element (DPE) found on a class of Drosophila TATA-less promoters and known to bind the homologue of human TAF(II)70 (a component of TFIID), can functionally substitute for DAS in the context of the strict late UL38 promoter in spite of no obvious sequence similarity. Although Drosophila DPE-containing promoters do not require a TATA box, the element does not remove the requirement for a TATA box when functioning in the HSV promoter. Next, we demonstrate that hTAF(II)70, interacts in a sequence specific manner with DAS as predicted from the fact that DPE binds Drosophila TBP. These results suggest that multiple TFIID/promoter interactions are important in the activation of HSV-1 late gene expression upon viral DNA replication. We propose that such interactions could be favored upon viral DNA replication since TFIID concentrates to viral transcription foci that form during the later stages of infection.

Analysis of factors influencing kinetics of herpes simplex virus transcription utilizing recombinant virus.

The herpes simplex virus type 1 (HSV-1) transcription program is a regulated cascade in which early and late phases of gene expression are separated by viral DNA replication. While promoters controlling expression of transcripts encoding immediate-early proteins contain virus-specific cis-acting elements, these are in the context of cellular promoter elements, and the promoters controlling expression of other viral transcripts contain only cellular cis-acting elements. We had developed and continue to refine a general method for the production of recombinant viruses in which modified promoters can be inserted into nonessential loci within the viral genome through homologous recombination. This approach has been especially useful in defining the features of model promoters of the various kinetic classes. Our work suggests that class-specific differences in promoter architecture are critical factors in the ability of the cellular transcription machinery to form stable preinitiation complexes at various phases of infection and, thus, mediate kinetic class-specific transcription. Early (beta) promoters contain a TATA box and upstream activation elements while sequences downstream of the TATA homology are dispensible for transcription. Late transcripts can be catagorized as either leaky-late (beta gamma) or strict late (gamma) depending on whether they are readily detectable prior to viral DNA replication. Promoters controlling both types are clearly distinct from early ones in that sequences near the transcription start site which resemble consensus mammalian initiator elements are required along with the TATA box and activator elements. Strict late promoters do not contain elements upstream of the TATA box but include what appears to be a class specific element downstream of the transcription start site.

Purification and characterization of a cellular protein that binds to the downstream activation sequence of the strict late UL38 promoter of herpes simplex virus type 1.

Previous work on the strict late (gamma) UL38 promoter of herpes simplex virus type 1 identified three cis-acting elements required for wild-type levels of transcription: a TATA box at -31, a consensus mammalian initiator element at the transcription start site, and a downstream activation sequence (DAS) at +20 to +33. DAS is found in similar locations on several other late promoters, suggesting an important regulatory role in late gene expression. In this communication, we further characterize the interaction between DAS and a cellular protein which is found in both uninfected and infected nuclear extracts. This protein was purified from HeLa nuclear extracts and identified as the DNA binding component (Ku heterodimer) of DNA-dependent protein kinase (DNA-PK) by peptide mapping. Highly purified DNA-PK was able to stimulate UL38 transcription in vitro approximately 10-fold. DAS is similar in sequence to another element, nuclear regulatory element 1 (NRE1) of the glucocorticoid-responsive mouse mammary tumor virus long terminal repeat. NRE1 is known to specifically bind Ku in the absence of DNA ends. We demonstrated that NRE1 is able to substitute for DAS in the UL38 promoter to activate transcription as measured by in vitro transcription and in vivo during infection of tissue culture cells with recombinant virus. Also, we found that the binding of DNA-PK to DAS involves the bases demonstrated to be important in UL38 transcription and that the 70-kDa subunit of Ku binds to DAS.

Functional modules important for activated expression of early genes of herpes simplex virus type 1 are clustered upstream of the TATA box.

Functional analysis of two promoters controlling early herpes simplex virus type 1 (HSV-1) transcripts encoding the UL37 and UL50 (dUTPase) proteins are described in this report. Transcripts expressed under the control of these promoters were found to be expressed early regardless of the position of the transcription unit within the viral genome. Despite this, wt dUTPase mRNA was 6-10 times more abundant than the UL37 transcript both in wt and recombinant viruses. This same difference in transcript abundance was seen when a reporter gene (beta-galactosidase) was controlled by the two promoters in recombinant viruses in the heterologous glycoprotein C (gC) locus. Thus, both the kinetics and relative abundance of UL50 and UL37 transcripts are a direct function of their respective promoter regulatory elements. Characterization of mutated UL37 and UL50 promoters in recombinant viruses showed that the functional modules important for expression from these promoters are concentrated upstream of the transcription start site; however the extent and composition of these modules in terms of the cis-acting elements they contain was different for each. For the UL37 promoter, both a HiNF-P factor binding site (-53 to -58 bp) and the TATA homology (-22 to -27) were required for any detectable expression, while an Sp1 binding site at -123 augmented this but was not absolutely required. In contrast, the only functional elements crucial for expression from the UL50 promoter were the TATA box (-25 to -31) and an Sp1 binding site at -117 bp relative to the cap site. Despite differences in detail, when the functional architecture of these two early promoters were compared to the extensively characterized HSV-1 thymidine kinase (UL23) promoter, class-specific similarities are clearly apparent.

The herpes simplex virus type 1 VP5 promoter contains a cis-acting element near the cap site which interacts with a cellular protein.

The promoter controlling the expression of the transcript encoding the major herpes simplex virus type 1 capsid protein (VP5, UL19) extends only 60 bases or so from a functional Sp1 site at --48 to include a cis-acting element 3' of the transcript start site. In the present communication, we report the generation of recombinant viruses bearing mutations between --6 and + 8 relative to the cap site in the VP5 promoter controlling expression of a reporter gene. Analysis of the effects of these mutations upon reporter gene expression along with the results of protein binding assays demonstrates that this cap transcription element functionally interacts with a cellular protein of a normal size of 40 kDa. Thus, like the strict late UL38 promoter characterized earlier, the late VP5 promoter has the essential properties of a cellular promoter.