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Herpes simplex virus Research

How Do HSV-1 Promoters Mediate the Kinetics of Transcription Observed?

Based upon our results to date, we conclude that major factors in the regulation of HSV gene expression are template restriction and differential promoter recognition.  Thus, even though a major feature of HSV productive infection is the global activation of transcription by viral regulatory proteins, regulation of gene expression is accomplished through the selective ability of the virus to restrict transcription to specific architectural motifs at various times following infection.

promoters

The cis-acting elements identified as diagnostic of various kinetic classes of HSV promoters are all cellular elements; thus, their action should be similar to those observed operating in the general transcriptional environment of the uninfected cell. This argues that (in agreement with available data) virus-mediated effects upon the transcriptional milieu of the cell are global.

Early HSV Promoter Architecture Provides A Clue As To How Transcriptional Shut-off Occurs

thymidine kinase promoter

As was shown with the thymidine kinase promoter, early HSV promoters are representative of a large class of cellular TATA-box containing promoters.  We suggest that the absence of transcription factor binding sites downstream of the TATA box of early promoters is important in the mechanism of shut-off of such promoters following viral genome replication.

early expression

We argue that for reasons yet to be determined, the stabilization of TFIID complexes through TATA box binding in concert with upstream accessory proteins is inadequate to allow appreciable transcription in the context of a replicating transcription template sequestered in replication compartments.

Promoters Controlling Expression of Late HSV Genes Have Stabilizing Elements Downstream of the TATA Box

late promoter

The cis-acting elements which function downstream of the TATA box in late HSV promoters are also recognizable as cellular elements.  Sequence analysis of the VP16 promoter cap region shows that it has identity with the "strong" Inr elements of the murine terminal deoxynucleotidyl transferase gene (TdT) and the mouse ribonucleotide reductase R1 promoters with the consensus sequences of (-2) YYANT/AYY(+5).  While, the VP5 promoter cap sequences are quite divergent from this consensus, it shares significant homology with cap sequences in the HIV LTR which are known to interact with cellular transcription factors.

ul38 promoter

sequence comparisonThe UL38 promoter which is so characteristic of other g promoters, has an architecture similar to that of the promoter regulating expression of the glial fibrillary acidic protein (GFAP).  The GFAP promoter contains the same three cis-acting elements- TATA box, initiator element, and downstream element positioned between +10 and +40; additionally, the relative contribution of each of these elements to activity of the GFAP promoter is essentially identical to the situation observed with UL38.

late expression

Our studies are consistent with a model of late gene expression in which the TATA box and sequences near the transcriptional start site (Inr sequences) interact together with proteins to form a stable pre-initiation complex. Here, the presence of upstream transcription factor binding sites can serve to stabilize this interaction with leaky-late promoters, while DAS elements fulfill this function in strict late promoters.

A central feature of this model is that the interactions with TFIID in the region between the TATA box and, potentially, the Inr can efficiently occur even when the transcription template is sequestered in replication compartments late in infection.

In the working model proposed, the DAS element of strict late promoters could function either alone to enhance the binding of components of the TFIID complex or via the action of a stabilizing protein--DAS binding factor (DBF).  Such a protein would not have access to strict late promoters at early times after infection, possibly due to other (non)specific protein interactions with the promoter.

Demonstration that DBF is a Portion of Cellular DNA-PK

multiple roles of DNA-pk

Since DBF is potentially a significant transcription factor important not only in HSV gene expression but also for cellular transcription by RNA polymerase II we purified DBF in order to study the function of the protein in vitro, determining the mode of activation of the protein by HSV infection, and identifying the protein or proteins it interacts with.  Our results demonstrate that DBF is a portion (the DNA binding or Ku subunit) of the ubiquitous and multi-functional cellular protein DNA-PK.

pk study

Addition of this purified protein to nuclear extracts markedly increases transcription rates from DAS-containing promoters in in vitro transcription assays.  Further, we have found that DAS has sequence homology with the NRE-1 element which binds the Ku heterodimer and represses glucocorticoid induced transcription from the murine mammary tumor virus LTR.  NRE-1 is fully functional in activating expression when substituted for DAS in the UL38 promoter.

HSV Promoters Functioning in Unmodified Cells are Also Cellular in Organization and Structure

While the similarities between cellular and HSV core promoters (which function in a wide variety of  cells in various states of differentiation) is striking, this similarity also extends to those viral promoters whose function is linked to specific features of viral gene expression in highly differentiated tissue.  For example, the latency-associated promoter requires a number of cellular elements defining its neuronal activity in order for it to function in latently infected neurons.  Also, while the immediate-early promoters function in the absence of any virus-induced modification of the infected cell by virtue of their containing specific enhancers which bind the VP16 transcriptional activator, they do so via a cis-acting sequence which binds cellular NF-1.  It is this protein which serves as an adaptor to allow VP16 to bind viral DNA.