The Homepage of Dr. Edward K. Wagner 
border corner

Herpes simplex virus Research

Analysis of Herpes Simplex Virus Promoters

Herpes Simplex Virus Promoters as a Guide to Gene Regulation

Herpes simplex virus uses its 100 plus genes in complex and fascinating ways to ensure its continued success as a ubiquitous human pathogen.  The interaction between virus and host runs the gamut from the almost entirely passive latent state where viral genomes are maintained as severely transcriptionally restricted "guest" genomes in non-replicating neurons to the active destruction of the host cell where as much as half of the total mass of cellular DNA is made up of viral genomes.

Each stage in the replication of the virus is characterized by the expression of a specific set of viral transcripts, and each individual transcript in the set or class is controlled by the action its own class-specific promoter.  While each type of viral promoter is characterized by a specific set of virus-induced conditions for its maximal expression, each promoter is clearly representative of one or another general types of cellular promoters.

In essence, then, control of virus gene expression during the replication cycle is mediated by virus-encoded proteins modifying the transcriptional environment of the host cell so that expression of mRNA controlled by one or another class of viral promoter is favored.  Understanding those class-specific features of individual viral promoters provides a critical foundation in the continuing construction of a self-consistent and predictive understanding of the interaction between viral and cellular genes taking place during normal and abnormal patterns of viral infection and pathogenesis.

The rationale for using recombinant viruses for the experimental analysis of the early/late switch during productive infection by HSV

In order to identify specific cis-acting sequences defining the kinetic classes of promoters controlling transcript expression we have extensively mutated selected promoters of each kinetic class.  Such analysis is designed to reveal whether a general pattern of functional architecture can be identified as being of central importance in either time-based differences in promoter availability on the viral template, or in a differential ability of the cellular transcriptional environment to recognize promoters of different kinetic classes.

An important basic experimental question is what is the best method for analysis of the defined alterations made?; HSV promoters controlling expression of all classes of transcripts are active in in vitro transcription with uninfected cell extracts.  These extracts do not exhibit any evidence of the early/late or latent/productive state-associated restrictions of transcription seen in virus infection.  This means that no specific viral functions are required to "unmask" late promoters, and that time-correlated changes in viral transcription are not a result of a global change in the gross activity or population of cellular transcription factors induced by virus infection.

We also know that transcripts of all kinetic classes are randomly distributed throughout the viral genome, so their differential expression cannot be mediated by the active repression of one or another extended region of the viral genome during the productive cycle.

Taken together, these considerations mean that the study of elements involved in differential gene expression during virus replication requires the introduction of modifications into the viral genome itself.  This is true even though study of the interaction between isolated viral promoters and uninfected cellular transcription machinery have been of great value in the identification and determination of the general properties of HSV promoter.

We developed standard methods for introducing viral promoters into two regions of the HSV genome.  These regions are the glycoprotein C (gC or UL44) locus in the long unique region (UL) of the genome, and the area encompassing the promoter and 5' portion of the latency associated transcription unit (LAT) within the long repeat regions (RL) of the genome.  Neither contain genes which are essential for replication in cultured cell.

Some features and advantages of our approach are as follows:

1 The locations of insertion of modified promoters are in genes which are dispensable for efficient productive replication in cultured cells.

2 The activity of the wt promoter in its "normal" location can be co-assayed with any modifications induced.  This provides an invaluable internal ontrol.

3 Modifications which might influence levels of promoter activity in a general rather than specific way can be made in the vicinity of the inserted promoter without disrupting other viral genes.

4 All constructs with modified promoters express very similar or identical mRNAs.

5 Any promoter can be assayed under standard and equivalent conditions.  This means that late promoters encoding capsid proteins can be readily studied in the same context as early promoters, etc.  Importantly, variations in complementing cell lines are avoided.

6 All recombination is essentially equivalent; therefore, the possibility of introducing deleterious mutations by the recombination event itself, while not eliminated, is equivalent for all promoters and constructs studied.

Recombination cassettes

The cassettes were constructed by inserting the appropriate region of HSV DNA containing sufficient sequence on either side of the reporter construct to ensure efficient recombination into a convenient plasmid using a SalI site at each end.  The unique SalI sites also allow the viral insertion sequences to be easily separated from bacterial DNA sequences prior to transfection.

recombination vectors Much of our work is done using the b-galactosidase reporter in the cassette, terminated with a bi-functional SV40 cleavage/poly(A) site.  This is inserted downstream of a unique XbaI within flanking viral DNA sequences.  There is a unique KpnI site in the b-galactosidase gene downstream of the translation initiation signal.  Thus, modified viral (or any) promoters can be constructed in separate plasmids as XbaI-KpnI cassettes and then inserted into the appropriate recombination vector.

To generate recombinant viruses, infectious viral DNA from any appropriate viral strain and the recombination fragment are cotransfected by standard procedures into 60 mm dishes of rabbit skin cells.  Cells are then incubated until they demonstrate cytopathic effects (CPE) and then they are harvested. The approach outlined generally yields 1--5% recombinants which is plenty for screening.

screening graphicVirus generated from the transfected cells are diluted to limiting levels and then used to infect cultures of rabbit skin cells or Vero cells.  Virus containing the reporter gene is then screened for in infected cultures by hybridization.  Following this preliminary screen, positive cultures are subjected to further screening until the isolate is pure, as judged by lack of evidence of wt genomes, this takes at least three rounds of plaque purification.  Depending upon the efficiency of transfection and the motivation of the principals, generation of recombinant virus stocks can take as little as two weeks.

additional screening