Post transcriptional regulation of HSV gene expression and studies of the mutagenic properties of HSV-1

During herpes simplex virus (HSV) infection expression of the different classes of viral genes is co-ordinately regulated and sequentially ordered. The three major classes of genes - immediate-early (IE), early and late can be separated on the basis of the kinetics of their expression and requirements for ongoing DNA synthesis. At the core of this regulation are three of the IE proteins IE110, IE175 and IE63. Both IE175 and IE63 are essential for viral growth and IE110 while non-essential does confer growth advantage in cell culture. While much is known about transcriptional control of gene expression and the interplay of these three IE proteins, little is known of any post- transcriptional regulatory mechanisms that may be employed by the virus. Recent interest has focused on this area and the aim of this study has been the investigation of a virus induced factor, termed LPF (late processing factor), which was previously shown to selectively increase the 3' processing efficiency of the herpes simplex virus type-2 (HSV-2) UL38 late poly(A) site. In this study an in vitro 3' processing assay was used to examine the 3' processing efficiencies of a selection of herpes simplex virus type-1 (HSV-1) poly(A) sites from the three temporal classes of genes. It was demonstrated that LPF selectively increased processing at the poly(A) sites of two late HSV-1 genes while having no effect on the processing efficiencies of another four poly(A) sites from the IE and early classes of genes. In addition both LPF responsive poly(A) sites were shown to be inherently less efficient 3' processing sites that the non-responsive sites. No common factor, which could be responsible for the reduced efficiency of these two sites, was identified by sequence or two dimensional structural analysis. Examination of the protein binding properties of each of the test poly(A) site RNAs revealed that three protein bands A, B and C were consistently bound to the poly(A) site RNAs. These proteins were of similar size to components of the 3' processing complex required for efficient cleavage and polyadenylation of precursor mRNAs. In addition the level of protein binding was shown to be increased by HSV infection. As stated previously the HSV-1 IE63 gene is essential for viral growth and it has been shown by a number of studies to be required for late gene expression. There is evidence that it can exert this influence at both the transcriptional and post- transcriptional levels and also that it may or may not influence late gene expression via an effect on DNA synthesis. In this study IE63 has been shown to be required for LPF activity and the expression of selected true-late genes (UL38, UL44 and US 11). IE63 also appears to be required for the efficient expression of three additional genes, two early genes UL29 and UL42 components of the viral DNA synthesis machinery and the IE gene lEllO. The evidence presented here supports a central role for IE63 in the regulation of gene expession. At least part of the mechanism of this regulation is at the post-transcriptional level mediated by its ability to increase the processing efficiency of selected poly(A) sites, with specific deficiencies in individual poly(A) sites making them targets for regulation. It is clear this is not the whole story and that IE63 may exert an additional influence via regulation of viral DNA synthesis. HSV-1 and HSV-2 can transform mammalian cells to a tumourigenic phenotype and it has been proposed that transformation occurs via a hit-and-run mechanism since the continued presence of viral DNA or proteins is not required for the maintenance of the transformed state. One way in which such a hit-and-run mechanism might operate is by increasing the frequency of mutations and there is evidence that HSV-1 can act as a mutagen. The aim of the second part of this study was to identify the properties of HSV-1 which could induce such mutations using a mutagenesis assay based on the shuttle vector pZ189. This study was the continuation of work initiated in the Institute by P. Clarke and the first step was to increase the efficiency of transformant recovery in the assay. To this end each aspect - plasmid preparation, transfection, infection and tranformation was optimized in turn and transformant recovery increased 3 to 5-fold. Using this assay HSV-1 infection was shown to increase the mutation frequency by 2.5-fold, however this was not considered to be significantly different from the spontaneous mutation frequency and further use of the assay to determine the mutagenic properties of HSV-1 was not pursued.