A Mutational Analysis of the Structure and Function of the Herpes Simplex Virus Immediate Early Protein Vmw175

Herpes simplex virus type 1 (HSV-1) expresses three main classes of genes (immediate-early [IE], early [E] and late [L]) in a temporal cascade upon infection of tissue culture cells. These three groups of genes can also be defined by the sensitivity of their expression to metabolic inhibitors of protein or DNA synthesis. IE genes are transcribed in the absence of de novo protein synthesis and their transcription is stimulated by a component of the virus particle, Vmw65. IE gene products are required for the activation of later classes of viral genes, whose expression varies in sensitivity to inhibitors of viral DNA replication. Early genes are expressed at maximal levels in the absence of DNA synthesis, whilst true late genes are critically dependent on replication for expression. The predominant transcriptional regulatory protein specified by HSV-1 is the IE protein Vmw175, whose functions are essential for virus growth. Studies of viruses with temperature sensitive lesions in this protein have shown that Vmw175 is a complex multifunctional protein required for the transcriptional activation of many HSV-1 promoters and the repression of its own transcription. Cloned Vmw175 is a promiscuous transactivator of transcription of RNA polymerase type II promoters. In addition cloned Vmw175 represses transcription from its own promoter, probably by binding to a specific target sequence at the start site for transcription. The aim of the work described in this thesis was to investigate the relationship between the structure and function of this protein, and to define which regions of the protein are involved in each of its various activities. A panel of plasmid-borne in-frame insertion and deletion mutants of the gene encoding Vmw175 were constructed and assayed for their ability to regulate transcription in transient transfection assays. Fusions of HSV promoters to the chloramphenicol acetyl transferase gene were used to assay the ability of each mutant to transactivate an HSV early promoter (that of the gene encoding glycoprotein gD) and to repress the promoter of its own gene, IE3, in transiently transfected cells. By this approach it was possible to define the regions of the Vmw175 amino acid sequence that are required for transcriptional activation and repression. Large stretches of the protein are relatively unimportant for either function, while the regions most sensitive to disruption correlate to sequences conserved between Vmw175 and VZV 140K, the corresponding transactivating protein of another alphaherpesvirus, varicella-zoster virus. The region from amino acids 275 to 495 is particularly important for both repression and transactivation; whilst that from around 840 to 1100 seems to be more important for transactivation than repression. A monoclonal antibody directed against Vmw175 was used to visualize expression and localization of Vmw175 in transfected cells by indirect immunofluorescence. This provided confirmation that each of the mutant plasmids expressed Vmw175. Each of the 39 insertion mutant proteins exhibited a pattern of nuclear localization indistinguishable from that of wild-type, but when amino acids 682-774 were deleted a signal essential for nuclear localization was lost. A strong candidate for a nuclear localization signal centres around amino acid 728 and is strongly conserved in the VZV homologue. This sequence PREGRKRKSP contains four consecutive arginine (R) and lysine (K) residues and is related to a signal in the SV40 large T antigen required for nuclear localization. Vmw175 is known to bind directly to a number of HSV-1 sequences, some of which contain the consensus sequence ATCGTC. The binding of Vmw175 to this sequence at the transcriptional start site of IE gene 3 is thought to be involved in the mechanism of autoregulation. In order to correlate the transcriptional activity of each mutant with its ability to bind to DNA, the site specific DNA binding activity of each mutant was assayed. In these experiments nuclear extracts of transfected cells were incubated with a DNA probe spanning the IE3 cap site and protein complexes detected using the gel retardation technique. The results show that a critical region of Vmw175, amino acid residues 275-495, includes structures which are essential for specific DNA binding, transactivation and repression