Identification of the Cellular Proteins Which Interact With the Essential HSV-1 Protein IE63

Herpesviruses are involved in a range of prominent medical or veterinary diseases making this one of the most significant virus families. The HSV-1 protein IE63 (ICP27) is essential for viral replication, and is the only HSV-1 IE protein which has homologues in every herpesvirus sequenced so far which infect mammals and birds, indicative of its key regulatory role and making it an excellent antiviral target. The aim of this study was to screen a library of cellular proteins to identify those which interacted with HSV-1 IE63. This information was likely to shed light on the mode of action of this protein. The yeast two-hybrid system was used to identify interacting proteins. This system exploits the ability of a 'bait' protein fused to the binding domain of a yeast transcription factor to interact with a target protein fused to the activation domain of the same yeast transcription factor, bringing the two domains into close enough proximity to reconstitute activity of the transcription factor and subsequent transcription of reporter genes in yeast cells. Proteins that do not interact do not permit activation of reporter gene expression. In this study, a truncated IE63 protein was used to screen proteins expressed from an uninfected HeLa cell cDNA library. The protein was truncated because it was found that that the N-terminal ten amino acids of IE63 caused transcription of reporter genes in the absence of an interacting protein. The screen identified eight cellular proteins which interacted with IE63, four of which were of initial interest. These were heterogeneous nuclear ribonucleoprotein K (hnRNP K), casein kinase II (CKII) p subunit, and the splicing factors Spliceosome Associated Protein 145 (SAP 145) and p32:- these interactions were all subsequently confirmed in virus infected cells by others in our laboratory. Truncation mutants of IE63 were constructed and using these in the two-hybrid assay, the regions of IE63 required for these various interactions were mapped as well as the IE63 region required for dimersation. Similarly, using hnRNP K truncations, regions required for interaction with IE63 were identified A region containing the zinc finger domain of IE63, located towards the C-terminus, was required for interaction with the CKIip subunit, with SAP 145 and for IE63 dimerisation. Dimerisation would permit IE63 to form interactions with multiple partners in a complex of proteins with RNA, allowing cross-talk between these molecules. An IE63 region involved in the interaction with hnRNP K contained a five amino acid sequence (SADET) which is well conserved throughout the alphaherpesviruses, and which is present in a region of hnRNP K required for interaction with IE63 and is necessary for the nuclear export of hnRNP K. This region of IE63 could have a role in nuclear export, perhaps recognising a nucleoporin also recognised by hnRNP K. A further IE63 region with runs of prolines was identified as involved in interactions with hnRNP K and CKIip, and was the only region necessary for the interaction with p32. It is highly probable that IE63 inhibits splicing by interacting with SAP 145 and/or p32, and it appears that CKII is recruited by IE63 to phosphorylate itself, hnRNP K and probably other proteins. The function of hnRNP K in uninfected cells is not completely understood but it is thought that the protein has a role in transcription, pre- mRNA processing, RNA transport and translation. It is proposed that IE63 may alter transcription, mRNA processing and transport in the HSV-1 infected cell via its interaction with hnRNP K. The study also demonstrated that IE63 can form interactions with a transcriptional activator ALY, a glutamine-rich protein, the protein human JTV-1 of unknown function and the nucleoporin RIPl. Furthermore, the nucleoporin CRMl which interacts with HIV-1 Rev and facilitates its export to the cytoplasm did not interact with IE63 in the two-hybrid assay, suggesting that IE63 and Rev may use different pathways to cross the nuclear pore. Finally, the HHV-8 (Kaposi's sarcoma herpesvirus) ORF57 homologue of IE63, which also acts post-transcriptionally, did not interact with the hnRNP K, CKIip, SAP 145 and p32 clones identified in the library screen suggesting that these distantly related homologues, which belong to different herpesvirus subgroups, are likely to possess certain different activities. (Abstract shortened by ProQuest.)