The roles of ND10 proteins ATRX and hDaxx in the regulation of herpesvirus infection

Protection against viruses is provided by the host innate, adaptive and cellular intrinsic immunities. Cellular intrinsic immunity, or intrinsic defence, against herpesviruses is in part enacted by components that constitute nuclear substructures known as ND10. ND10 (also called PML nuclear bodies after its major organising component) are dynamic nuclear domains that contain various cellular proteins, including PML itself, Sp100, hDaxx and ATRX. In the early stages of infection, herpesvirus genomes and sites of immediate early transcription become associated with ND10 and their components. Representative herpesviruses such as an alphaherpesvirus HSV-1 and a betaherpesvirus HCMV encode for strong transcriptional activators, namely the Immediate Early protein ICP0 and the tegument protein pp71, respectively. These proteins are known to counteract cellular intrinsic defence mechanisms. In the context of HSV-1 and HCMV infections, the major ND10 component PML has been identified as an important constituent of cellular intrinsic defence. In addition, a number of research studies have demonstrated that Sp100 and hDaxx contribute to these processes during infection with HSV-1 and HCMV, respectively. The general hypothesis of the present study implies that cellular chromatin-associated factors within ND10 may act to repress viral gene expression. The two representative ND10 components present a particular interest for the current investigation based on the facts that: (i) hDaxx is a transcriptional co-repressor; (ii) ATRX is a chromatin-remodelling enzyme; and (iii) ATRX and hDaxx interact with each other to form a chromatin-remodelling complex with repressive properties. The purpose of the present study was therefore to investigate the roles of these two proteins in HSV-1 and HCMV infection. By using virus mutants incapable of efficient stimulation of Immediate Early gene expression (ICP0-null HSV-1 and pp71-null HCMV) it was possible to analyse the contribution of ATRX and hDaxx to the repression mechanism that occurs in the absence of these viral transactivators. An RNA interference approach was utilised for generating cell lines depleted of ATRX or hDaxx in order to assess their roles in viral infectivity. In addition, cell lines reconstituted with wt hDaxx, and ATRX-interaction or SUMO-interaction deficient hDaxx mutants were constructed in order to study the contribution of these functional elements to the role of hDaxx in the repression of ICP0-null mutant HSV-1. The key findings presented in the current study can be summarised as follows: (1) ATRX contributes to the intrinsic resistance against HCMV infection, and this mechanism is strongly counteracted by viral pp71; (2) A chromatin-remodelling complex formed between ATRX and hDaxx contributes to the efficient repression of ICP0-null HSV-1 genomes, thereby constituting a part of anti-HSV-1 intrinsic cellular defence. ICP0 counteracts this process, and the possible mechanisms of ICP0 action are proposed. These data provide the first evidence for the role of ATRX in viral infection and in addition demonstrate a role for hDaxx in the regulation of HSV-1 infection. The strong indication that ATRX and hDaxx act as a complex opens a possibility of chromatin-dependent repression of ICP0-null HSV-1 genomes. Whether ATRX and hDaxx contribute to the repression of pp71-null HCMV genomes as a complex is yet to be established. In summary, the conclusion of the studies presented in this thesis suggests regulatory roles of ND10-localised chromatin remodelling proteins ATRX and hDaxx in cellular anti-herpesvirus intrinsic resistance mechanisms.EThOS - Electronic Theses Online ServiceGBUnited Kingdo