Transcriptional control of icp0 and its effect on hsv-1 replication

Resumen

Herpes simplex virus-1 (HSV-1) is one of the most common infectious agents found in humans, although only about a third of infected individuals exhibit symptoms such as cold sores and facial lesions. Its enveloped virions contain a 152 kilobases (kb) linear, double stranded (ds) DNA genome. HSV-1 replicates profusely in epithelial cells but in neurons, enters into latency, from which it can be reactivated to lytic replication by different external cues. The mechanism by which HSV-1 switches between these two life cycle modes is not known. An understanding of this will not only improve understanding of viral physiology but also enhance development of improved tools for viral vector-mediated gene therapy. During productive replication HSV-1 synthesizes approximately 75 proteins out of which five are translated immediately after infection. These are the immediate early (IE) proteins known as ICP0, ICP4, ICP22, ICP27 and ICP47 (ICP = infected cell protein). ICP0 is the first translated viral product and possesses multiple functions for the efficient progression of productive lytic infection. This thesis aimed to contribute to the understanding of the HSV-1 lytic-latent transition by testing if artificial control over ICP0 transcription can be used to regulate HSV-1 replication. The ICP0 gene is duplicated in the HSV-1 genome; the internal repeat (IR) and terminal repeat (TR) regions each encode one copy. In the present study, I removed one copy of ICP0 by deleting the IR region after which components of the Tetracycline-inducible system were inserted into the remaining copy to impose artificial transcriptional control of the gene. The IR-deleted mutant constructed using BAC technology confirms that the IR deletion does not affect viral replication in Vero cells. My results also indicate that the remaining copy of the ICP0 gene in TR becomes a critical determinant of productive infection when a second gene in this repeat, that of ICP34.5, is additionally deleted. My study further investigates the ability to regulate viral replication through the consensus TAATGARAT element which has a key role in ICP0 promoter function and reports that distal displacement of the TAATGARAT element away from its natural position diminishes immediate-early ICP0 expression but results in surprisingly high late protein accumulation, altered protein cleavage and cytoplasmic translocation, all of which result in impaired viral growth. These defects could be reduced by repositioning the TAATGARAT motif to decrease the displacement but not by binding of tetracycline-responsive regulators to the artificially inserted tetO sites. The results further suggest that the HSV-1 proteins ICP0 and ICP4 can interfere with the tetracycline-responsive system and thereby disrupt the regulation. The study in this thesis supports the role of ICP0 as a critical switch component in HSV-1 productive infection but shows that high levels of ICP0 do not necessarily assure viral growth and may even be detrimental when expression kinetics are altered. Not only simulatory, but also inhibitory effects are mediated by the ICP0 promoter TAATGARAT element to impose complex dynamic control on ICP0 expression, and this cannot be reproduced by simply tethering VP16 to the promoter such as with the tetracycline-responsive system. Most importantly, the present study indicates that: (1) simple ON and OFF switching of ICP0 transcription does not result in efficient viral replication which is dependent on correct ICP0 kinetics and (2) the tetracycline-responsive system may not accurately control ICP0 transcription due to interference by the ICP0 protein itself.