Investigating the Dynamic Aspects of Herpes Simplex Virus 1 Latency

Abstract

Herpes simplex virus type 1 (HSV-1) is a common, persistent pathogen. Common disease manifestations of HSV-1 infection can be as mild as cold sores, however, severe forms of the disease can be fatal. Herpes encephalitis, herpes keratitis and neonatal herpes, are associated with significant life-long illness and, in some cases mortality. The biology of HSV-1 is characterised by phases of productive, and latent infection with episodic reactivation events. Latency temporally dominates over the course of infection and allows the viral genome to persist in the absence of productive virus formation. There is a growing appreciation that HSV-1 latency is not entirely quiescent, contrary to the traditional silent perspective, and this view is explored in the thesis. The use of the ROSA26R/Cre mouse system allows for historic assessment of viral activity. After infection with viruses that express Cre, permanent expression of beta-galactosidase (b-gal) occurs in the infected cells which is an indirect marker of viral activity. Previous research demonstrated that a lytic promoter gB (UL27) can drive protein expression in latency, based on the accumulation of b-gal+ cells in latency. However, the promoter-Cre cassette was placed in an ectopic location in the viral genome - the UL3 and UL4 intergenic region. Importantly, the UL3/UL4 region is closer to genomic repeat regions that are known to be transcriptionally active in latency. We asked if the activity in latency is dependent on the position of the promoter-Cre cassettes in the viral genome. To investigate this, we inserted promoter gB-Cre cassette in two independent regions - the UL26/UL27 region, away from the repeat area, and the UL55/UL56 region, closer to the repeat region. We found that the gB promoter could make protein in latency only when it was placed in the location closer to the repeat region. To explore further, we looked at the native promoters of UL3, UL27 and UL56 in their respective genomic locations. By using the 'self-cleaving' T2A sequence, the promoters drive the native gene and cre at the same level and the proteins are separated post-translationally. The results largely recapitulated what was observed for ectopic promoters in the different genomic locations. This set of experiments also comprehensively demonstrated that lytic promoters in their native location can produce protein during HSV-1 latency. HSV-1 latency is governed by both host and viral factors, including miRNAs. Due to the redundancy in miRNA activity and unknown roles of many viral miRNAs, we explored the global role of both host and viral miRNAs in latency. This was achieved by removing Dicer, which is an essential enzyme for the miRNA biogenesis pathway, in a novel in vivo model. We targeted Dicer only in infected cells by knocking out the functional protein using an HSV-1 that expresses Cre. Surprisingly, loss of Dicer impacted the latent phase whereas acute HSV-1 pathogenesis was not altered. We observed almost a 61% decrease in HSV-1 genomes between days 30 and 150 after infection, when Dicer was removed from infected cells. Additionally, the reactivation potential of the virus was severely affected in the absence of a functional Dicer. We further explored if the loss in HSV-1 genomes and reactivation potential in the absence of Dicer was due to the death of infected neurons. However, infected neurons survived in the absence of Dicer. Altogether, this suggests that Dicer is required for the active maintenance of HSV-1 latency. Further, Dicer, and the miRNA biogenesis pathway, are potential therapeutic targets to eliminate latent HSV. Show more