Developing an in vitro model for investigating the effects of the HSV-1 latency-associated ncRNAs in human neurons

Abstract

Herpes simplex virus 1 (HSV-1) persists for the lifetime of the host due to establishing latency in sensory neurons. During latency, the only abundantly transcribed HSV-1 gene is the latency-associated transcript (LAT), which is processed into the 1.5kb or 2.0kb major LAT intron and several microRNAs. These latency-associated non-coding RNAs (ncRNAs) have been reported to impact the establishment, maintenance and reactivation from latency. However, the molecular mechanisms of these ncRNAs are not fully characterised, especially in the context of human neurons. In this study an in vitro model was developed to investigate the roles of the latency-associated ncRNAs in human neurons by establishing a method to differentiate SH-SY5Y cells into neurons and deliver the latency-associated ncRNAs. To achieve this, the cells were infected with a replication-defective HSV-1 mutant, in1382, that establishes a quiescent infection in which LAT is strongly expressed. Alternatively, lentiviruses were engineered to express the first 3.1kb of LAT, without or with mutations in splice sites that prevents splicing of the major LAT intron, or five HSV-1 microRNAs, shown to be abundant in latently infected human ganglia. To investigate how the latency-associated ncRNAs affects the human neuronal transcriptome, RNA-Seq was performed on uninfected versus infected/transduced differentiated SH-SY5Y cells. Over 1500 significantly differentially expressed genes (DEGs) were identified, including 179 DEGs that overlapped following quiescent infection or lentivirus delivery of the latency-associated ncRNAs. A subset of these gene changes was validated by PCR. Various cellular pathways and functions were found to be associated with the DEGs found. This included neuroprotection, control of the Wnt pathway, regulation of the cell-cycle and links to neurodegeneration. Some of these functions have been implicated in previous HSV-1 latency data. This work provides insight into putative roles of the latency-associated ncRNAs that could have implications on how HSV-1 latency affects human neurons.