Functional characterization of herpes simplex virus type-2 microRNA-H3 in human induced pluripotent stem cells-derived peripheral sensory neurons

Abstract

Human Simplex Virus Type-2 (HSV-2) is a type of ubiquitous α-herpes virus causing genital herpes. After primary lytic infection in the mucosal epithelium, HSV-2 establishes latency within the sensory neurons. Viral latency is characterized by the repression of lytic gene expression whereas latency associated transcript (LAT) is the only viral transcript being expressed. To facilitate viral infection, HSV acquires mechanisms to inhibit host’s cell death. In another HSV serotype, Human Simplex Virus Type-1 (HSV-1), HSV-1 LAT was found to promote human neuronal cell survival in many studies. However, even HSV-2 is biologically similar to HSV-1, the anti-apoptotic ability of HSV-2 LAT in human neuron remains unclear. There is a lack of studies in HSV-2 LAT and human neuronal cell death. It would be interesting to find out if HSV-2 LAT has similar anti-apoptotic function. Several HSV-2 encoded microRNAs (miRNAs) are found within the LAT locus. Some of them are highly expressed during viral latent infection. I hypothesized these abundantly expressed LAT-encoded miRNAs may function in a way to facilitate viral latency. As many other herpesviruses encode microRNAs to inhibit host’s cell death, it is fascinating to study the role of the LAT-encoded miRNAs and their relation to host cell death. My study focused on the most abundantly expressed miRNAs during latency, the miR-H3. Peripheral sensory neurons (PSNs) are the site where HSV-2 establishes latency, I aimed to characterize the function of HSV-2 miR-H3 in human induced pluripotent stem cells (iPSCs)-derived PSNs. Bioinformatic analysis predicted miR-H3 targets several key signaling molecules in apoptosis and necroptosis, including Apaf1, Casp8, Casp10 and RIPK1 etc. More importantly, sequence alignment analysis demonstrated that miR-H3 binding sites within targets’ 3’ untranslated region (UTR) can only be found in human or primates. To consolidate the target prediction, association between miR-H3 and putative targets was demonstrated by dual luciferase reporter assays and Ago2 RNA-IP experiments. I also established the iPSCs-derived PSNs as a physiologically relevant cell model to study HSV-2 infection and miR-H3. Protocols used for the differentiation were optimized to ensure over 90% of successful differentiation. With the use of the model system, I was first to show susceptible HSV-2 infection in iPSCs-derived PSNs. Meanwhile, a miR-H3 stably expressing iPSC-derived PSNs were generated to study the role of miR-H3 in cell death. Neurons’ viability was estimated after the induction of cell death. In the presence of miR-H3, significant decrease in the percentage of propidium iodide (PI) positive cells was observed after challenging the cells with camptothecin or nutrient depletion. My findings suggested HSV-2 LAT may possess ability to inhibit cell death via the action of its encoded miR-H3. This is crucially important for a better understanding in HSV-2 virology and the development of therapeutics against HSV-2 infection.