Bioinformatically-predicted varicella zoster virus small non-coding RNAs are expressed in lytically-infected epithelial cells and neurons

Herpesviruses use both host and viral small non-coding RNAs, including microRNAs (miR) to modulate infection. In our previous study, 24 potential VZV-encoded sncRNAs were predicted by bioinformatic analyses of NGS data, seven of which were confirmed to be expressed in infected human fibroblasts and neurons using stem-loop quantitative reverse transcription PCR (SL-PCR). Transfection of a locked nucleotide (LNA) antagonist to one of the VZVsncRNA modulated viral spread, consistent with its having a role in the life-cycle of VZV. In the present study, we surveyed for the expression of all 24 of the predicted VZVsncRNAs by SL-PCR in cells infected by VZV. 23 of the 24 predicted sequences were detected in VZV-infected ARPE19 cells and 19 of the 24 sequences in infected human neurons. We then examined the role of a frequently detected VZVsncRNA2 (5/8 experiments) and infrequently detected VZVsncRNA20 (1/5 experiments) on VZV lytic replication. LNA antagonists to VZVsncRNAs were transfected into VZV-infected cells, focus size was measured using live cell-imaging, and standard plaque assays performed. The antagonist to VZVsncRNA20 significantly and consistently enhanced focus growth and plaque assays revealed a significant increase in plaques formed by cells from antagonist-treated wells. By contrast the antagonist to VZVsncRNA2 had no effect on viral growth. Intriguingly, our preliminary results show that at least some of the VZVsncRNAs are detected in the medium using protocols for isolation of exosomes. This study is a further step in understanding the additional complexity of herpesvirus replication that sncRNAs add to expression control by proteins.