Its sequence is included in a monocistronic form of ORF10, a bicistronic form ORF 9-10 and a tricistronic form ORF9A-9-10 and may be cleaved from all of them without having an actual physiological role. infected human neurons generated by two methods from embryonic stem cells. BIO We also show BIO that blocking one of two newly-tested VZV-encoded sncRNA using Itgbl1 locked nucleotide antagonists significantly increased viral replication. These findings suggest that further study of VZV encoded sncRNA could provide an additional level of regulation into the life cycle of this pathogenic human herpesvirus. 1Introduction A recent focus of the herpesvirus field has been the discovery of non-coding and microRNAs and how they may control viral growth, latency and reactivation (reviewed in (Cullen, 2011),(Piedade and Azevedo-Pereira, 2016). Varicella Zoster virus (VZV, human herpesvirus-3) is usually a pathogenic neurotropic human alphaherpesvirus, causing varicella (chickenpox) on primary contamination and herpes zoster (shingles) upon reactivation from the latency in the peripheral nervous system. The study of how VZV growth might be regulated by non-coding RNAs has lagged that of other human herpesviruses. Two published NGS studies of latently infected human post-mortem ganglia failed to reveal any sequences with the characteristics of miRNAs encoded by VZV (Umbach et al., 2009), (Depledge et al., 2018)). However, a recent study of the VZV transcriptome using long-read NGS has detected dozens of non-coding RNAs (Prazsk et al., 2018). A recent study of enriched viral RNA from human ganglia has suggested that latency is usually associated with multiple spliced transcripts that potentially encode small non-coding RNAs (Depledge et al., 2018). We recently reported that NGS analyses of small (<200 nucleotides, nt) RNA in lytically-infected cultured human fibroblasts and neurons revealed at least 24 sequences of 22-24nt encoded by VZV, one of which was predicted to fold into BIO a miR structure (Markus et al., 2017). The sequences of these potential small non-coding RNAs were predicted based on a novel bioinformatic analysis using manual alignment of comparable sequences from multiple reads. That study confirmed the presence of 7 of these putative VZVsncRNA using stem-loop qRT-PCR (SL-PCR) in VZV infected human fibroblasts. NGS counts representing all the predicted VZVsncRNA were also detected in BIO small RNA extracted from human embryonic stem-cell (hESC) derived neurons infected productively with VZV, although presence of only one was confirmed by SL-PCR. hESC-derived neurons latently-infected with VZV also yielded NGS reads for several of the VZVsncRNA. Given the current interest in the roles of multiple types of viral non-coding RNA in gene control of expression of the herpesvirus life-cycle, it is important to further investigate which of the NGS reads of small RNAs could be demonstrated to be expressed in VZV-infected cells using an independent assay. We report here the results of a survey for the predicted VZVsncRNA using Taqman SL-PCR for all of the 24 VZVsncRNA we predicted to be encoded by the virus in samples of small RNA (<200nt) extracted from productively infected ARPE19 cells, which are highly permissive for VZV replication and from productively infected human neurons derived from human embryonic stem cells (hESC, (Pomp et al., 2005), (Birenboim et al., 2013)). In order to investigate whether expression of VZV-encoded sncRNA may contribute to the regulation of VZV replication, we measured infectious focus growth (Markus et al., 2017) and performed plaque assays with VZV-infected ARPE19 cells transfected with specific locked-RNA antagonists to two of these VZVsncRNA. 1.?Materials and Methods Viruses, infection and cells. The VZV used in these studies were a recombinant virus expressing GFP as an N terminal fusion to ORF66 (VZV66GFP), derived from parent of Oka cosmids as detailed previously (Erazo et al., 2008). A similar virus expressing monomeric red fluorescent protein (mRFP) linked to the N terminus of ORF66, VZV66RFP, was made by recombineering of a self-excisable VZV BAC originally described in (Tischer et al., 2007) and generated as detailed previously (Markus BIO et al., 2017). Contamination of ARPE19 cells (ATCC) and neurons was performed using cell-associated or cell-free virus obtained from sonicates of infected cells, either as the low velocity supernatant or the pelleted debris fraction, as detailed previously (Sloutskin and Goldstein, 2014). Cells were harvested when at least 60% of cells were fluorescent, typically 4-5 days for ARPE cells and 6-7 days for neuronal cultures. The human embryonic stem cell (hESC) line H9 (WA09) was maintained on STO feeder cells in Nutristem (Biological Industries, Israel) medium and differentiated to neurons using two methods. The first method used PA6 stromal cell induction (Kawasaki et al., 2000) as described in detail (Pomp et al., 2005). The.