VZV SMALL NON-CODING RNA #9 INHIBITS VIRAL REPLICATION IN PRODUCTIVE INFECTION

*Equal contribution

BACKGROUND

Varicella-Zoster virus (VZV) is a human alphaherpesvirus that causes varicella as the primary infection, while the reactivation from latency results in painful Zoster. Small noncoding RNAs (sncRNA) are important actors in modulating gene expression in many herpesviruses. Putative VZV encoded sncRNA were discovered recently by NGS (Markus et al., 2017) and a physiological effect of changing expression of one of the 23 sequences (VZVsnc23) was demonstrated. We here demonstrate that reducing expression of an additional putative VZVsncRNA increases productive infection by the virus.

METHODS:

ARPE19 cells were infected at low MOI with VZV expressing mCherrry fused to ORF66. At 24hpi, cells were transfected with a locked RNA antagonist against VZVsncRNA9. Entire wells were photographed at multiple time points and the images “stitched” to generate a single image. The viral spread was determined by measuring the fluorecent area occupied by individual infected foci (FOI). For direct measurement of viral yield, 8dpi wells was trypsinized, cells were harvested seeded at 10 ten-fold over ARPE19 cells. At 5 dpi foci were counted and virus yield was determined.

RESULTS AND CONCLUSIONS

Transfection of an antagonist to VZVsncRNA9 increased FOI growth in three independent biological repeats (p<0.05). The effect of the antagonist was confirmed by infectious foci assays that showed double the amount of infectious virus generated in wells treated with the antagonist in comparison to a control locked RNA.

These results demonstrate that another VZV-encoded sncRNA reduces viral replication. Preliminary experiments using inhibitors to another 5 predicted VZVsncRNA have shown that at least one enhances viral replication. Thus, VZV replication appears to be modulated by virally-encoded sncRNAs, similar to the rest of the herpesvirus family. The mechanisms of action of VZVsncRNA are now under investigation. These findings may support development of novel therapies for chickenpox and zoster targeting VZVsncRNA pathways pharmacologically.