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Studies of Infection and Experimental Reactivation by Recombinant VZV with Mutations in Virally-Encoded Small
Punam Bisht1, Biswajit Das1, Tatiana Borodianskiy-Shteinberg1
1Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.
Abstract:
Locked-nucleotide analog antagonists (LNAA) to four varicella zoster virus small non-coding RNA (VZVsncRNA 10-13) derived from the mRNA of the open reading frame (ORF) 61 gene individually reduce VZV replication in epithelial cells and fibroblasts. To study the potential roles VZVsncRNA 10-13 have in neuronal infection we generated two recombinant VZV; one in which 8 nucleotides were changed in VZVsncRNA10 without altering the encoded residues of ORF61 (VZVsnc10MUT) and a second containing a 12-nucleotide deletion of the sequence common to VZVsncRNA12 and 13, located in the ORF61 mRNA leader sequence (VZVsnc12-13DEL). Both were developed from a VZV BAC with a green fluorescent protein (GFP) reporter fused to the N terminal of the capsid protein encoded by ORF23. The growth of both mutant VZV in epithelial cells and fibroblasts was similar to that of the parental recombinant virus. Both mutants established productive infections and experimental latency in neurons derived from human embryonic stem cells (hESC). However, neurons that were latently infected with both VZV mutant viruses showed impaired ability to reactivate when given stimuli that successfully reactivated the parental virus. These results suggest that these VZVsncRNA may have a role in VZV latency maintenance and/or reactivation. The extension of these studies and confirmation of such roles could potentially inform the development of a non-reactivating, live VZV vaccine.
Insights
Varicella zoster virus (VZV) small non-coding RNAs (VZVsncRNAs) 10-13 are implicated in viral latency. Disrupting these VZVsncRNAs impairs VZV reactivation from neuronal latency, suggesting a role in maintaining VZV latency.
Area of Science:
- Virology
- Neuroscience
- Molecular Biology
Background:
- Varicella zoster virus (VZV) establishes lifelong latency in neurons.
- Small non-coding RNAs (VZVsncRNAs) derived from ORF61 mRNA are known to influence VZV replication.
- The role of VZVsncRNAs 10-13 in VZV neuronal infection and latency remains unclear.
Purpose of the Study:
- To investigate the role of VZVsncRNAs 10-13 in VZV neuronal infection, latency, and reactivation.
- To generate and characterize recombinant VZV mutants with altered VZVsncRNA 10, 12, and 13 sequences.
Main Methods:
- Generation of recombinant VZV expressing GFP, with specific mutations in VZVsncRNA10 (VZVsnc10MUT) and a deletion in VZVsncRNA12-13 (VZVsnc12-13DEL).
- Assessment of viral growth in epithelial cells and fibroblasts.
- Induction of productive infection and experimental latency in human embryonic stem cell-derived neurons (hESCs).
- Evaluation of viral reactivation from latent neurons.
Main Results:
- Mutant VZV strains (VZVsnc10MUT and VZVsnc12-13DEL) exhibited growth kinetics similar to the parental virus in non-neuronal cells.
- Both mutant viruses successfully established productive infections and experimental latency in hESC-derived neurons.
- Neurons latently infected with VZV mutants demonstrated significantly impaired reactivation compared to neurons infected with the parental virus.
Conclusions:
- VZVsncRNAs 10-13 play a crucial role in the maintenance and/or reactivation of VZV latency in neurons.
- Targeting these VZVsncRNAs could offer novel strategies for VZV therapeutic interventions.
- Further research may inform the development of non-reactivating VZV vaccines.
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