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Studies of Neurotropism and Endonuclease Targeting of Varicella Zoster Virus Using a Cultured Human Neuron System

Wu, Betty (2022) Studies of Neurotropism and Endonuclease Targeting of Varicella Zoster Virus Using a Cultured Human Neuron System. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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The alphaherpesvirus varicella zoster virus (VZV) causes varicella (chickenpox) after primary infection, and herpes zoster (shingles) during reactivation from latency in sensory ganglia. Its 125kb dsDNA genome encodes several transactivators, some of which are virion-incorporated and facilitate initiation of lytic infections. Two abundant virion-associated transactivators are encoded by ORFs 10 and 62, homologs of HSV VP16 and ICP4, respectively. VP16 is essential for virion formation and promotes immediate early gene transcription, facilitating exit from latency. However, ORF10 is dispensable in cell culture, though it is important for skin infections; its role in neurons is unknown. VZV virions also contain abundant IE62 (ICP4 is a minor HSV virion component). We hypothesized ORF10 protein may have overlapping transactivation functions with virion IE62 upon infection, perhaps in cell type-specific roles. To test this, recombinant viruses deleted for ORF10 with and without an ORF62 mutation preventing IE62 virion incorporation were evaluated for epithelial and neuronal spread in culture. VZV lacking one or both virion transactivators were successfully isolated in epithelial cells and replicated similarly to wild type. VZV lacking virion IE62 was slightly impaired for epithelial replication, but severely impaired in human neurons, indicating a neuron-specific role. Unexpectedly, when ORF10 was subsequently deleted, neuronal growth was restored. Partially purified virion analyses suggested ORF10 deletion causes novel compensatory mechanisms to still incorporate virion IE62 for efficient neuronal spread, despite continued presence of the ORF62 mutation that normally prevents virion accumulation.
The second part of this thesis addresses the targeting of the essential duplicated ORF62/71 genes by AAV vector-delivered CRISPR/Cas9, to effectively limit VZV replication and spread in epithelial and neuronal cultures. We show that a single treatment with ORF62-targeting Cas9 AAV effectively reduced VZV progeny virus in multiple epithelial cell types and lytically infected neurons. In targeting latent and reactivating infections, these constructs greatly reduced production of infectious virus from reactivation, though not latent genome loads. Taken together, our results support a novel important role of virion IE62 in neuronal infections, and the potential of AAV-delivered genome editing tools as an antiviral strategy to limit VZV epithelial and neuron growth, reducing the reactivated viral burden.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Wu, Bettybww20@pitt.edubww20
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairDeLuca, Neal
Thesis AdvisorKinchington, Paul
Committee MemberByrne, Leah
Committee MemberHoma, Fred
Committee MemberAlbers, Kathryn
Committee MemberSarkar, Saumendra
Date: 21 April 2022
Defense Date: 26 May 2022
Approval Date: 9 August 2022
Submission Date: 31 May 2022
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 180
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Microbiology and Immunology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: Neuron, Virion, Varicella Zoster Virus, Herpesvirus, Transactivator, CRISPR, Endonuclease, Antiviral
Date Deposited: 10 Aug 2022 00:09
Last Modified: 10 Aug 2022 00:09


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