Varicella-zoster virus early infection but not complete replication is required for the induction of chronic hypersensitivity in rat models of postherpetic neuralgia

Abstract

Herpes zoster, the result of varicella-zoster virus (VZV) reactivation, is frequently complicated by difficult-to-treat chronic pain states termed postherpetic neuralgia (PHN). While there are no animal models of VZV-induced pain following viral reactivation, subcutaneous VZV inoculation of the rat causes long-term nocifensive behaviors indicative of mechanical and thermal hypersensitivity. Previous studies using UV-inactivated VZV in the rat model suggest viral gene expression is required for the development of pain behaviors. However, it remains unclear if complete infection processes are needed for VZV to induce hypersensitivity in this host. To further assess how gene expression and replication contribute, we developed and characterized three replication-conditional VZV using a protein degron system to achieve drug-dependent stability of essential viral proteins. Each virus was then assessed for induction of hypersensitivity in rats under replication permissive and nonpermissive conditions. VZV with a degron fused to ORF9p, a late structural protein that is required for virion assembly, induced nocifensive behaviors under both replication permissive and nonpermissive conditions, indicating that complete VZV replication is dispensable for the induction of hypersensitivity. This conclusion was confirmed by showing that a genetic deletion recombinant VZV lacking DNA packaging protein ORF54p still induced prolonged hypersensitivities in the rat. In contrast, VZV with a degron fused to the essential IE4 or IE63 proteins, which are involved in early gene regulation of expression, induced nocifensive behaviors only under replication permissive conditions, indicating importance of early gene expression events for induction of hypersensitivity. These data establish that while early viral gene expression is required for the development of nocifensive behaviors in the rat, complete replication is dispensable. We postulate this model reflects events leading to clinical PHN, in which a population of ganglionic neurons become abortively infected with VZV during reactivation and survive, but host signaling becomes altered in order to transmit ongoing pain.

Varicella‐Zoster virus ORF9 is an antagonist of the DNA sensor cGAS

“…#thpd‐komydTHP1 Dual WTInvivogenCat. #thpd‐nfisTHP1 IRF3 KOThis studyN/ATHP1 MAVS KOThis studyN/ATHP1 STING KOGift from Soren Paludan (Aarhus University, Denmark) (Holm et al , 2016)N/ATHP1 TBK1 KOGift from Soren Paludan (Aarhus University, Denmark) (Holm et al , 2016)N/AVZV rOkaGift from Jeffrey Cohen (NIH, Bethesda, USA) (Cohen & Seidel, 1993)N/AVZV‐ORF9 ecDHFR Gift from Paul R. Kinchington (University of Pittsburgh, USA) (Warner et al , 2021)N/AVZV ORF9‐V5 Gift from Catherine Sadzot (University of Liege, Belgium) (Riva et al , 2013)N/A Recombinant DNA hRIG‐I‐CARDGift from Andreas Pichlmair (Technical University Munich, Germany)N/Ap125‐LucGift from T. Fujita (Kyoto University, Japan) (Yoneyama et al , 2004)N/Ap8.91Gift from Greg Towers (University College London)N/ApcDNA/FLAG/hcGASThis studyN/ApcDNA/HA/hSTINGThis studyN/ApCMV‐3tag‐KSHV‐ORF52Gift from Fanxiu Zhu (Florida State University, USA) (Wu et al , 2015)N/ApCMV‐VSV‐GGift from Bob WeinbergAddgene_8454pDONR/ORF22AGift from Jurgen Haas (University of Edinburgh, UK) (Uetz et al , 2006)N/ApDONR/ORF22BGift from Jurgen Haas (University of Edinburgh, UK) (Uetz et al , …”

“…The virus was a kind gift from Catherine Sadzot (University of Liege, Belgium;Riva et al, 2013). VZV-ORF9 ecDHFR is a recombinant virus, in which an E. coli dihydrofolate reductase degron motif was fused C-terminally to the coding sequence of ORF9 (University of Pittsburgh, USA;Warner et al, 2021).…”

Antiviral Targeting of Varicella Zoster Virus Replication and Neuronal Reactivation Using CRISPR/Cas9 Cleavage of the Duplicated Open Reading Frames 62/71

“…The virus was generated from the BACs that were just detailed after their transfection into MeWo cells, using Lipofectamine 3000 (#L3000015, Thermo Fisher Scientific) with 2.5 µg of purified BAC DNA and 100 ng each of plasmids expressing ORF61 and ORF62 DNA under the constitutive hCMV immediate-early promoter, as detailed previously [64,65]. Upon the appearance of fluorescent plaques, infected cultures were amplified by trypsinization and replated with uninfected ARPE-19 cells until they exhibited >80% cytopathic effect.…”

“…Viruses were further amplified for a minimum of six passages to self-excise the BAC replicon elements and restore ORF71. Virus stocks were generated from ARPE-19-infected cultures showing >80% cytopathic effect, after treating cultures with 0.01 mM mitomycin C for 3-4 h. at 37 • C prior to harvesting for cryopreservation, as detailed previously [65].…”

“…Virus growth curves were determined as detailed previously [64,65], initiating infections in confluent 6-wells with approximately 400 pfu of virus (per 0.5 × 10 6 cells in a single well) from cryopreserved, pre-titered, mitomycin C-treated aliquots of infected ARPE-19 cells. All assays were performed in triplicate.…”