Accepted

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.

The methyltransferase-like 3 (Mettl3) is a key component of the large N6-adenosine-methyltransferase complex in mammalian responsible for RNA N6-methyladenosine (m6A) modification, which plays an important role in gene post-transcription modulation. Although RNA m6A is enriched in mammalian neurons, its regulatory function in nociceptive information processing remains elusive. Here, we reported that Complete Freund's Adjuvant (CFA)-induced inflammatory pain significantly decreased global m6A level and m6A writer Mettl3 in the spinal cord. Mimicking this decease by knocking down or conditionally deleting spinal Mettl3 elevated the levels of m6A in ten-eleven translocation methylcytosine dioxygenases 1 (Tet1) mRNA and TET1 protein in the spinal cord, leading to production of pain hypersensitivity. By contrast, overexpressing Mettl3 reversed a loss of m6A in Tet1 mRNA and blocked the CFA-induced increase of TET1 in the spinal cord, resulting in the attenuation of pain behavior. Furthermore, the decreased level of spinal YT521-B homology domain family protein 2 (YTHDF2), an RNA m6A reader, stabilized upregulation of spinal TET1 because of the reduction of Tet1 mRNA decay by the binding to m6A in Tet1 mRNA in the spinal cord after CFA. This study reveals a novel mechanism for downregulated spinal cord METTL3 coordinating with YTHDF2 contributes to the modulation of inflammatory pain through stabilizing upregulation of TET1 in spinal neurons.

To assess the prevalence of antiplexin D1 antibodies (plexin D1-immunoglobulin G [IgG]) in small fiber neuropathy (SFN) and the effects of these antibodies in vivo.