Role of the JNK Pathway in Varicella-Zoster Virus Lytic Infection and Reactivation

Abstract

Mechanisms of neuronal infection by varicella-zoster virus (VZV) have been challenging to study due to the relatively strict human tropism of the virus and the paucity of tractable experimental models. Cellular mitogen-activated protein kinases (MAPKs) have been shown to play a role in VZV infection of nonneuronal cells, with distinct consequences for infectivity in different cell types. Here, we utilize several human neuronal culture systems to investigate the role of one such MAPK, the c-Jun N-terminal kinase (JNK), in VZV lytic infection and reactivation. We find that the JNK pathway is specifically activated following infection of human embryonic stem cell-derived neurons and that this activation of JNK is essential for efficient viral protein expression and replication. Inhibition of the JNK pathway blocked viral replication in a manner distinct from that of acyclovir, and an acyclovir-resistant VZV isolate was as sensitive to the effects of JNK inhibition as an acyclovir-sensitive VZV isolate in neurons. Moreover, in a microfluidic-based human neuronal model of viral latency and reactivation, we found that inhibition of the JNK pathway resulted in a marked reduction in reactivation of VZV. Finally, we utilized a novel technique to efficiently generate cells expressing markers of human sensory neurons from neural crest cells and established a critical role for the JNK pathway in infection of these cells. In summary, the JNK pathway plays an important role in lytic infection and reactivation of VZV in physiologically relevant cell types and may provide an alternative target for antiviral therapy. Varicella-zoster virus (VZV) has infected over 90% of people worldwide. While primary infection leads to the typically self-limiting condition of chickenpox, the virus can remain dormant in the nervous system and may reactivate later in life, leading to shingles or inflammatory diseases of the nervous system and eye with potentially severe consequences. Here, we take advantage of newer stem cell-based technologies to study the mechanisms by which VZV infects human neurons. We find that the c-Jun N-terminal kinase (JNK) pathway is activated by VZV infection and that blockade of this pathway limits lytic replication (as occurs during primary infection). In addition, JNK inhibition limits viral reactivation, exhibiting parallels with herpes simplex virus reactivation. The identification of the role of the JNK pathway in VZV infection of neurons reveals potential avenues for the development of alternate antiviral drugs.

Extracellular vesicles: novel vehicles in herpesvirus infection

“…VZV can cause chickenpox among the elderly and immunocompromised individuals during acute infection (Ahmed et al, 2015). Although primary chickenpox is typically self-limited, VZV can remain dormant in the nervous system and then is reactivated in a weak host, leading to shingles or inflammation (Kurapati et al, 2017). Given that T cells play a crucial role in disseminating VZV to the skin and ganglia in the early period of VZV infection, a nonhuman primate model of VZV infection was used to demonstrate VZV-T cell interactions.…”

Human stem cell derived sensory neurons are positioned to support varicella zoster virus latency

“…By six weeks of differentiation, >90% of cells are positive for peripherin and Brn3a, >80% are positive for Islet 1 and >50% are positive for Na v 1.7 and Na v 1.8, indicating a mature sensory neuron phenotype (top panel in Fig 1C ). In contrast, our human ESC-derived neurons (16, 18) represent a mixed population (human mixed neurons; HMNs) that express CNS (central nervous system) markers such as GABA A receptor, Glycine receptor, and CTIP1 along with robust expression of the pan-neuronal markers Map2 and Tuj1 (16, 18). Expression of the sensory neuronal markers peripherin, Brn3a, Islet 1, and Na v 1.8 was rarely (<1% of cells) observed in HMNs (lower panel in Fig 1C ).…”

“…Following characterization of HSN, we first attempted to infect them with VZV via standard conditions (16, 18). HSNs and HMNs differentiated for six to seven weeks were exposed to cell-free rVZV LUC BAC (20), which contains a GFP cassette that is expressed upon infection.…”

“…We next examined the establishment of latency in HSNs. We took advantage of a microfluidic platform that we have previously developed that allows for axonal infection of neurons, a factor that appears critical for developing an in vitro latent state (16, 18) ( Fig 5A ). In this platform, neuronal cell bodies are cultured in the somal compartment and allowed to extend axons into the fluidically restricted axonal compartment.…”

“…More recently, in vitro neuronal models of VZV infection utilizing human neural stem cells (12) and human embryonic stem cells (ESC) (13) have been developed. We and others have used such models to study various aspects of VZV neuropathogenesis, including characterization of transcriptional changes in infected cells, demonstration of viral axonal transport, exploration of the role of the cellular JNK pathway in viral infection, and establishment of models of viral latency and reactivation (13–18).…”