Non-Infectious Extracellular Vesicles from Varicella Zoster Virus-Infected Neurons Suppress Antiviral Responses and Facilitate Secondary Pathologies

Varicella zoster virus (VZV) is a highly pervasive human alphaherpesvirus responsible for causing varicella (chickenpox) upon primary infection and subsequently establishing lifelong latency in ganglionic sensory neurons. In more than 95% of adults, VZV remains dormant until it reactivates, often due to aging or immunosuppression, to produce herpes zoster (shingles). Shingles, characterized by a painful, unilateral dermatomal rash, predominantly occurs in the thoracic region. Beyond the immediate discomfort and complications of shingles, such as post-herpetic neuralgia and myelitis, VZV reactivation poses significant systemic health risks, including heightened susceptibility to stroke and other neurological complications. These systemic effects are not fully understood and represent a significant challenge in managing VZV-associated diseases. A particularly puzzling aspect of VZV reactivation is its ability to cause complications in areas of the body distant from the initial infection site. The mechanisms underlying these remote pathologies are not well-defined. Researchers have traditionally focused on direct cell-to-cell spread of the virus and local immune evasion strategies employed by VZV. However, the virus’s ability to affect distant tissues and organs suggests the involvement of more complex and systemic processes. One potential mechanism involves small extracellular vesicles (sEVs), which are membranous structures released by cells to facilitate intercellular communication by transporting proteins, lipids, and nucleic acids. Recent studies have shown that viruses can hijack the host’s sEV machinery to propagate infection and modulate host immune responses. Yet, the specific role of non-infectious sEVs released from VZV-infected neurons in viral spread and the development of distant complications has not been studied extensively.

The study led by Professors Ravi Mahalingam and Andrew Bubak, published in the Journal of Virology, aims to fill this critical gap in understanding. By employing advanced proteomic and transcriptomic analyses, the researchers sought to characterize the content and impact of sEVs released from VZV-infected human sensory neurons. They hypothesized that these sEVs might carry viral and host-derived factors capable of modulating immune responses and contributing to VZV pathogenesis at sites remote from the primary infection. To investigate the role of small extracellular vesicles (sEVs) in VZV pathogenesis, the researchers conducted a series of detailed experiments combining advanced proteomic and transcriptomic analyses. Their primary aim was to characterize the content of sEVs released from VZV-infected human sensory neurons and to understand their impact on host cells. The first step involved isolating sEVs from human sensory neurons (huSNs) infected with VZV. The researchers used nanoparticle tracking analysis to measure the size and concentration of sEVs. They found that VZV-infected huSNs released smaller vesicles compared to those from uninfected cells, with a mode size of approximately 166 nm versus 194 nm. This size reduction, although statistically significant, indicated a potential shift in sEV populations associated with disease states. Importantly, the concentration of sEVs did not differ significantly between VZV-infected and mock-infected cells, suggesting that VZV infection alters the vesicle size but not the overall production rate. To determine whether these sEVs were infectious, primary human brain vascular adventitial fibroblasts (HBVAFs) were exposed to VZV-derived sEVs. No viral plaques, cytopathic effects, or VZV glycoprotein B (gB) antigen staining were observed, indicating that these sEVs were non-infectious. This was in stark contrast to direct VZV infection of HBVAFs, which showed robust viral plaques and VZV gB staining within three days. This finding was critical as it demonstrated that VZV sEVs, although non-infectious, might still play a significant role in modulating host cellular responses.

The researchers further explored whether VZV sEVs could suppress innate antiviral responses. HBVAFs exposed to VZV sEVs showed a blunted IFNβ release upon stimulation with poly(I), a synthetic analog of double-stranded RNA that activates interferon responses. This suppression of IFNβ, a crucial component of the antiviral defense, highlighted the potential of VZV sEVs to modulate immune responses in the absence of direct viral infection. To investigate the mechanisms of this immune modulation, RNA sequencing was performed on HBVAFs treated with VZV sEVs. Analysis revealed significant changes in gene expression, with 110 differentially expressed genes (DEGs) identified. Notably, pathways related to extracellular matrix organization, cell adhesion, and communication were significantly enriched. Moreover, genes involved in interferon signaling, such as IFITM1 and OAS3, were downregulated, providing further evidence of the immunosuppressive nature of VZV sEVs.  Mass spectrometry analysis of the sEVs’ protein content revealed the presence of the VZV immediate-early 62 (IE62) protein along with host proteins associated with immunosuppression and vascular disease. For example, proteins like vimentin, which negatively regulates type 1 interferon responses, and integrin beta 1, involved in viral entry, were enriched in VZV sEVs. Additionally, miRNA analysis showed an enrichment of miRNAs such as hsa-miR-146a-5p, known for its role in suppressing the antiviral type 1 interferon response. This combination of viral and host factors within the sEVs pointed to a sophisticated strategy employed by VZV to evade immune detection and potentially facilitate secondary pathologies.

To understand the in vivo implications of these findings, the researchers performed experiments on mice. C57BL/6 mice were intranasally inoculated with VZV sEVs followed by a secondary infection with herpes simplex virus type-1 (HSV-1). Histological analysis of the olfactory epithelium (OE) showed a substantial decrease in resident macrophages in VZV sEV-treated mice, which was not observed in mock-treated animals. This suppression of macrophages led to increased HSV-1 invasion into the olfactory bulb (OB) and central nervous system (CNS), demonstrating that VZV sEVs can create a hyper-permissive environment for secondary infections. Further experiments demonstrated that VZV IE62 alone could recapitulate the immunosuppressive effects observed with VZV sEVs. Transfection of HBVAFs with a plasmid encoding VZV IE62 resulted in decreased expression of interferon-stimulated genes and reduced secretion of IFNβ. This confirmed that VZV IE62, found in sEVs, plays a pivotal role in suppressing the host’s innate antiviral response. Pathway enrichment analysis of the differentially expressed proteins and miRNAs in VZV sEVs highlighted significant associations with viral replication, vascular disease, and immune response pathways. These included pathways related to viral mRNA translation, immune response modulation, and conditions like stroke and blood coagulation. Such insights suggest that VZV sEVs could contribute to the systemic complications observed following VZV reactivation.

The study conducted by Professors Ravi Mahalingam and Andrew Bubak presents groundbreaking insights into the pathogenesis of varicella zoster virus (VZV), particularly focusing on the role of small extracellular vesicles (sEVs) released from VZV-infected neurons. The findings challenge the conventional understanding of VZV infection by highlighting a novel mechanism of immune modulation and systemic disease facilitation through non-infectious sEVs. The discovery that VZV can use non-infectious sEVs to suppress the host’s antiviral type 1 interferon response before direct infection significantly advances our understanding of viral immune evasion strategies. This suggests that VZV has evolved a sophisticated method to preemptively dampen immune responses, thereby facilitating viral spread and secondary infections. Moreover, the non-infectious yet pathogenic nature of VZV sEVs introduces a new dimension to the clinical management of VZV-related diseases. Traditionally, the absence of detectable virions in distant tissues has made it difficult to associate certain systemic complications with VZV reactivation. This study suggests that pathogenic effects can occur through sEV-mediated pathways, independent of direct viral presence. This insight could lead to the development of diagnostic tools that detect sEV-associated biomarkers, offering earlier and more accurate diagnosis of VZV-related complications. Furthermore, understanding the specific components of VZV sEVs, such as the immediate-early 62 (IE62) protein and immunosuppressive miRNAs, opens new avenues for therapeutic interventions. Targeting these sEV components or their pathways could mitigate the systemic effects of VZV reactivation. For instance, therapies aimed at blocking the release or action of VZV sEVs might reduce the risk of stroke, myocardial infarction, and secondary infections in individuals with VZV reactivation.

Given the high prevalence of VZV infection and reactivation, the study’s findings have significant public health implications. Improved diagnostic and therapeutic strategies could reduce the burden of VZV-related diseases, particularly in aging and immunocompromised populations. Understanding the role of sEVs in systemic complications could lead to better preventive measures and more effective management of VZV outbreaks. Overall, the experiments conducted by Mahalingam and Bubak provide compelling evidence that VZV sEVs, despite being non-infectious, carry immunomodulatory cargo capable of altering host immune responses and facilitating secondary infections. This study significantly advances our understanding of VZV pathogenesis by revealing a novel immune evasion strategy employed by the virus, highlighting the critical role of sEVs in mediating systemic disease beyond the primary site of infection.