Differentiated Human SH-SY5Y Cells Provide a Reductionist Model of Herpes Simplex Virus 1 Neurotropism

Abstract

Neuron-virus interactions that occur during herpes simplex virus (HSV) infection are not fully understood. Neurons are the site of lifelong latency and are a crucial target for long-term suppressive therapy or viral clearance. A reproducible neuronal model of human origin would facilitate studies of HSV and other neurotropic viruses. Current neuronal models in the herpesvirus field vary widely and have caveats, including incomplete differentiation, nonhuman origins, or the use of dividing cells that have neuropotential but lack neuronal morphology. In this study, we used a robust approach to differentiate human SH-SY5Y neuroblastoma cells over 2.5 weeks, producing a uniform population of mature human neuronal cells. We demonstrate that terminally differentiated SH-SY5Y cells have neuronal morphology and express proteins with subcellular localization indicative of mature neurons. These neuronal cells are able to support a productive HSV-1 infection, with kinetics and overall titers similar to those seen in undifferentiated SH-SY5Y cells and the related SK-N-SH cell line. However, terminally differentiated, neuronal SH-SY5Y cells release significantly less extracellular HSV-1 by 24 h postinfection (hpi), suggesting a unique neuronal response to viral infection. With this model, we are able to distinguish differences in neuronal spread between two strains of HSV-1. We also show expression of the antiviral protein cyclic GMP-AMP synthase (cGAS) in neuronal SH-SY5Y cells, which is the first demonstration of the presence of this protein in nonepithelial cells. These data provide a model for studying neuron-virus interactions at the single-cell level as well as via bulk biochemistry and will be advantageous for the study of neurotropic viruses Herpes simplex virus (HSV) affects millions of people worldwide, causing painful oral and genital lesions, in addition to a multitude of more severe symptoms such as eye disease, neonatal infection, and, in rare cases, encephalitis. Presently, there is no cure available to treat those infected or prevent future transmission. Due to the ability of HSV to cause a persistent, lifelong infection in the peripheral nervous system, the virus remains within the host for life. To better understand the basis of virus-neuron interactions that allow HSV to persist within the host peripheral nervous system, improved neuronal models are required. Here we describe a cost-effective and scalable human neuronal model system that can be used to study many neurotropic viruses, such as HSV, Zika virus, dengue virus, and rabies virus.

Contemporary Circulating Enterovirus D68 Strains Have Acquired the Capacity for Viral Entry and Replication in Human Neuronal Cells

“…These differentiated cells are characterized by the formation of extensive neurites, as well as the induction of neuron-specific enzymes, receptors, and neurotransmitters ( 42 ). Differentiated SH-SY5Y cells have been used as models for neuron-virus interactions ( 66 ) including EV-A71 ( 54 , 55 ), chikungunya virus ( 60 ), and varicella-zoster virus ( 59 ). Our results show that both differentiated and undifferentiated SH-SY5Y can be infected with neurotropic strains of EV-D68, suggesting neurotropic strains of EV-D68 can invade both immature and mature neuronal cells.…”

Filoviruses Can Efficiently Infect Human Neuron-Like Cells Without Genetic Adaptation

“…The human neuroblastoma cell lines SH-SY5Y and BE(2)-M17 are two of the best-characterised cell lines for studies into human neurological disease, such as Alzheimer’s and Parkinson’s diseases (30–35), and neurovirology (39–46, 49, 52). Previous studies have confirmed their suitability as in vitro models of human neurons due to their expression of human neuron-specific markers, and the fact that their neuron-like phenotypes can be further improved through the use of chemical differentiation agents, such as RA and staurosporine (36–38, 43).…”

“…Both differentiated and undifferentiated SH-SY5Y and BE(2)-M17 cells have been previously used for in vitro studies with a range of neurotropic viruses, including herpes viruses (39–41), neurotropic enteroviruses (42, 43), chikungunya virus (44), poliovirus (45), various orthobunyaviruses (46), henipaviruses (47, 48), and Zika virus (49). In a number of these studies, growth in SH-SY5Y and BE(2)-M17 cells differed based upon whether the strains involved were virulent or attenuated in vivo , with neurovirulent viruses reaching higher titres than attenuated ones (43, 45).…”

Host cell targeting with peptide candidates derived from a HSV-1 entry protein

“…Cellular uptake of Cf-HSV peptides into host cell model [4][5][6], SH-SY5Y human neuroblastoma cells [7][8][9] was characterised by the enhanced intracellular fluorescence measured by BD LSR II flow cytometer (Coherent Sapphire laser; λ = 488 nm, 22 mW) (incubation time: 3h; channel FITC LP505; BP 530/30), data were analysed with FACSDiva 5.0 software. All measurements were performed in duplicates, and the Cf-positive (Cf+) live cells with standard error of the mean was presented ( Figure 1).…”