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 in vitroIMPORTANCE 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.

Keywords: HSV-1; SH-SY5Y; SK-N-SH; cGAS; differentiation; herpes simplex virus; infection; neuron; virus.

FIG 1

Terminally differentiated neuronal SH-SY5Y cells are morphologically distinct from undifferentiated SH-SY5Y cells and SK-N-SH cells. (A) Terminally differentiated SH-SY5Y cells appear neuronal after 2.5 weeks of differentiation. At 2 days postplating, mixed cell morphologies are visible in undifferentiated SH-SY5Y cells (B) and in the parent cell line SK-N-SH (C). Black arrows indicate epithelial-like cells, and red arrowheads indicate neuronal-like cells. (D) Traced outlines of representative cells, approximately 1.5 times their actual size. Scale bars represent 100 μm.

FIG 2

Terminally differentiated SH-SY5Y neuronal cells show distinct localization of cytoskeletal and synaptic markers. Differentiation of SH-SY5Y cells for 2.5 weeks yields morphologically distinct localization of neuronal proteins (B, D, and F), in contrast to that seen in undifferentiated SH-SY5Y cells (A, C, and E). Higher magnification of differentiated SH-SY5Y neurites shown in phase-contrast (G) reveals further details of microtubule and synaptic vesicle staining (H). The 2.5-week differentiation process includes the addition of RA, neurotrophic factors, and ECM proteins. MAP-2, microtubule associated protein 2; NF-H, unphosphorylated neurofilament heavy chain; p-NF-H, phosphorylated neurofilament heavy chain; SV2, synaptic vesicle protein 2; Nucleus, nuclear/DNA stain. Images were taken with an Olympus FV10i confocal microscope at a magnification of ×60, and the scale bars represent 20 μm. Images in panels G and H were taken with an additional 2.6× digital zoom, and the scale bars represent 10 μm.

FIG 3

Neuronal differentiation significantly impacts expression of GAP-43, SY38, and p-AKT. (A) Proteins involved in neuronal differentiation, growth, and signaling are expressed at high levels in terminally differentiated SH-SY5Y neuronal cells. The neuronal growth-associated protein GAP-43 and synaptic vesicle marker SY38 are expressed at significantly higher levels in differentiated SH-SY5Y neuronal cells than in the undifferentiated progenitor cells. The cytoplasmic DNA sensor cGAS, as part of the intrinsic immune response, is expressed in both the undifferentiated and neuronal SH-SY5Y cells. While AKT levels appear equal in undifferentiated and neuronal SH-SY5Y cells, p-AKT levels are significantly higher in differentiated neuronal cells. HSV-1 glycoprotein E (gE) serves as a positive control for viral infection. GAPDH serves as a loading control. Images are representative of three biological replicates. Twenty micrograms of protein was loaded for each cell population. (B) Quantification of the effects of differentiation on protein expression levels in uninfected SH-SY5Y cells that are either undifferentiated (red) or neuronally differentiated (blue). The signal of each protein target was normalized and expressed relative to GAPDH loading levels. p-AKT signal was normalized to AKT. Multiple t tests were performed with alpha at 5.0%. *, P = 0.03; **, P = 0.01; ***, P = 0.002. (C) HSV-1 infection has a significant impact on SY38 and AKT protein levels in SH-SY5Y cells. With the exception of p-AKT induction in undifferentiated SH-SY5Y cells and SY38 expression in differentiated SH-SY5Y neuronal cells, most protein levels are unchanged following HSV-1 infection. Results are presented as fold change of expression following infection. One-sample t tests were performed on each protein target against a hypothetical uninfected value of 1. *, P = 0.03; **, P = 0.007. See the text for protein descriptions and Materials and Methods for antibody details.

FIG 4

Neuronal SH-SY5Y cells release less extracellular virus during lytic HSV-1 infection. (A) Single-step growth curves of viral replication kinetics in differentiated SH-SY5Y neuronal cells (blue), undifferentiated SH-SY5Y cells (red), and SK-N-SH cells (gray). Each cell type was infected with HSV-1 McKrae at an MOI of 10. Titers are plotted as log PFU per 100,000 cells. Cell-free viral titers, cell-associated viral titers, and combined total viral titers include three biological replicates. A 2-way analysis of variance (ANOVA) was performed for each graph: for cell-free virus, *, P = 0.0229; ***, P = 0.004; for cell-associated virus, *, P = 0.0452. Each graph displays the average from the three replicates, and the error bars represent the standard errors of the means (SEM). (B) Time-lapse images reveal morphological changes in differentiated SH-SY5Y neuronal cells, undifferentiated SH-SY5Y cells, and SK-N-SH cells during infection with HSV-1 McKrae at a high MOI (10). Initial changes in neuronal cell morphology begin at 6 hpi, with clear neurite retraction by 12 hpi. At 24 hpi, neuronal morphology is markedly different and few neurites remain. Following viral infection in the undifferentiated SH-SY5Y cells and SK-N-SH cells, morphological changes are not visible until 12 hpi. By 24 hpi, the undifferentiated SH-SY5Y cells appear to all be infected and rounded up. The SK-N-SH cells appear to be more robust, with only about 50% of cells rounded up by 24 hpi. The scale bar represents 25 μm. Images are representative of those from three biological replicates. (C) Procaspase 3 expression data demonstrate cleavage and activation of caspase 3 in differentiated SH-SY5Y neuronal cells, undifferentiated SH-SY5Y cells, and SK-N-SH cells over a time course of 24 hpi with HSV-1 McKrae (MOI of 10). Sorbitol-induced osmotic shock serves as a positive control for activation of caspase 3. Blots are representative of those from two biological replicates.

FIG 5

Neuronal SH-SY5Y cells reveal distinct phenotypes of cell-to-cell spread for different strains of HSV-1. Following a low-MOI infection with sample collection at 24, 48, and 72 hpi, neuronal SH-SY5Y cells (A) but not undifferentiated SH-SY5Y cells or Vero cells (B) distinguish between two phenotypically distinct viral strains. For these multistep growth curves, all three cell types were infected with HSV-1 McKrae (green) or HSV-1 KOS (orange) at an MOI of 0.01 in the presence of 0.1% pooled human serum. Data include three biological replicates, and error bars represent the SEM. Multiple t tests were performed for neuronal SH-SY5Y cells, with alpha of 5.0%. *, P = 0.04.

FIG 6

Differentiated neuronal cells have a unique pattern of cGAS expression compared to MRC5 cells before and during HSV-1 infection. Differentiated neuronal cells express the antiviral protein cGAS in the cytoplasm and in neurites both before and after HSV-1 infection, while MRC5 cells express cGAS and IFI16 in the nucleus following HSV-1 infection. (A) Terminally differentiated SH-SY5Y neuronal cells were infected at an MOI of 10 with HSV-1 McKrae and then fixed and processed for cGAS expression at 3 hpi or 6 hpi. Scale bars represent 20 μm. The zoomed-in images were acquired with a 168× objective, and scale bars represent 10 μm. (B) MRC5 fetal lung fibroblasts were infected at an MOI of 10 with HSV-1 McKrae and then fixed and processed for immunofluorescence at 3 hpi. Scale bars represent 20 μm. The zoomed-in images were acquired with a 156× objective, and scale bars represent 10 μm. Images were taken with an Olympus FV10i confocal microscope with a 60× objective.