Lund Human Mesencephalic (LUHMES) Neuronal Cell Line Supports Herpes Simplex Virus 1 Latency In Vitro

Abstract

Lund human mesencephalic (LUHMES) cells are human embryonic neuronal precursor cells that can be maintained as proliferating cells due to the expression of a tetracycline-regulatable (Tet-off) v-myc transgene. They can be differentiated to postmitotic neurons by the addition of tetracycline, glial cell-derived neurotrophic factor (GDNF), and dibutyryl cAMP. We demonstrate that these cells can be infected with herpes simplex virus 1 (HSV-1) at a multiplicity of infection (MOI) of 3 with the majority of cells surviving. By 6 days postinfection, there is a loss of lytic gene transcription and an increase in the numbers of neurons that express the latency-associated transcripts (LATs). Importantly, the virus can then be reactivated by the addition of a phosphoinositide 3-kinase inhibitor, which has previously been shown to reactivate HSV-1 in rat neuron cultures. While rodent primary culture neuron systems have been described, these are limited by their lack of scalability, as it is difficult to obtain more than 500,000 neurons to employ for a given experiment. Several recent papers have described a human dorsal root ganglion (DRG) neuron culture model and human induced pleuripotent stem cell (iPSC) neuron culture models that are scalable, but they require that the presence of an antiviral suppression be maintained following HSV-1 infection. The human LUHMES cell model of HSV-1 infection described here may be especially useful for studying HSV-1 latency and reactivation on account of its scalability, its amenability to maintenance of latency without the continual use of antiviral inhibitors, and its latent gene expression profile which mirrors many properties observed in vivo, importantly, the heterogeneity of cells expressing the LATs.IMPORTANCE Herpes simplex virus (HSV) is responsible for significant morbidity in humans due to its ability to cause oral and genital lesions, ocular disease, and encephalitis. While antivirals can attenuate the severity and frequency of disease, there is no vaccine or cure. Understanding the molecular details of HSV latency and reactivation is key to the development of new therapies. One of the difficulties in studying HSV latency has been the need to rely on establishment of latent infections in animal models. While rodent primary neuron culture models have shown promise, they yield relatively small numbers of latently infected neurons for biochemical and molecular analyses. Here we present the use of a human central nervous system (CNS)-derived conditionally proliferating cell line that can be differentiated into mature neurons and latently infected with HSV-1. This model shows promise as a scalable tool to study molecular and biochemical aspects of HSV-1 latency and reactivation in human neurons.

Keywords: latency; neuron.

FIG 1

Conditionally immortalized proliferating LUHMES cells can be uniformly differentiated into postmitotic neurons. (A to C) Bright-field micrographs showing LUHMES neuronal cultures in the undifferentiated proliferative phase (A) and then following 7 days of differentiation (B), with the boxed inset magnified (C). (D to I) Immunofluorescence micrographs showing that differentiated LUHMES cells are positive for neuronal-specific markers. Green, neurofilament H (NF-H) (D to F) and synaptophysin (Syn) (H and I); red, βIII-tubulin (G). (I) Magnified image, with arrows indicating synaptophysin-positive punctate staining and intermediate III vimentin filaments in purple. Nuclei stained with DAPI are shown in all immunofluorescence images. Scale bars, 20 μm.

FIG 2

Differentiated LUHMES cultures are permissive for HSV-1 infection. (A) Infection of 5-day-postdifferentiated LUHMES cells with 17syn⁺ at an MOI of 3 results in robust expression of the viral immediate early (ICP4) and early (ICP8) proteins as shown by Western blotting (numbers indicate days p.i.; M, mock control). (B) HSV-1 genome copy number was measured by qPCR over a 15-day time course of 17syn⁺ infection of LUHMES cells and found to be maintained during the course of the infection. (C) The production of the HSV-1 latency-associated transcript (LAT) was measured by RT-qPCR and found to be abundantly expressed during the course of the experiment, with highest expression seen early following infection (day 3 to 5 p.i.), tapering off around day 8 p.i., and then maintained at levels fairly constant for the remainder of the experiment. (D) Three additional HSV-1 transcripts representing different kinetic phases of the viral life cycle were measured by RT-qPCR: ICP4 (immediate early), TK (early), and the late gene gC. HSV-1 transcript levels were calculated following normalization to GAPDH and to no-RT controls and are represented as fold change over levels in mock-infected cells (where the mock-infected cell value was set to 1).

FIG 3

Establishment of a timeline for HSV-1 latency and reactivation by in situ detection of HSV-1 LATs and ICP4 RNA transcripts. (A) Experimental timeline of neuronal differentiation, infection, latency, and reactivation. (B) LUHMES neuronal cultures were plated and differentiated on coverslips for 5 days and then infected with 17syn⁺ at an MOI of 3 in the presence of 50 μM acyclovir (ACV). Forty-eight hours later, the medium was changed to medium without ACV and infection allowed to proceed, with harvesting of coverslips for RNAScope analysis on the indicated days. RNAScope analysis was performed per the manufacturer’s recommendation, and probes for LAT and ICP4 transcripts were designed by ACD. HSV-1 LATs were detected from the C2 channel (red) and the HSV-1 lytic transcripts (ICP4) were detected from the C1 channel (green). Reactivation was induced by incubation of day 11 (p.i.) cultures (labeled as 13d P.I. + 48h WM) for 48 h with the PI3K pathway inhibitor wortmannin (1 μM final concentration).

FIG 4

Lytic HSV-1 proteins are abundantly expressed in LUHMES neuronal cultures following acute infection and are reduced during latency. Differentiated, postmitotic LUHMES neurons were infected with 17syn⁺ at an MOI of 3 and analyzed by immunofluorescence for expression of viral lytic proteins over a time course of 7 days. (A to E) During the acute infection, ICP4 (shown in green) is abundantly expressed in almost all the neurons as large well-developed centers in the nucleus (1 to 4 days p.i.). (F) Mock-infected LUHMES cells. (G to I) Punctate, nuclear, chromatin-associated ICP8 (shown in green) is also evident during the acute infection (2 to 3 days p.i.). (J to L) By 6 to 7 days p.i., a dramatic reduction in both ICP4 (J and K) and ICP8 (L) expression is seen. Scale bars represent 20 μm. Neurofilaments are stained for βIII-tubulin (red); nuclei are stained with DAPI (blue).

FIG 5

HSV-1 genomes were characterized by FISH. LUHMES cells were plated and differentiated on coverslips as described in Materials and Methods and infected with 17syn⁺ at an MOI of 3 in the presence of 50 μM ACV. Coverslips were harvested at 24 h p.i. and neuronal cultures analyzed for the presence of HSV-1 genomes using a biotin-labeled HSV-1 probe followed by amplification with HRP-streptavidin and Alexa Fluor 594 tyramide (AF594). (A) The majority of neuronal nuclei display evidence of harboring HSV-1 genomes (compare mock with 24 h p.i.). Genomes are shown in magenta and DAPI-stained nuclei in blue; scale bar, 20 μm. (B) Total nuclei (DAPI) and replication centers (AF594) were quantified by counting each from 7 fields of cells, and the data are represented as average counts per field. The proportion of neuronal nuclei containing at least one genome-positive signal was 87.4% ± 15.9%.