A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Summary

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

Abstract

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.

Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA⁺ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor¹. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

Protocol

1. Isolation and Culture of SCG Neurons from Rat Embryos

To provide a useful context for understanding this protocol, and for a comprehensive discussion of earlier literature that established methods of SCG neuron culture, including the basis for SCG in vitro culture, plate-coating substrates, and the components of serum-free media, the reader is referred to references^2-4.

1. The use of rats as a source for SCG neurons was conducted in accordance with NIH guidelines under an active protocol approved by the Institutional Animal Care & Use Committee (IACUC).
2. Before commencing the dissec....

Discussion

This primary neuron culture and infection system provides a simple and effective method to explore the molecular mechanisms underlying HSV-1 latency and reactivation. The system faithfully recapitulates the accepted hallmarks of latency defined in both human infections and in live-animal models. When the virus is latent in the SCG cultures, infectious particles and viral lytic gene products cannot be detected. The only viral gene product detected in latently-infected neurons is the non-coding LAT trans.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
70μm nylon filter( cell strainer)	BD Biosciences	352350
1x Hank’s Balanced Salt Solution (HBSS-/-)	Invitrogen	14175	w/o CaCl2 and MgCl2
1x Minimum Essential Media (MEM)	Invitrogen	11095-080
5-Fluoro-2’-deoxyuridine	Sigma-Aldrich	F0503	prepare 20 mM stock in 1x MEM; store at -20°C
96-well flat well bottom TC plates	Corning	3599
Acyclovir	Calbiochem	114798	prepare 31 mM stock in DMSO; store at -20°C
Aphidicolin	Calbiochem	178273	prepare 10 mM stock in DMSO; store at -20°C
B-27 Supplement	Invitrogen	17504-44
Collagenase	Sigma-Aldrich	C2674	prepare 10 mg/ml stock in HBSS-/-; store at -20°C
D-(+)-Glucose	Sigma-Aldrich	G6152	prepare 40% stock in H2O; filter sterilize & store at 4°C
L-Glutamine	Invitrogen	25030-081
Laminin	Sigma-Aldrich	L2020	prepare 1 mg/ml stock in H2O; quick-freeze 20 μl aliquats & store at -80°C; dilute to 2 μg/ml working conc. in sterile H2O
Leibovit’z L-15 media	Invitrogen	11415
Nerve Growth Factor	Harlan Laboratories	BT.5017	prepare 50 μg/ml stock in HBSS-/-; store at -80°C
Neurobasal medium	Invitrogen	12348
Phosphonoacetic acid (PAA)	Sigma-Aldrich	P6909	prepare 75 mg/ml stock in H2O; store at -20°C
Poly-D-lysine hydrobromide	Sigma-Aldrich	P0899	prepare 20 mg/ml stock in H2O; store at -20°C
Rat-tail collagen	EMD Millipore	08-115	Concentration varies with supply lot; store at 4°C and dilute to 0.66 mg/ml working conc. in sterile H2O
Trichostatin A	Sigma-Aldrich	T8552	prepare 1 mM stock in DMSO; store at -20°C
Trypsin 2.5%	Invitrogen	15090-04