Summary

Первичная система культуры нейронов по изучению простого герпеса латентности вирусов и реактивации

Published: April 02, 2012
doi:

Summary

В протоколе описываются эффективные и воспроизводимые модели системы для изучения вируса простого герпеса типа 1 (HSV-1) задержки и реактивации. Анализ использует однородную симпатических нейронов культуры и позволяет молекулярной вскрытия вируса нейронных взаимодействий, используя различные инструменты, включая РНК-интерференции и экспрессии рекомбинантных белков.

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. Выделение и культуры СКГ нейроны из эмбрионов крыс Чтобы обеспечить полезный контекст для понимания этого протокола, а также для всестороннего обсуждения ранее литературы, установленных методов культуры SCG нейронов, в том числе на основе СКГ в пробирке культуру, пли?…

Discussion

Это первичной культуре нейронов и инфекции система обеспечивает простой и эффективный метод для изучения молекулярных механизмов, лежащих в основе HSV-1 и задержкой реактивации. Система точно повторяет принятое признаки задержки определен в обоих инфекций человека и…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

Мы благодарим рецензентов за их вдумчивые предложения, которые помогли улучшить эту рукопись. Эта работа была поддержана грантами на MVC (NS21072, HD23315), ACW (GM61139, S10RR017970) и IM (AI073898, GM056927) из NIH. MK была частично поддержана грантом NIH обучение (5T32 AI007180).

Materials

Reagent Company Catalog# 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 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 C2674 prepare 10 mg/ml stock in HBSS-/-; store at -20°C
D-(+)-Glucose Sigma G6152 prepare 40% stock in H2O; filter sterilize & store at 4°C
L-Glutamine Invitrogen 25030-081  
Laminin Sigma 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 P6909 prepare 75 mg/ml stock in H2O; store at -20°C
Poly-D-lysine hydrobromide Sigma P0899 prepare 20 mg/ml stock in H2O; store at -20°C
Rat-tail collagen 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 T8552 prepare 1 mM stock in DMSO; store at -20°C
Trypsin 2.5% Invitrogen 15090-04  

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Citazione di questo articolo
Kobayashi, M., Kim, J., Camarena, V., Roehm, P. C., Chao, M. V., Wilson, A. C., Mohr, I. A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation. J. Vis. Exp. (62), e3823, doi:10.3791/3823 (2012).

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