Using Primary SCG Neuron Cultures to Study Molecular Determinants of HSV-1 Latency and Reactivation
We describe a primary neuronal culture system suitable for molecular characterization of herpes simplex virus type 1 (HSV-1) infection, latency, and reactivation. While several alternative models are available, including infections of live animal or explanted ganglia, these are complicated by the presence of multiple cell types, including immune cells, and difficulties in manipulating the neuronal environment. The highly pure neuron culture system described here can be readily manipulated and is ideal for molecular studies that focus exclusively on the relationship between the virus and host neuron, the fundamental unit of latency. As such this model allows for detailed investigations of both viral and neuronal factors involved in the establishment and maintenance of HSV-1 latency and in viral reactivation induced by defined stimuli.
Key wordsHSV-1 Latency Reactivation SCG neuron culture In vitro system Lentiviral delivery RNA interference
We thank past members of the Mohr and Wilson Labs for establishing these protocols as well as Moses Chao for his continuous support of our latency studies and for teaching us about the role of nerve growth factor in sustaining sympathetic neurons. This work was funded by NIH grants GM107257 (T.T.H.), GM056927, AI073898 (I.M.), AI130618 (A.W.), and funds from the V Foundation for BRCA Cancer Research to T.T.H.
- 5.Camarena V, Kobayashi M, Kim JY, Roehm PC, Perez R, Gardner J, Wilson AC, Mohr I, Chao MV (2010) Nature and duration of growth factor signaling through receptor tyrosine kinases regulates HSV-1 latency in neurons. Cell Host Microbe 8:320–330. https://doi.org/10.1016/j.chom.2010.09.007CrossRefPubMedPubMedCentralGoogle Scholar
- 10.Cliffe AR, Arbuckle JH, Vogel JL, Geden MJ, Rothbart SB, Cusack CL, Strahl BD, Kristie TM, Deshmukh M (2015) Neuronal stress pathway mediating a histone methyl/phospho switch is required for Herpes simplex virus reactivation. Cell Host Microbe 18:649–658. https://doi.org/10.1016/j.chom.2015.11.007CrossRefPubMedPubMedCentralGoogle Scholar
- 17.Harlow E, Lane D (1988) Antibodies, a laboratory manual. Cold Spring Harbor Press, Cold Spring Harbor, NYGoogle Scholar
- 18.Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Press, Cold Spring Harbor, NYGoogle Scholar
- 19.Hu H-L, Shiflett LA, Kobayashi M, Chao MV, Wilson AC, Mohr I, Huang TT (2019) TOP2ß-dependent nuclear DNA damage shapes extracellular growth factor responses via dynamic AKT phosphorylation to control virus latency. Mol Cell 74:1–15. https://doi.org/10.1016/j.molcel.2019.02.032CrossRefGoogle Scholar
- 20.Linderman JA, Kobayashi M, Rayannavar V, Fak JJ, Darnell RB, Chao MV, Wilson AC, Mohr I (2017) Immune escape via a transient gene expression program enables productive replication of a latent pathogen. Cell Rep 18:1312–1323. https://doi.org/10.1016/j.celrep.2017.01.017CrossRefPubMedPubMedCentralGoogle Scholar