Neuritic Transport of Herpes Simplex Virus in Rat Sensory Neurons in vitro. Effects of Substances Interacting with Microtubular Function and Axonal Flow [Nocodazole, Taxol and Erythro-9-3-(2-hydroxynonyl)adenine]

Abstract

SUMMARYHerpes simplex virus type 1 and a fluorescein-labelled lectin (wheat germ agglutinin) were selectively transported to nerve cell bodies located in the inner compartment of a two-chamber tissue culture system after the application of virus or lectin to the neuritic processes in the outer culture compartment. Taxol, which stabilizes and alters intracellular arrangements of microtubules, and nocodazole, which disrupts microtubules, both inhibited this retrograde axonal transport of viral particles and lectin. The transport was also inhibited by erythro-9-3-(2-hydroxynonyl)adenine (EHNA), which blocks ATPases. However, EHNA was also an effective inhibitor of infection with the virus in non-neuronal cells (GMK AH-1). The nature of the action(s) of EHNA on neuritic transport of the virus is therefore less clear.The pathogenesis of herpes simplex virus (HSV) infections involves axonal transport of virus and viral subunits by sensory neurons in the anterograde as well as the retrograde direction. Neurites of dissociated dorsal root ganglion cells have the capacity to internalize the virus (Ziegler & Herman, 1980), presumably by fusion of the viral envelope with the neuritic plasma membrane (Lycke et al., 1984). Since nucleocapsids are detectable in neurites exposed to HSV and are seen around nuclear pores in HSV-infected cell lines (Batterson et al., 1983), we assume that they are involved in retrograde axonal transport of the virus. The mechanisms of this axonal transport of HSV remain to be clarified.We now report the effects on neuritic transport of HSV of three substances capable of inhibiting intracellular transport of endosomes, namely nocodazole, which causes disruption of microtubules, and taxol, which promotes assembly of microtubules (Herman & Albertini, 1984); the third drug, erythro-9-3-(2-hydroxynonyl)adenine (EHNA) (Bouchard et al., 1981) is supposed to inhibit the function of the microtubule-associated ATPase dynein and can inhibit the retrograde movement of organelles in axons. EHNA is considered to have less effect on anterograde axonal transport (Forman et al., 1983).We have used a two-chamber culture system allowing us to infect the neuritic extensions of rat sensory neurons cultured in vitro, without exposing the cell bodies to the virus. For evaluation of the results the neuritic transfer of HSV was compared with the transport of wheat germ agglutinin (WGA).The two-chamber system employed has been described previously (Ziegler & Herman, 1980). Briefly, 35 mm collagen-coated plates were scratched on the bottom, fitted in the centre with an 8 mm cloning cylinder kept in place by silicone high vacuum grease and seeded in the cloning cylinder with trypsin-dissociated dorsal root ganglion (DRG) cells. The cells were obtained by dissecting eight to ten embryos of 16 to 17 day pregnant Sprague-Dawley rats. Trypsinization was performed for 30 min at 37 °C using 0.25~ trypsin (porcine pancreas, type 2, Sigma) in a Ca z÷-and MgZ+-free Hanks' buffer pH 7.2. For the first 2 days, the cells were cultured ...

HSV trafficking and development of gene therapy vectors with applications in the nervous system

“…102,103 The rapid movement of HSV in a retrograde manner would require association with a minusend-directed motor molecule to propel the nucleocapsids toward the minus end of the microtubule network. Ye et al 81 demonstrated that the HSV UL34 protein Reactivation from latency and anterograde transport to the nerve terminal…”

Microtubule polarity in the peripheral processes of trigeminal ganglion cells: relevance for the retrograde transport of herpes simplex virus

“…Previous studies in which microtubule-depolymerizing drugs were used to inhibit transport of HSV did not assess the metabolic activity of the drug-exposed neurons (Kristensson et al, 197 1;Openshaw et al, 1978;Kristensson et al, 1986;Bosem et al, 1990). Therefore, it is impossible to know whether transport was inhibited due to the loss of microtubules or more severe cell destruction.…”

“…Previous studies indicate that HSV takes advantage of the host neuron's natural machinery for retrograde axonal transport, using microtubules for translocation from the nerve terminals in the periphery to the cell bodies of dorsal root ganglion neurons in rive (Kristensson et al,197 1). Microtubules and translocator molecules have also been implicated in the transport of HSV from the neurites to the cell bodies of dorsal root ganglion cells in vitro (Kristensson et al, 1986). However, no study has yet provided quantitative evidence of the dependence of HSV transport on intact microtubules in metabolically active neurons.…”

The Anterograde Transport of Rabies Virus in Rat Sensory Dorsal Root Ganglia Neurons

“…Rabies virus infects a great variety of neuronal cell lines (Clark, 1978;Honda et al, 1984;Iwasaki & Clark, 1977;Tsiang et al, 1983a) as well as primary dissociated neuron cultures (Lycke & Tsiang, 1987;Tsiang et al, 1983aTsiang et al, , b, 1986. Whereas conventional neuronal cultures are less appropriate for the investigation of viral transport, neurons cultivated in a compartmentalized culture system as described by Campenot (1977) will demonstrate convincingly the transport of herpes simplex virus in vitro (Kristensson et al, 1986;Lycke et al, 1984;Ziegler & Herman, 1980) and the retrograde transport of both fixed and street rabies viruses in cultured rat sensory neurons (Lycke & Tsiang, 1987;Tsiang, 1988). In this system, herpes and rabies viruses were transported in a retrograde fashion with a velocity in the range of 50 mm/day (herpes virus) and 25 mm/day (rabies virus) (Lycke et al, 1984;Lycke & Tsiang, 1987).…”