Interferon alpha induces establishment of alphaherpesvirus latency in sensory neurons in vitro - PubMed (original) (raw)

Interferon alpha induces establishment of alphaherpesvirus latency in sensory neurons in vitro

Nick De Regge et al. PLoS One. 2010.

Abstract

Background: Several alphaherpesviruses, including herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), establish lifelong latency in neurons of the trigeminal ganglion (TG). Although it is thought that efficient establishment of alphaherpesvirus latency is based on a subtle interplay between virus, neurons and the immune system, it is not clear which immune components are of major importance for the establishment of latency.

Methodology/principal findings: Here, using an in vitro model that enables a natural route of infection, we show that interferon alpha (IFNalpha) has the previously uncharacterized capacity to induce a quiescent HSV-1 and PRV infection in porcine TG neurons that shows strong similarity to in vivo latency. IFNalpha induced a stably suppressed HSV-1 and PRV infection in TG neurons in vitro. Subsequent treatment of neurons containing stably suppressed virus with forskolin resulted in reactivation of both viruses. HSV and PRV latency in vivo is often accompanied by the expression of latency associated transcripts (LATs). Infection of TG neurons with an HSV-1 mutant expressing LacZ under control of the LAT promoter showed activation of the LAT promoter and RT-PCR analysis confirmed that both HSV-1 and PRV express LATs during latency in vitro.

Conclusions/significance: These data represent a unique in vitro model of alphaherpesvirus latency and indicate that IFNalpha may be a driving force in promoting efficient latency establishment.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Productive replication of PRV and HSV-1 in porcine TG neurons.

Confocal images of TG neuronal cultures in the inner chamber at 24hpi with PRV (A,B) and 48hpi with HSV-1 (D,E) stained for neurofilament (red) and late viral antigens (green) (bar = 50 µm). Percentage of neurons with axons growing out to the outer chamber that show viral antigens at 24hpi with PRV (C) and 48hpi with wt HSV-1 (F, left bar) and beta-galactosidase activity at 24hpi with SΔUS5-LacZ HSV-1 (F, right bar). Data represent the mean ± s.e.m. of three independent experiments.

Figure 2

Figure 2. IFNalpha induces a reactivatable, latent PRV and HSV-1 infection in porcine TG neurons.

Percentage infected neurons that are late viral antigen positive at 1, 5 and 8dpi with PRV (A) and at 2, 5 and 12dpi with HSV-1 (B) in the presence or absence of 500 U/ml IFNalpha. For the neurons fixed at 8dpi with PRV and 12dpi with HSV-1, medium containing IFNalpha was washed out at 5dpi and replaced with new culture medium or new culture medium supplemented with forskolin (200 µM). Data represent the mean ± s.e.m. of three independent experiments.

Figure 3

Figure 3. PRV and HSV-1 express LATs during in vitro latency.

(A,B) RT-PCR analysis of actin and viral immediate early (IE180 and ICP0), late (gB and gD) and LAT transcript RNA isolated from neuronal cultures that were either mock infected, productively infected with PRV (A, 1dpi) or HSV-1 (B, 2dpi), or latently infected with PRV (A, 5dpi with IFNalpha) or HSV-1 (B, 9dpi, 4 days post IFNalpha withdrawal). For each condition three different samples were analyzed and representative gels are shown. For HSV-1, two samples of 9dpi, 4 days post IFNalpha withdrawal are shown, one without and one with detectable ICP0 transcript expression. Specific bands are marked with a black arrowhead. (C) Percentage of infected neurons positive for LAT promoter-driven beta-galactosidase at 2 and 5dpi with HSV-1 LbetaA in the presence or absence of 500 U/ml IFNalpha. Data represent the mean ± s.e.m. of three independent experiments. (D) Light microscopic images of uniform (i,ii) and focal (iii) LAT promoter-driven beta-galactosidase distribution during the acute stage (2dpi without IFNalpha, i, ii) or the latent stage (5dpi with IFNalpha, iii) of infection with HSV-1 LbetaA. Arrows point to infected non-neuronal cells (i), dashed line marks contour of neuronal cell body in (iii) (bar = 20 µm).

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