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. 2008 Oct;82(19):9555-63.
doi: 10.1128/JVI.01174-08. Epub 2008 Jul 30.

Association of the cellular coactivator HCF-1 with the Golgi apparatus in sensory neurons

Gaelle Kolb  1 , Thomas M Kristie
Affiliations

Association of the cellular coactivator HCF-1 with the Golgi apparatus in sensory neurons

Gaelle Kolb et al. J Virol. 2008 Oct.

Abstract

HCF-1 is a cellular transcriptional coactivator that is critical for mediating the regulated expression of the immediate-early genes of the alphaherpesviruses herpes simplex virus type 1 and varicella-zoster virus. HCF-1 functions, at least in part, by modulating the modification of nucleosomes at these viral promoters to reverse cell-mediated repressive marks and promote activating marks. Strikingly, HCF-1 is specifically sequestered in the cytoplasm of sensory neurons where these viruses establish latency and is rapidly relocalized to the nucleus upon stimuli that result in viral reactivation. However, the analysis of HCF-1 in latently infected neurons and the protein's specific subcellular location have not been determined. Therefore, in this study, the localization of HCF-1 in unstimulated and induced latently infected sensory neurons was investigated and was found to be similar to that observed in uninfected mice, with a time course of induced nuclear accumulation that correlated with viral reactivation. Using a primary neuronal cell culture system, HCF-1 was localized to the Golgi apparatus in unstimulated neurons, a unique location for a transcriptional coactivator. Upon disruption of the Golgi body, HCF-1 was rapidly relocalized to the nucleus in contrast to other Golgi apparatus-associated proteins. The location of HCF-1 is distinct from that of CREB3, an endoplasmic reticulum-resident HCF-1 interaction partner that has been proposed to sequester HCF-1. The results support the model that HCF-1 is an important component of the viral latency-reactivation cycle and that it is regulated by association with a component that is distinct from the identified HCF-1 interaction factors.

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Figures

FIG. 1.
FIG. 1.
Localization and transport of HCF-1 in neurons of latently infected mice. (A) Trigeminal ganglia of mock-infected and latently infected mice were fixed, sectioned, and stained for HCF-1 at various times postexplantation (0 to 72 h). LI-0, latently infected and rapidly fixed; LI-6, latently infected and fixed at 6 h postexplantation. (B) The number of ganglia counted and the percentage of neurons exhibiting a nuclear accumulation (% Nuc) of HCF-1 are listed at the various times postexplantation. The results are graphically represented. Exp-1 and Exp-2 represent two independent time course experiments.
FIG. 2.
FIG. 2.
A DRG primary neuronal cell culture system. FC-DRG were dissociated and plated in the presence of NGF and mitotic inhibitors. After 14 days in culture, the neuronal network was visualized using bright-field microscopy or were stained with DAPI (4′,6′-diamidino-2-phenylindole; blue) and anti-neurofilament (red) and visualized by confocal microscopy (right panel).
FIG. 3.
FIG. 3.
HCF-1 does not colocalize with nuclear, mitochondrial, or ER markers in unstimulated neurons. Cultures of DRG neurons were costained for HCF-1 (green), DNA (DAPI [4′,6′-diamidino-2-phenylindole]; blue), and the indicated subcellular markers.
FIG. 4.
FIG. 4.
HCF-1 is localized at the Golgi apparatus in unstimulated DRG neurons. Cultures of DRG neurons were costained for HCF-1 (green), DNA (DAPI [4′,6′-diamidino-2-phenylindole]; blue), and the 58K or GM130 Golgi apparatus markers.
FIG. 5.
FIG. 5.
HCF-1 is localized at the Golgi apparatus in neurons of cryo-sectioned mouse trigeminal ganglia. Trigeminal ganglia of mock-infected (MI) or latently infected (LI) mice were rapidly explanted, snap-frozen, cryo-sectioned, and stained for HCF-1 (green), DNA (DAPI [4′,6′-diamidino-2-phenylindole]; blue), and the Golgi apparatus (58K; red). Three-dimensional reconstructions of Z-stack sections are shown.
FIG. 6.
FIG. 6.
Brefeldin A disruption of the Golgi apparatus results in nuclear accumulation of HCF-1. Cultures of DRG neurons were mock treated (M) or treated with brefeldin A (BFA) for 2 h, fixed, and stained for HCF-1 (A; green), CREB3 (B; green), DNA (DAPI [4′,6′-diamidino-2-phenylindole]; blue), and Golgi apparatus (58K) or nuclear (RNAP II) markers.
FIG. 7.
FIG. 7.
HCF-1 does not fractionate with the nucleus or cytoplasmic membranes of DRG neurons. Cultures of DRG neurons were fractionated as described in Materials and Methods into soluble cytoplasmic (C) and particulate (P) fractions containing the nucleus and cytoplasmic membranes. Fractions were resolved in 4 to 20% denaturing polyacrylamide gels and Western blotted using anti-HCF-1, anti-RNAP II, and anti-PDI antibodies. E, whole cell extract; MW, positions of molecular weight markers (in thousands).
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