Neuregulin-1 (Nrg-1) contains an intracellular domain (Nrg-ICD) that translocates into the nucleus, where it may regulate gene expression upon neuronal depolarization. However, the identity of its target promoters and the mechanisms by which it regulates transcription have been elusive. Here we report that, in the mouse cochlea, synaptic activity increases the level of nuclear Nrg-ICD and upregulates postsynaptic density protein-95 (PSD-95), a scaffolding protein that is enriched in post-synaptic structures. Nrg-ICD enhances the transcriptional activity of the PSD-95 promoter by binding to a zinc-finger transcription factor, Eos. The Nrg-ICD-Eos complex induces endogenous PSD-95 expression in vivo through a signaling pathway that is mostly independent of gamma-secretase regulation. This upregulation of PSD-95 expression by the Nrg-ICD-Eos complex provides a molecular basis for activity-dependent synaptic plasticity.

The maturation and envelopment of varicella-zoster virus (VZV) was studied in infected human embryonic lung fibroblasts. Transmission electron microscopy confirmed that nucleocapsids acquire an envelope from the inner nuclear membrane as they enter the perinuclear-cisterna-rough endoplasmic reticulum (RER). Tegument is not detectable in these virions; moreover, in contrast to the mature VZV envelope, the envelope of VZV in the RER is not radioautographically labeled in pulse-chase experiments with [3H]mannose, and it lacks gpI immunoreactivity and complex oligosaccharides. This primary envelope fuses with the RER membrane (detected in cells incubated at 20 degrees C), thereby releasing nucleocapsids to the cytosol. Viral glycoproteins, traced by transmission electron microscopy radioautography in pulse-chase experiments with [3H]mannose, are transported to the trans-Golgi network (TGN) by a pathway that runs from the RER through an intermediate compartment and the Golgi stack. At later chase intervals, [3H]mannose labeling becomes associated with enveloped virions in post-Golgi locations (prelysosomes and plasma membrane). Nucleocapsids appear to be enveloped by wrapping in specialized cisternae, identified as the TGN with specific markers. Tegument-like material adheres to the cytosolic face of the concave surface of TGN sacs; nucleocapsids adhere to this protein, which is thus trapped between the nucleocapsid and the TGN-derived membrane that wraps around it. Experiments with brefeldin A suggest that tegument may bind to the cytosolic tails of viral glycoproteins. Fusion and fission convert the TGN-derived wrapping sacs into an inner enveloped virion and an outer transport vesicle that carries newly enveloped virions to cytoplasmic vacuoles. These vacuoles are acidic and were identified as prelysosomes. It is postulated that secreted virions are partially degraded by their exposure to the prelysosomal internal milieu and rendered noninfectious. This process explains the cell-associated nature of VZV in vitro; however, the mechanism by which the virus escapes diversion from the secretory pathway to the lysosomal pathway in vivo remains to be determined.

Envelope glycoproteins of varicella zoster virus (VZV) contain mannose 6-phosphate (Man6P) residues.We now report that Man6P competitively and selectively inhibits infection of cells in vitro by cell-free VZV; furthermore, dephosphorylation of VZV by exposure to alkaline phosphatase rapidly destroys infectivity. Cells are also protected from VZV in a concentration-dependent manner by heparin (ED50 = 0.23 ,Lg/ml; 95% confidence limits = 0. 16-0.26 Viral entry may involve more than a single receptor. Entry of HSV, for example, requires both initial attachment and subsequent internalization (6-8). Different glycoproteins of the HSV envelope have been implicated in each of these steps. Adsorption of HSV is the responsibility of glycoprotein C (gC),The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. and mutant virions from which gC has been deleted are unable to bind to target cells (9, 10). In contrast, antibodies to gD do not inhibit the attachment of virions, but they do prevent the penetration of cells to which the virions have adsorbed (10). If gC and gD each interact with a different receptor, then the cellular protein responsible for adsorption would be different from that which mediates penetration. The cell surface molecules that interact either with gC or gD have not been identified, although gC of HSV-1 (9), like glycoproteins of other herpesviruses (11, 12), binds to heparin-like molecules. Genetic (6) and biochemical (7) studies of mutant CHO cell lines, defective in glycosaminoglycan (GAG) biosynthesis (13, 14), also support the idea that a cell surface heparan sulfate proteoglycan (HSPG) is required for the attachment and subsequent infection of cells by HSV. It is plausible that the uptake of VZV, like that of HSV, involves more than a single step and more than one type of receptor. The current experiments were undertaken to test the hypotheses that adsorption of VZV is mediated by HSPG and that a receptor for which Man6P is a ligand plays a subsequent role in viral entry. MATERIALS AND METHODSConfluent monolayers of human embryonic lung fibroblasts (HELFs) and cell-free VZV were prepared as described (2). To determine the stage of viral infection affected by Man6P, HELF monolayers were exposed to cell-free VZV [>25 plaqueforming units (pfu)/ml]. Man6P (20 mM final concentration) was added at various intervals following inoculation. Cultures were then incubated for 3 or 4 days, after which plaques were counted. To analyze the effect of phosphorylated monosaccharides on the expression of gpl on the surfaces of infected cells, HELF monolayers were infected with -50 pfu of cell-free VZV in medium containing Man6P (10-20 mM) or glucose 1-phosphate (GlclP; 10-20 mM). Neither monosaccharide was added to controls. HELFs were cultured for 3 or 4 days and VZV gpl was quantified by RIA. For this assay, infected cells were exposed to a murine mon...

The induction of a long-term hyperexcitability (LTH) in vertebrate nociceptive sensory neurons (SNs) after nerve injury is an important contributor to neuropathic pain in humans, but the signaling cascades that induce this LTH have not been identified. In particular, it is not known how injuring an axon far from the cell soma elicits changes in gene expression in the nucleus that underlie LTH. The nociceptive SNs of Aplysia (ap) develop an LTH with electrophysiological properties after axotomy similar to those of mammalian neurons and are an experimentally useful model to examine these issues. We cloned an Aplysia PKG (cGMP-dependent protein kinase; protein kinase G) that is homologous to vertebrate type-I PKGs and found that apPKG is activated at the site of injury in the axon after peripheral nerve crush. The active apPKG is subsequently retrogradely transported to the somata of the SNs, but apPKG activity does not appear in other neurons whose axons are injured. In the soma, apPKG phosphorylates apMAPK (Aplysia mitogen-activated protein kinase), resulting in its entry into the nucleus. Surprisingly, studies using recombinant proteins in vivo and in vitro indicate that apPKG directly phosphorylates the threonine moiety in the T-E-Y activation site of apMAPK when the -Y-site contains a phosphate. We used inhibitors of nitric oxide synthase, soluble guanyl cyclase, or PKG after nerve injury, and found that each prevented the appearance of the LTH. Moreover, blocking apPKG activation prevented the nuclear import of apMAPK. Consequently, the nitric oxide-PKG-MAPK pathway is a potential target for treatment of neuropathic pain.

When the nuclear localization signal peptide (sp) of the SV 40 large T antigen was coupled to human serum albumin (HSA), rhodaminated (r), and microinjected into axons of Aplysia neurons in vitro, the rHSA-sp was conveyed through the axon to the cell body and then into the nucleus (Ambron et al., 1992). But since rHSA-sp is an artificial construct, we needed to determine whether naturally occurring nuclear proteins use this pathway. We therefore injected calf thymus histone H-1 and Xenopus oocyte nucleoplasmin into axons. By 3 hr both were retrogradely transported and targeted into the nucleus, though histone H-1 less efficiently than rHSA-sp or nucleoplasmin. In contrast, neither rHSA, nor rHSA conjugated to a peptide with a random distribution of basic amino acids, was transported or imported. To see if proteins that use the pathway remain intact, we coupled sp to HRP. When injected into varicosities, the HRP-sp was transported/imported to the nucleus, where it was enzymatically active. A key issue was to determine whether endogenous proteins use this pathway. Consequently, axoplasm was extruded from Aplysia nerves and the proteins were fractionated by size. SDS-PAGE and Western blots showed that two fractions contained proteins that were recognized by an affinity-purified antibody to sp: fraction 3 included sp83, and fraction 4 contained sp75. In addition, these two proteins were found in nuclei isolated from neurons. To assess transport, the total proteins in the fractions were rhodaminated and injected into varicosities. Fraction 3, but not fraction 4, contained protein that was transported through the axon to the nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)