Herpes simplex virus (HSV) and other alphaherpesviruses must move from sites of latency in ganglia to peripheral epithelial cells. How HSV navigates in neuronal axons is not well understood. Two HSV membrane proteins, gE/gI and US9, are key to understanding the processes by which viral glycoproteins, unenveloped capsids, and enveloped virions are transported toward axon tips. Whether gE/gI and US9 function to promote the loading of viral proteins onto microtubule motors in neuron cell bodies or to tether viral proteins onto microtubule motors within axons is not clear. A lphaherpesviruses depend upon highly evolved mechanisms to move from mucosal epithelial tissues within neuronal axons to ganglia where latency is established. Following reactivation from latency, virus particles move from ganglia back to peripheral tissues for spread to other hosts. This anterograde transport involves fast axon transport involving microtubules and kinesin motors that propel viral particles from neuron cell bodies (in ganglia) over large distances to axon tips.Depending upon the strain of alphaherpesvirus and the type of neuron, anterograde transport can apparently involve either fully assembled virions or unenveloped capsids (reviewed in references1, 2, and3). Fully assembled, enveloped virions or "Married" particles (4) are produced by capsid envelopment in the cytoplasm of neuron cell bodies, while "Separate" (4) unenveloped capsids (lacking viral glycoproteins) become enveloped at or near axon tips. Early electron microscopy (EM) studies produced evidence for Separate herpes simplex virus (HSV) capsids in human and rat neuronal axons (5-7). Other, more recent EM studies observed a mixture of Separate capsids (25%) and Married particles for two HSV strains (8), but this ratio was reversed, so that 70% of the particles in axons were Separate particles with another HSV strain (T. Mettenleiter, personal communication). Our antibody staining of HSV-infected human neuroblastoma cells produced evidence for mainly Separate capsids and distinct glycoprotein-containing vesicles (4, 9, 10). EM and fluorescent protein analyses of pig pseudorabies virus (PRV) strongly support only Married transport (11)(12)(13)(14). A study involving a "two-color" HSV recombinant expressing a fluorescent glycoprotein and capsids concluded that most HSV anterograde transport involved Married particles (15). Using another "two-color" HSV recombinant expressing fluorescent capsids and glycoproteins gB, we concluded that a majority of capsids moving in rat superior cervical ganglion (SCG) neurons were Separate particles (60%) (16). Thus, we believe that both modes of transport are possible and, in fact, occur.HSV and PRV express two membrane proteins, gE/gI and US9, which are key to the understanding of anterograde transport in neuronal axons (reviewed in references 2 and3). gE/gI is a heterodimer, with both gE and gI required for function, and possesses both substantial extracellular domains and ϳ100-amino-acid (aa) cytoplasmic domains with acidic clusters, ...

Following reactivation from latency, there are two distinct steps in the spread of herpes simplex virus (HSV) from infected neurons to epithelial cells: (i) anterograde axonal transport of virus particles from neuron bodies to axon tips and (ii) exocytosis and spread of extracellular virions across cell junctions into adjacent epithelial cells. The HSV heterodimeric glycoprotein gE/gI is important for anterograde axonal transport, and gE/gI cytoplasmic domains play important roles in sorting of virus particles into axons. However, the roles of the large (ϳ400-residue) gE/gI extracellular (ET) domains in both axonal transport and neuron-to-epithelial cell spread have not been characterized. Two gE mutants, gE-277 and gE-348, contain small insertions in the gE ET domain, fold normally, form gE/gI heterodimers, and are incorporated into virions. Both gE-277 and gE-348 did not function in anterograde axonal transport; there were markedly reduced numbers of viral capsids and glycoproteins compared with wildtype HSV. The defects in axonal transport were manifest in neuronal cell bodies, involving missorting of HSV capsids before entry into proximal axons. Although there were diminished numbers of mutant gE-348 capsids and glycoproteins in distal axons, there was efficient spread to adjacent epithelial cells, similar to wild-type HSV. In contrast, virus particles produced by HSV gE-277 spread poorly to epithelial cells, despite numbers of virus particles similar to those for HSV gE-348. These results genetically separate the two steps in HSV spread from neurons to epithelial cells and demonstrate that the gE/gI ET domains function in both processes. IMPORTANCEAn essential phase of the life cycle of herpes simplex virus (HSV) and other alphaherpesviruses is the capacity to reactivate from latency and then spread from infected neurons to epithelial tissues. This spread involves at least two steps: (i) anterograde transport to axon tips followed by (ii) exocytosis and extracellular spread from axons to epithelial cells. HSV gE/gI is a glycoprotein that facilitates this virus spread, although by poorly understood mechanisms. Here, we show that the extracellular (ET) domains of gE/gI promote the sorting of viral structural proteins into proximal axons to begin axonal transport. However, the gE/gI ET domains also participate in the extracellular spread from axon tips across cell junctions to epithelial cells. Understanding the molecular mechanisms involved in gE/gI-mediated sorting of virus particles into axons and extracellular spread to adjacent cells is fundamentally important for identifying novel targets to reduce alphaherpesvirus disease.A lphaherpesviruses, such as herpes simplex virus (HSV) and varicella-zoster virus (VZV), have evolved specialized mechanisms enabling virus spread in epithelial and neuronal tissues. Primary infection involves entry into skin or mucosal epithelial cells, followed by rapid virus spread between these cells. During this phase of virus replication and spread, viruses enter sensory...

In Drosophila, the Polycomb-group constitutes a set of structurally diverse proteins that act together to silence target genes. Many mammalian Polycomb-group proteins have also been identified and show functional similarities with their invertebrate counterparts. To begin to analyze the function of Polycomb-group proteins in Xenopus development, we have cloned a Xenopus homolog of Drosophila Polycomblike, XPcl1. XPcl1 mRNA is present both maternally and zygotically, with prominent zygotic expression in the anterior central nervous system. Misexpression of Pcl1 by RNA injection into embryos produces defects in the anterior central nervous system. The forebrain and midbrain contain excess neural tissue at the expense of the ventricle and include greatly thickened floor and roof plates. The eye fields are present but Rx2A, an eye-specific marker, is completely repressed. Overexpression of Pcl1 in Xenopus embryos alters two hindbrain markers, repressing En-2 and shifting it and Krox-20 in a posterior direction. Similar neural phenotypes and effects on the En-2 expression pattern were produced by overexpression of three other structurally unrelated Polycomb-group proteins: M33, XBmi-1, and mPh2. These observations indicate an important role for the Polycomb-group in regulating gene expression in the developing anterior central nervous system.

Ift172 encodes a gene product that is part of a complex that mediates intraflagellar transport (IFT), a process necessary for the genesis and maintenance of cilia. Genetic studies in mice have offered evidence that Ift172 also plays a role in hedgehog signaling. Disruption of Ift172 in mice is associated with lethality at about embryonic day 11, limiting studies to understand the role for Ift172 in later development and the adult. To further our understanding of the later roles of Ift172, we have generated mice with a conditional allele for Ift172. We have confirmed the phenotype of the disrupted allele by using CRE expression directed by the prx1 enhancer to disrupt the conditional Ift172 allele in the developing limb. Keywords Intraflagellar transport; Hedgehog; Limb Development; CRE recombinaseThe intraflagellar transport protein, IFT172, is a member of a complex of proteins that are required for genesis and maintenance of primary cilia (Eggenschwiler and Anderson 2007). Disruption of intraflagellar transport by mutation of Ift genes is associated with several human pathologies including polycystic kidney disease (Yoder 2007). Mutation of the genes for the IFT proteins IFT88 or IFT172 also blocks hedgehog signaling (Huangfu et al. 2003). Subsequent studes have demonstrated that many hedgehog signaling components, including the Gli transcriptional regulators, must localize to the primary cilium for proper signaling to occur (Eggenschwiler and Anderson 2007). We originally cloned the cDNA encoding IFT172 in a yeast two-hybrid screen for proteins that interact with the LIM domains of the LIMhomeodomain transcription factor Lhx3 (Howard and Maurer 2000). Disruption of Ift172 causes lethality in mice at about embryonic day 11 (Huangfu et al. 2003;Gorivodsky et al. 2009). To further our understanding of the possible involvement of Ift172 in Lhx3-mediated pituitary signaling we have generated mice harboring a conditional allele for Ift172. This mouse model circumvents the embryonic lethality of the Ift172 null by allowing the tissue-specific or temporal disruption of Ift172. Materials and methods Construction of the Ift172 gene targeting vectorGenomic DNA containing the Ift172 gene was obtained by screening a λGT-10 library of 129/ SvJ gemonic DNA. A targeting vector in which exons 2 and 3 are flanked by loxP sites and which also contains neomycin and chloramphenicol resistance cassettes flanked by frt sites was prepared by homologous recombination in E. coli (Lee et al. 2001). Details of the construction of the targeting vector are available on request.The targeting vector was tested for proper design and function by subjecting it to FLP-mediated recombination in the bacterial stain EL250 and CRE mediated recombination in the bacterial strain EL350 (Lee et al. 2001). Generation of Mice Carrying the Conditional and Disrupted Ift172 AllelesThe completed targeting vector was linearized and electroporated into 129/SvJ embryonic stem cells and recombinant cells were selected with G418. Recombinant embryonic stem...