Paul W. Howard

A composite Ets/Pit-1 binding site in the prolactin gene can mediate transcriptional responses to multiple signal transduction pathways.

Binding sites for the tissue-specific transcription factor, Pit-1, are required for basal and hormonally induced prolactin gene transcription. Although Pit-1 is phosphorylated in response to several signaling pathways, the mechanism by which Pit-1 contributes to hormonal induction of gene transcription has not been defined. Recent reports suggest that phosphorylation of Pit-1 may not be required for hormonal regulation of the prolactin promoter. Analysis of the contribution of individual Pit-1 binding sites has been complicated due to the fact that some of the elements appear to be redundant. To better understand the role of Pit-1 sites in mediating hormonal regulation of the prolactin gene, we have performed enhancer tests using the three most proximal Pit-1 binding sites of the rat prolactin gene which are designated the 1P, 2P, and 3P sites. The results demonstrate that multimers of the 3P Pit-1 binding site are much more responsive to several hormonal and intracellular signaling pathways than multimers of the 1P or 2P sites. The 3P DNA element was found to contain a consensus binding site for the Ets family of proteins. Mutation of the Ets binding site greatly decreased the ability of epidermal growth factor, phorbol esters, Ras, or the Raf kinase to induce reporter gene activity. Mutation of the Ets site had little effect on basal enhancer activity. In contrast, mutation of the consensus Pit-1 binding site in the 3P element essentially abolished all basal enhancer activity. Overexpression of Ets-1 in GH3 pituitary cells enhanced both basal and Ras induced activity from the 3P enhancer. These data describe a composite element in the prolactin gene containing binding sites for two different factors and the studies suggest a mechanism by which Ets proteins and Pit-1 functionally cooperate to permit transcriptional regulation by different signaling pathways.

Anterograde Transport of Herpes Simplex Virus Capsids in Neurons by both Separate and Married Mechanisms

ABSTRACT Anterograde transport of herpes simplex virus (HSV) from neuronal cell bodies into, and down, axons is a fundamentally important process for spread to other hosts. Different techniques for imaging HSV in axons have produced two models for how virus particles are transported in axons. In the Separate model, viral nucleocapsids devoid of the viral envelope and membrane glycoproteins are transported in axons. In the Married model, enveloped HSV particles (with the viral glycoproteins) encased within membrane vesicles are transported in the anterograde direction. Earlier studies of HSV-infected human neurons involving electron microscopy (EM) and immunofluorescence staining of glycoproteins and capsids supported the Separate model. However, more-recent live-cell imaging of rat, chicken, and mouse neurons produced evidence supporting the Married model. In a recent EM study, a mixture of Married (75%) and Separate (25%) HSV particles was observed. Here, we studied an HSV recombinant expressing a fluorescent form of the viral glycoprotein gB and a fluorescent capsid protein (VP26), observing that human SK-N-SH neurons contained both Separate (the majority) and Married particles. Live-cell imaging of rat superior cervical ganglion (SCG) neuronal axons in a chamber system (which oriented the axons) also produced evidence of Separate and Married particles. Together, our results suggest that one can observe anterograde transport of both HSV capsids and enveloped virus particles depending on which neurons are cultured and how the neurons are imaged.

Herpes Simplex Virus Membrane Proteins gE/gI and US9 Act Cooperatively To Promote Transport of Capsids and Glycoproteins from Neuron Cell Bodies into Initial Axon Segments

ABSTRACT 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. One impediment to understanding how HSV gE/gI and US9 function in axonal transport relates to observations that gE−, gI−, or US9− mutants are not absolutely blocked in axonal transport. Mutants are significantly reduced in numbers of capsids and glycoproteins in distal axons, but there are less extensive effects in proximal axons. We constructed HSV recombinants lacking both gE and US9 that transported no detectable capsids and glycoproteins to distal axons and failed to spread from axon tips to adjacent cells. Live-cell imaging of a gE−/US9− double mutant that expressed fluorescent capsids and gB demonstrated >90% diminished capsids and gB in medial axons and no evidence for decreased rates of transport, stalling, or increased retrograde transport. Instead, capsids, gB, and enveloped virions failed to enter proximal axons. We concluded that gE/gI and US9 function in neuron cell bodies, in a cooperative fashion, to promote the loading of HSV capsids and vesicles containing glycoproteins and enveloped virions onto microtubule motors or their transport into proximal axons.

Human Cytomegalovirus gH/gL Forms a Stable Complex with the Fusion Protein gB in Virions

Human cytomegalovirus (HCMV) is a ubiquitous virus that is a major pathogen in newborns and immunocompromised or immunosuppressed patients. HCMV infects a wide variety of cell types using distinct entry pathways that involve different forms of the gH/gL glycoprotein: gH/gL/gO and gH/gL/UL128-131 as well as the viral fusion glycoprotein, gB. However, the minimal or core fusion machinery (sufficient for cell-cell fusion) is just gH/gL and gB. Here, we demonstrate that HCMV gB and gH/gL form a stable complex early after their synthesis and in the absence of other viral proteins. gH/gL can interact with gB mutants that are unable to mediate cell-cell fusion. gB-gH/gL complexes included as much as 16–50% of the total gH/gL in HCMV virus particles. In contrast, only small amounts of gH/gL/gO and gH/gL/UL128-131 complexes were found associated with gB. All herpesviruses express gB and gH/gL molecules and most models describing herpesvirus entry suggest that gH/gL interacts with gB to mediate membrane fusion, although there is no direct evidence for this. For herpes simplex virus (HSV-1) it has been suggested that after receptor binding gH/gL binds to gB either just before, or coincident with membrane fusion. Therefore, our results have major implications for these models, demonstrating that HCMV gB and gH/gL forms stable gB-gH/gL complexes that are incorporated virions without receptor binding or membrane fusion. Moreover, our data is the best support to date for the proposal that gH/gL interacts with gB.

Herpes simplex virus gE/gI extracellular domains promote axonal transport and spread from neurons to epithelial cells.

ABSTRACT 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 (∼400-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 wild-type 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. IMPORTANCE An 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.

Generation of mice with a conditional allele for Ift172

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.

Interaction of mouse TTC30/DYF-1 with multiple intraflagellar transport complex B proteins and KIF17.

Intraflagellar transport (IFT) is a microtubule based system that supports the assembly and maintenance of cilia. Genetic and biochemical studies have identified two distinct complexes containing multiple proteins that are part of the IFT machinery. In this study we prepared mouse pituitary cells that expressed an epitope-tagged IFT protein and immuno-purified the IFT B complex from these cells. Mass spectrometry analysis of the isolated complex led to identification of a number of well known components of the IFT B complex. In addition, peptides corresponding to mouse tetratricopeptide repeat proteins, TTC30A1, TTC30A2 and TTC30B were identified. The mouse Ttc30A1, Ttc30A2, Ttc30B genes are orthologs of Caenorhabditis elegans dyf-1, which is required for assembly of the distal segment of the cilia. We used co-immunoprecipitation studies to provide evidence that, TTC30A1, TTC30A2 or TTC30B can be incorporated into a complex with a known IFT B protein, IFT52. We also found that TTC30B can interact with mouse KIF17, a kinesin which participates in IFT. In vitro expression in a cell-free system followed by co-immunoprecipitation also provided evidence that TTC30B can directly interact with several different IFT B complex proteins. The findings support the view that mouse TTC30A1, TTC30A2 and TTC30B can contribute to the IFT B complex, likely through interactions with multiple IFT proteins and also suggest a possible link to the molecular motor, KIF17 to support transport of cargo during IFT.

Expression of the Synaptotagmin I Gene Is Enhanced by Binding of the Pituitary-Specific Transcription Factor, POU1F1

The POU1F1 transcription factor (also known as Pit-1/GHF1) is required for development of pituitary cells that secrete prolactin, GH, and TSH. Presumably, POU1F1 regulates the expression of multiple genes required for expansion and differentiation of these pituitary cell lineages. However, only a few genes regulated by POU1F1 have been identified. In the present studies we have identified synaptotagmin I (Syt1) as a target gene for POU1F1 in GH(3) pituitary cells. Chromatin immunoprecipitation assays have provided evidence that POU1F1 binds close to the Syt1 exon that contains the initiation codon. Although this exon has previously been considered to be located far from the transcription initiation site, transcript mapping in GH(3) cells indicates that Syt1 mRNA synthesis is initiated close to the mapped POU1F1-binding site. POU1F1 knockdown studies using a short hairpin RNA vector have provided evidence that POU1F1 plays a role in stimulating expression of the endogenous Syt1 gene. Transfection studies with a Syt1-luciferase reporter gene are consistent with the presence of an internal, POU1F1-regulated promoter in the Syt1 gene. In vitro binding studies have provided further evidence for a POU1F1-binding site within this region of the Syt1 gene. Overall the studies provide evidence that Syt1 is a target gene regulated by POU1F1 in GH(3) pituitary cells. Because SYT1 has been extensively studied as an important transducer of Ca(2+) signaling in regulated secretion, it seems likely that activation of Syt1 gene expression is part of a mechanism mediating POU1F-induced differentiation of pituitary cells.

Transcription intermediary factor 1γ decreases protein expression of the transcriptional cofactor, LIM-domain-binding 1

LIM-domain-binding 1 (LDB1) is a cofactor that participates in formation of regulatory complexes involving transcription factors containing LIM domains as well as other factors. We have examined the ability of transcriptional intermediary factor 1gamma (TIF1gamma) to decrease LDB1 expression. An expression vector for TIF1gamma was found to decrease expression of LDB1. A mutation which disrupts the ubiquitin ligase activity of TIF1gamma was found to block the ability of TIF1gamma to decrease LDB1 expression. Proteasome inhibitors were also able to block TIF1gamma effects on LDB1. Immunoprecipitation studies provided evidence that LDB1 interacts with TIF1gamma in intact cells. Knockdown of TIF1gamma in zebrafish embryos led to increased expression of LDB1 providing evidence for a physiological role of TIF1gamma in regulating LDB1 expression. Reporter gene assays demonstrated that TIF1gamma can alter the activity of LIM-homeodomain transcription factor-responsive promoters. These studies are consistent with a model in which TIF1gamma acts to ubiquitinate LDB1 leading to degradation of LDB1 and changes in transcription of LDB1-dependent promoters.

Herpes Simplex Virus gE/gI and US9 Promote both Envelopment and Sorting of Virus Particles in the Cytoplasm of Neurons, Two Processes That Precede Anterograde Transport in Axons

ABSTRACT Herpes simplex virus (HSV) anterograde transport in neuronal axons is vital, allowing spread from latently infected ganglia to epithelial tissues, where viral progeny are produced in numbers allowing spread to other hosts. The HSV membrane proteins gE/gI and US9 initiate the process of anterograde axonal transport, ensuring that virus particles are transported from the cytoplasm into the most proximal segments of axons. These proteins do not appear to be important once HSV is inside axons. We previously described HSV double mutants lacking both gE and US9 that failed to transport virus particles into axons. Here we show that gE− US9− double mutants accumulate large quantities of unenveloped and partially enveloped capsids in neuronal cytoplasm. These defects in envelopment can explain the defects in axonal transport of enveloped virions. In addition, the unenveloped capsids that accumulated were frequently bound to cytoplasmic membranes, apparently immobilized in intermediate stages of envelopment. A gE-null mutant produced enveloped virions, but these accumulated in large numbers in the neuronal cytoplasm rather than reaching cell surfaces as wild-type HSV virions do. Thus, in addition to the defects in envelopment, there was missorting of capsids and enveloped particles in the neuronal cytoplasm, which can explain the reduced anterograde transport of unenveloped capsids and enveloped virions. These mechanisms differ substantially from existing models suggesting that gE/gI and US9 function by tethering HSV particles to kinesin microtubule motors. The defects in assembly of gE− US9− mutant virus particles were novel because they were neuron specific, in keeping with observations that US9 is neuron specific. IMPORTANCE Herpes simplex virus (HSV) and other alphaherpesviruses, such as varicella-zoster virus, depend upon the capacity to navigate in neuronal axons. To do this, virus particles tether themselves to dyneins and kinesins that motor along microtubules from axon tips to neuronal cell bodies (retrograde transport) or from cell bodies to axon tips (anterograde transport). This transit in axons is essential for alphaherpesviruses to establish latency in ganglia and then to reactivate and move back to peripheral tissues for spread to other hosts. Anterograde transport of HSV requires two membrane proteins: gE/gI and US9. Our studies reveal new mechanisms for how gE/gI and US9 initiate anterograde axonal transport. HSV mutants lacking both gE and US9 fail to properly assemble enveloped virus particles in the cytoplasm, which blocks anterograde transport of enveloped particles. In addition, there are defects in the sorting of virus particles such that particles, when formed, do not enter proximal axons.