Biomedical subjects

William T Ruyechan

Publications and source records attributed to William T Ruyechan.

16 recordsLinked to original sources

PubMed2006

Two alphaherpesvirus latency-associated gene products influence calcitonin gene-related peptide levels in rat trigeminal neurons.

Herpes simplex virus type-1 (HSV-1) initially infects mucoepithelial tissues of the eye and the orofacial region. Subsequently, the virus is retrogradely transported through the axons of the trigeminal sensory neurons. HSV-1 establishes a life-long latent infection in these neurons, during which the transcription of the viral genome is silent, except for the sequences encoding the latency-associated transcript (LAT). To determine if HSV-1 latency might affect calcitonin gene-related peptide (CGRP) expression in trigeminal sensory neurons, we transfected primary neuronal cultures of trigeminal ganglia from rat embryos with plasmids expressing LAT. In the presence of Bone Morphogenetic Protein-7 (BMP7), CGRP was expressed in 49% of sensory neurons. However, this percentage was reduced to 19% in neurons transfected with LAT expressing plasmids. We also found that transfection of the IE63 gene of varicella-zoster virus (VZV) reduced the percentage of trigeminal neurons containing CGRP. However, the observed effect of IE63 in contrast to that of LAT was completely reversed by treatment of cultures with MgCl2, which indicates that the effect of IE63 was due to increased release of CGRP from trigeminal neurons. We provide here the first evidence that HSV-1 LAT decreases the level of CGRP in trigeminal neurons. These effects may be important for reducing the neuroinflammatory response, thus protecting host neuronal cells during HSV-1 latency in trigeminal neurons. In contrast, increased release of CGRP in the presence of IE63 protein may contribute to the neuralgias associated with VZV infection.

Animals↗

PubMed2006

The varicella-zoster virus (VZV) ORF9 protein interacts with the IE62 major VZV transactivator.

The varicella-zoster virus (VZV) ORF9 protein is a member of the herpesvirus UL49 gene family but shares limited identity and similarity with the UL49 prototype, herpes simplex virus type 1 VP22. ORF9 mRNA is the most abundantly expressed message during VZV infection; however, little is known concerning the functions of the ORF9 protein. We have found that the VZV major transactivator IE62 and the ORF9 protein can be coprecipitated from infected cells. Yeast two-hybrid analysis localized the region of the ORF9 protein required for interaction with IE62 to the middle third of the protein encompassing amino acids 117 to 186. Protein pull-down assays with GST-IE62 fusion proteins containing N-terminal IE62 sequences showed that amino acids 1 to 43 of the acidic transcriptional activation domain of IE62 can bind recombinant ORF9 protein. Confocal microscopy of transiently transfected cells showed that in the absence of other viral proteins, the ORF9 protein was localized in the cytoplasm while IE62 was localized in the nucleus. In VZV-infected cells, the ORF9 protein was localized to the cytoplasm whereas IE62 exhibited both nuclear and cytoplasmic localization. Cotransfection of plasmids expressing ORF9, IE62, and the viral ORF66 kinase resulted in significant colocalization of ORF9 and IE62 in the cytoplasm. Coimmunoprecipitation experiments with antitubulin antibodies indicate the presence of ORF9-IE62-tubulin complexes in infected cells. Colocalization of ORF9 and tubulin in transfected cells was visualized by confocal microscopy. These data suggest a model for ORF9 protein function involving complex formation with IE62 and possibly other tegument proteins in the cytoplasm at late times in infection.

Amino Acid Sequence↗

PubMed2006

Herpes simplex virus type-1 latency inhibits dendritic growth in sympathetic neurons.

Herpes simplex virus type-1 (HSV-1) initially infects mucoepithelial tissues of the orofacial region, the eye and to a lesser extent the genitalia. Subsequently, the virus is retrogradely transported through the axons of the sensory and sympathetic neurons to their nuclei, where the virus establishes a life-long latent infection. During this latency period, the viral genome is transcriptionally silent except for a single region encoding the latency-associated transcript (LAT). LAT has been shown to affect apoptosis, but little else is known regarding its effects on neurons. To understand how HSV-1 latency might affect dendrites in sympathetic neurons, we transfected primary cultures of sympathetic neurons obtained from rat embryos, with LAT expressing plasmids. LAT inhibited initial dendritic growth and induced dendritic retraction in sympathetic neurons. Latent HSV-1 infection of cultured sympathetic neurons inhibited dendritic growth indicating that this is likely also a consequence of natural infection.

Animals↗

PubMed2006

Promoter activation by the varicella-zoster virus major transactivator IE62 and the cellular transcription factor USF.

The varicella-zoster virus major transactivator, IE62, can activate expression from homologous and heterologous promoters. High levels of IE62-mediated activation appear to involve synergy with cellular transcription factors. The work presented here focuses on functional interactions of IE62 with the ubiquitously expressed cellular factor USF. We have found that USF can synergize with IE62 to a similar extent on model minimal promoters and the complex native ORF28/29 regulatory element, neither of which contains a consensus IE62 binding site. Using Gal4 fusion constructs, we have found that the activation domain of USF1 is necessary and sufficient for synergistic activation with IE62. We have mapped the regions of USF and IE62 required for direct physical interaction. Deletion of the required region within IE62 does not ablate synergistic activation but does influence its efficiency depending on promoter architecture. Both proteins stabilize/increase binding of TATA binding protein/TFIID to promoter elements. These findings suggest a novel mechanism for the observed synergistic activation which requires neither site-specific IE62 binding to the promoter nor a direct physical interaction with USF.

Binding Sites↗

PubMed2005

Cell-type-dependent activation of the cellular EF-1alpha promoter by the varicella-zoster virus IE63 protein.

The varicella-zoster virus (VZV) IE63 protein is abundantly expressed during productive viral infection and is one of six gene products that appear to be expressed during latency. We have found that the IE63 protein can activate expression from the cellular EF-1alpha promoter in the absence of other viral proteins. The VZV IE62 protein, in contrast, was not found to transactivate this promoter. These data indicate that IE63 can function independently of the IE62 protein to positively influence the cellular transcription apparatus. We show that IE63 activation of the EF-1alpha promoter is cell type dependent and have examined the effects of point mutations important for IE63 phosphorylation and virus viability on this activation.

Binding Sites↗

PubMed2004

Combinatorial transcription of herpes simplex virus and varicella zoster virus immediate early genes is strictly determined by the cellular coactivator HCF-1.

The mammalian transcriptional coactivator host cell factor-1 (HCF-1) functions in concert with Oct-1 and VP16 to assemble the herpes simplex virus (HSV) immediate early (IE) transcription enhancer core complexes that mediate the high level transcription of these genes upon infection. Although this transcriptional model has been well characterized in vitro, the requirements and significance of the components have not been addressed. Oct-1 was previously determined to be critical but not essential for HSV IE gene expression. In contrast, RNA interference-mediated depletion of HCF-1 resulted in abrogation of HSV IE gene expression. The HSV IE gene enhancer domain is a model of combinatorial transcription and consists of the core enhancer and multiple binding sites for factors such as Sp1 and GA-binding protein. It was striking that HCF-1 was strictly required for VP16-mediated transcriptional induction via the core enhancer as well as for basal level transcription mediated by GA-binding protein and Sp1. HCF-1 was also found to be essential for the induction of varicella zoster virus IE gene expression by ORF10, the VZV ortholog of the HSV IE transactivator VP16, and the autostimulatory IE62 protein. The critical dependence upon HCF-1 demonstrates that this cellular component is a key factor for control of HSV and VZV IE gene expression by functioning as the common element for distinct factors cooperating at the IE gene enhancers. The requirements for this protein supports the model whereby the regulated transport of HCF-1 from the cytoplasm to the nucleus in sensory neurons may control IE gene expression and reactivation of these viruses from the latent state.

Animals↗

PubMed2004

Complete DNA sequence analyses of the first two varicella-zoster virus glycoprotein E (D150N) mutant viruses found in North America: evolution of genotypes with an accelerated cell spread phenotype.

Varicella-zoster virus (VZV) is considered to be one of the most genetically stable of all the herpesviruses. Yet two VZV strains with a D150N missense mutation within the gE glycoprotein were isolated in North America in 1998 and 2002. The mutant strains have an accelerated cell spread phenotype, which distinguishes them from all wild-type and laboratory viruses. Since the VZV genome contains 70 additional open reading frames (ORFs), the possibility existed that the phenotypic change was actually due to an as-yet-undiscovered mutation or deletion elsewhere in the genome. To exclude this hypothesis, the entire genomes of the two mutant viruses were sequenced and found to contain 124,883 (VZV-MSP) and 125,459 (VZV-BC) nucleotides. Coding single-nucleotide polymorphisms (SNPs) were identified in 14 ORFs. One missense mutation was discovered in gH, but none was found in gB, gI, gL, or gK. There were no coding SNPs in the major regulatory protein ORF 62. One polymorphism was discovered which could never have been anticipated based on current knowledge of herpesvirus genomics, namely, the origins of replication differed from those in the prototype strain but not in a manner expected to affect cell spread. When the two complete mutant VZV sequences were surveyed in their entirety, the most reasonable conclusion was that the increased cell spread phenotype was dependent substantially or solely on the single D150N polymorphism in glycoprotein gE. The genomic results also expanded the evolutionary database by identifying which VZV ORFs were more likely to mutate over time.

Aged↗

PubMed2004

The DNA element controlling expression of the varicella-zoster virus open reading frame 28 and 29 genes consists of two divergent unidirectional promoters which have a common USF site.

The mechanism of the divergent expression of the varicella-zoster virus (VZV) ORF 28 and ORF 29 genes from a common intergenic DNA element, the ORF 28/29 promoter, is of interest based on the observation that both genes are expressed during VZV lytic infection but only the ORF 29 gene is expressed in latently infected neurons. In the work presented here, expression driven by the ORF 28/29 intergenic region was examined. We found that the promoter activity towards the ORF 29 direction is more responsive to activation by the major viral transactivator IE62 than that towards the ORF 28 direction in the context of our experimental system. Analysis of the functional DNA elements involved in IE62 activation of the bidirectional ORF 28/29 regulatory element revealed that in both transfected and VZV-superinfected cells it is a fusion of two unidirectional promoters overlapping an essential USF binding site but with distinct TATA elements. A single TATA element directs expression in the ORF 28 direction, whereas the two TATA elements directing ORF 29 gene expression are alternatively and differentially utilized for transcription initiation. We also identified an Sp1 site localized proximal to the ORF 28 gene which functions as an activator element for expression in both directions. These results indicate that the ORF 28 and ORF 29 genes can be expressed either coordinately or independently and that the observed expression of only the ORF 29 gene during VZV latency may involve neuron-specific cellular factors and/or structural aspects of the latent viral genome.

Base Sequence↗

PubMed2003

Stage-specific translational efficiency and protein stability regulate the developmental expression of p37, an RNA binding protein from Trypanosoma brucei.

We have previously characterized two novel RNA binding proteins, p34 and p37, from Trypanosoma brucei. Their sequences do not show significant homology to other proteins but are highly homologous to one another. The p34 and p37 proteins are developmentally regulated, with p34 the predominant protein in the procyclic stage and p37 nearly exclusively expressed in the bloodstream cells. In vivo metabolic labeling of procyclic cells showed that p34 and p37 were differentially translated, with levels of p34 approximately fourfold higher than p37. The newly synthesized p34 and p37 exhibited differential stability in the procyclic stage. In vitro analysis confirmed this observation and further suggested that this differential stability may be due to a trypsin-like cysteine protease activity in procyclic extracts that selectively degraded the p37 protein. Taken together, these results indicate that the developmental regulation of the T. brucei RNA binding protein, p37, occurs at both translational and post-translational levels.

Animals↗

PubMed2003

Interaction between the varicella zoster virus IE62 major transactivator and cellular transcription factor Sp1.

The varicella zoster virus (VZV) IE62 protein is involved in the activation of expression of all three kinetic classes of VZV proteins. Analysis of the viral promoter for VZV glycoprotein I has shown that the cellular factor Sp1 is involved in or required for the observed IE62 mediated activation. Co-immunoprecipitation experiments show that the two proteins are present in a complex in VZV-infected cells. Protein affinity pull-down assays using recombinant proteins showed that IE62 and Sp1 interact in the absence of any other viral and cellular proteins. Mapping studies using GST-fusion proteins containing truncations of IE62 and Sp1 have delimited the interacting regions to amino acids 612-778 in Sp1 and amino acids 226-299 in IE62. The region identified in Sp1 is involved in DNA-binding, synergistic Sp1 activation, and Sp1 interaction with cellular transcription factors. The interacting region identified in IE62 overlaps with or borders on sites involved in interactions with the VZV IE4 protein and the cellular factors TBP and TFIIB. Assays using wild-type and mutant promoter elements indicate that Sp1 is involved in recruitment of IE62 to the gI promoter and IE62 enhances Sp1 and TBP binding.

DNA↗

PubMed2003

Cellular factors and IE62 activation of VZV promoters.

The varicella zoster virus IE62 protein transactivates promoters representing all three kinetic classes of viral genes. This is accomplished in conjunction with the general transcription machinery of the cell and specific cellular transcription factors that act at sites located within viral promoters. The incidence of a subset of specific recognition sites was examined for the ubiquitous cellular factors Sp1 and USF within the VZV genome. Evidence is also presented for a direct physical interaction between Sp1 and IE62.

Base Sequence↗

PubMed2003

Transcription factor USF, expressed during the entire phase of varicella-zoster virus infection, interacts physically with the major viral transactivator IE62 and plays a significant role in virus replication.

The expression of the genes of varicella-zoster virus (VZV) is regulated by self-encoded viral as well as cellular transcription factors. A potential candidate with an ability to influence the transcription of VZV genes is USF (upstream stimulatory factor), which recognizes the consensus E-box motif. Quantitative RT-PCR and immunoblot assays indicate stable expression of both USF1 and USF2 throughout infection. It was also found that USF binds to a variety of E-boxes (consensus and closely related motifs) within the promoters of ORF 8/9 (two elements), ORF 22 and ORF 67. Co-immunoprecipitation experiments and His-tag protein affinity pull-down assays indicate that a direct physical interaction occurs between USF and the major virus transactivator IE62. To study the general effects of USF in the replication of VZV, a cell line expressing a dominant-negative form of USF (A-USF), which inhibits binding of USF to its recognition sites, was created. A significant decrease in virus replication was detected when this cell line was infected with cell-free virus, indicating that USF is an important cellular factor that regulates the transcription of VZV genes.

Animals↗

PubMed2002

Physical and functional interaction between the varicella zoster virus IE63 and IE62 proteins.

The varicella zoster virus (VZV) IE63 protein is required for growth of the virus in cell culture and is expressed during both lytic and latent phases of VZV infection. We have investigated the physical and functional interaction of this protein with the major VZV transactivating protein IE62. The region of the IE63 protein required for interaction with the IE62 protein has been identified and encompasses the N-terminal 142 amino acids. We have found that the interaction is stable at physiological ionic strength. We have also shown that a portion of the IE63 and IE62 proteins colocalize in VZV-infected cells at both 15 and 48 h postinfection. IE63 was found to have no transcriptional activating or repressing activity within the context of a minimal VZV glycoprotein promoter. The presence of the IE63, however, upmodulated the IE62 transactivation of this promoter. Finally, we show that the IE63 protein can be coimmunoprecipitated with the cellular RNA polymerase II from infected cell extracts, indicating that it is present in a complex with that enzyme.

Binding Sites↗

PubMed2002

Two novel RNA binding proteins from Trypanosoma brucei are associated with 5S rRNA.

We have previously reported the identification of two closely related RNA binding proteins from Trypanosoma brucei which we have termed p34 and p37. The predicted primary structures of the two proteins are highly homologous with one major difference, an 18-amino-acid insert in the N-terminal region of p37. These two proteins have been localized to the nucleus based on immunofluorescence microscopy. To gain insight into their function, we have utilized UV crosslinking, coimmunoprecipitation, and sucrose density gradients to identify T. brucei RNA species that associate with p34 and p37. These experiments have demonstrated a specific interaction of both p34 and p37 with the 5S ribosomal RNA and indicate that other RNA species are unlikely to be specifically bound. This suggests a role for p34 and p37 in the import and/or assembly pathway of T. brucei 5S rRNA in ribosome biogenesis.

Animals↗

PubMed2002

Two families of RNA binding proteins from Trypanosoma brucei associate in a direct protein-protein interaction.

We have previously reported the identification of two closely related RNA binding proteins from Trypanosoma brucei, termed p34 and p37. The predicted primary structures of the two proteins are highly homologous with one major difference, an 18 amino acid insertion in the N-terminal region of p37. These two proteins are localized to the nucleus based on immunofluorescence microscopy. Recently, we have shown that p34 and p37 interact with T. brucei 5S rRNA. In order to gain further insight into their function, we have utilized protein affinity chromatography and immune capture approaches to identify T. brucei proteins which associate with p34 and p37. We demonstrate here an interaction of both p34 and p37 with the NOPP44/46 proteins, identified in T. brucei as a family of tyrosine-phosphorylated RNA binding proteins primarily localized to the nucleolus. This interaction was mapped to the RNA-binding region of p34/p37 and an acidic region of NOPP44/46 by protein affinity chromatography using recombinant deletion constructs of p34 and p37 and yeast two-hybrid analysis. These data may suggest a role for p34 and p37 and NOPP44/46 in the import and/or assembly pathway of T. brucei 5S rRNA in ribosome biogenesis.

Animals↗

PubMed2002

Memory cytotoxic T cell responses to viral tegument and regulatory proteins encoded by open reading frames 4, 10, 29, and 62 of varicella-zoster virus.

Cytotoxic T cell recognition of tegument and regulatory proteins encoded by open reading frames (ORFs) 4, 10, 29, and 62 of varicella-zoster virus (VZV) was evaluated using limiting dilution conditions to estimate the precursor frequencies of memory T cells specific for these proteins in immune subjects. Responder cell frequencies for ORFs 4, 10, and 62 gene products, which are virion tegument components and function as immediate early viral transactivating proteins, were equivalent. CTLp recognition of VZV proteins made in latently infected cells, which include ORF4 and ORF62 proteins, was not maintained preferentially when compared to ORF10 protein, which has not been shown to be expressed during latency. T cell recognition of ORF29 protein, the major DNA binding protein, which is expressed during replication but not incorporated into the virion tegument, was less common than responses to ORFs 4, 10, and 62 gene products. Older individuals had diminished numbers of memory CTLp that lysed autologous targets expressing IE62 protein; these responses were increased after immunization with live attenuated varicella vaccine to the range observed in younger adults. Adaptive immunity to VZV is characterized by a broad repertoire of memory CTL responses to proteins that comprise the virion tegument and regulate viral gene expression in infected cells.

Adult↗