Ann E Tollefson

Publications (34)129.2 Total impact

• Article: Identification of a previously unrecognized promoter that drives expression of the UXP transcription unit in the human adenovirus type 5 genome.

  Baoling Ying, Ann E Tollefson, William S M Wold
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  ABSTRACT: We previously identified an adenovirus (Ad) protein named U exon protein (UXP) encoded by a leftward-strand (l-strand) transcription unit. Here we identify and characterize the UXP promoter. Primer extension and RNase protection assays mapped the transcription initiation site at 32 nucleotides upstream of the UXP gene initiation codon. A series of viral mutants with mutations at two putative inverted CCAAT (I-CCAAT) boxes and two E2F sites were generated. With mutants lacking the proximal I-CCAAT box, the UXP mRNA level decreased significantly to 30% of the Ad type 5 (Ad5) mRNA level as measured by quantitative reverse transcription-PCR. Decreased UXP was also observed by immunoblotting and immunofluorescence. UXP mRNA and protein levels were similar to those of Ad5 for mutants lacking the distal I-CCAAT box or both putative E2F sites. Ad DNA levels were similar in mutant- and wild-type Ad5-infected cells during the late stage of infection, strongly suggesting that the decreased UXP mRNA and protein from mutants lacking the proximal I-CCAAT box was due to decreased promoter activity. Electrophoretic mobility shift assays (EMSA) indicated that a cellular factor binds specifically to the proximal I-CCAAT box of the UXP promoter. An in vitro luciferase reporter assay demonstrated that basal promoter activity lies between bp -158 and +30 of the transcription initiation site. No E1A-mediated promoter transactivation was observed in 293 cells compared with A549 cells. Thus, we propose that there is a previously unidentified Ad5 promoter that drives expression of the UXP transcription unit. This promoter is embedded within the gene for fiber, and it contains a proximal I-CCAAT box critical for UXP mRNA transcription.
  Journal of Virology 11/2010; 84(21):11470-8. · 5.40 Impact Factor
• Article: Functions and mechanisms of action of the adenovirus E3 proteins.

  Drew L Lichtenstein, Karoly Toth, Konstantin Doronin, Ann E Tollefson, William S M Wold
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  ABSTRACT: In the evolutionary battle between viruses and their hosts, viruses have armed themselves with weapons to defeat the host's attacks on infected cells. Various proteins encoded in the adenovirus (Ad) E3 transcription unit protect cells from killing mediated by cytotoxic T cells and death-inducing cytokines such as tumor necrosis factor (TNF), Fas ligand, and TNF-related apoptosis-inducing ligand (TRAIL). The viral protein E3-gp19 K blocks MHC class-I-restricted antigen presentation, which diminishes killing by cytotoxic T cells. The receptor internalization and degradation (RID) complex (formerly E3-10.4 K/14.5 K) stimulates the clearance from the cell surface and subsequent degradation of the receptors for Fas ligand and TRAIL, thereby preventing the action of these important immune mediators. RID also downmodulates the epidermal growth factor receptor (EGFR), although what role, if any, this function has in immune regulation is uncertain. In addition, RID antagonizes TNF-mediated apoptosis and inflammation through a mechanism that does not primarily involve receptor downregulation. E3-6.7 K functions together with RID in downregulating some TRAIL receptors and may block apoptosis independently of other E3 proteins. Furthermore, E3-14.7 K functions as a general inhibitor of TNF-mediated apoptosis and blocks TRAIL-induced apoptosis. Finally, after expending great effort to maintain cell viability during the early part of the virus replication cycle, Ads lyse the cell to allow efficient virus release and dissemination. To perform this task subgroup C Ads synthesize a protein late in infection named ADP (formerly E3-11.6 K) that is required for efficient virus release. This review focuses on recent experiments aimed at discovering the mechanism of action of these critically important viral proteins.
  International Reviews Of Immunology 08/2009; 23(1-2):75-111. · 3.43 Impact Factor
• Chapter: Construction of Mutations in the Adenovirus Early Region 3 (E3) Transcription Units

  Terry W. Hermiston, Ann E. Tollefson, William S. M. Wold
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  ABSTRACT: The E3 transcription unit of the well-studied subgroup C adenoviruses (Ad) (prototypic serotypes 2 [Ad2] and 5 [Ad5]) is located between map units 76–86 (see Fig. 1). The E3 region is surrounded by the genes for virion protein VIII and fiber, and is transcribed off the r-strand. The E3-region transcription unit is complex Extensive splicing controls expression of seven identified E3 proteins, four of which have functions that modify the host immune response to the viral infection (reviewed in ref. 1), Table 1) Fig 1Methods used to insert a mutated E3 region into the viral genome (A) Schematic illustrating some of the adenovirus transcription units expressed during early stages of infection (B) Construction of E3 mutations Ligation method Cleave cloned Ad2 EcoRI-D fragment containing a mutation with EcoRI, then ligate to EcoRI-cleaved Ad5 TP-DNA complex. Transfect ligation mixture into A549 cells and allow plaques to form. Overlap recombination method. Cotransfect cloned Ad2 KpnI-A fragment containing a mutation in the E3 region with EcoRI-cleaved Ad5 TP-DNA complex into A549 cells and plaques were allowed to form Table 1 Location of the E3 Genes Within the Ad5 and Ad2 Genomic Sequence Coding sequence Ad5 (2) Ad2 (3) E3 protein start stop start stop Function 12.5 K 27,858 28,179 27,919 28,220 Unknown 67K 28,547 28,736 28,630 28,819 Unknown gp 19 K 28,735 29,215 28,812 29,289 Inhibits Ad-specific cytotoxic T-lymphocyte recognition and killing of the virally infected cell by binding MHC class I molecules and inhibiting their transport to the cell surface 11.6K(ADP) a 29,491 29,770 29,468 29,771 Required for efficient cell lysis and release of adenovirus from infected cells (4,5) 10.4 K (RID α) b 29,784 30,057 29,781 30,054 10.4 K and 14.5 K proteins form a complex that. Prevents TNF cytolysis Clears epidermal growth factor and insulin receptors from the infected cell surface Clears Fas antigen from the infected cell surface (6) 14.5 K (RIDβ) b 30,062 30,458 30,059 30,449 14.7 K 30,453 30,837 30,444 30,828 Inhibits TNF-mduced apoptosis GTPase binding protein (7) a ADP, adenovirus death protein b RID, receptor internalization and degradation complex, made up of 10.4-K (RIDα) and 14 5-K (RIDβ) proteins
  01/2008: pages 11-24;
• Chapter: Preparation and Titration of CsCl-Banded Adenovirus Stock

  Ann E. Tollefson, Terry W. Hermiston, William S. M. Wold
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  ABSTRACT: An important step m the development of modern experimental virology was the development of the plaque assay, first with bacteriophage, then with eukaryotic viruses. In order to obtain quantitative, interpretable, and reproducible results, it is necessary to know how much virus is being used in the experiment. With adenoviruses, several approaches have generally been used to quantitate virus stocks. First, virus particles are counted, e.g., in an electron microscope (1,2). Another approach is to quantitate virion DNA by optical absorbance (2). The problem with these approaches is that many adenovirus particles are not infectious, perhaps because they have a defective complete genome or they lack fiber or some other protein. The second approach is to determine the number of plaque-forming units (PFU) per mL. Here, the analysis quantitates the number of virions capable of a full infectious cycle. This approach will be described in detail in this article.
  01/2008: pages 1-9;
• Article: Identification of a new human adenovirus protein encoded by a novel late l-strand transcription unit.

  Ann E Tollefson, Baoling Ying, Konstantin Doronin, Peter D Sidor, William S M Wold
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  ABSTRACT: A short open reading frame named the "U exon," located on the adenovirus (Ad) l-strand (for leftward transcription) between the early E3 region and the fiber gene, is conserved in mastadenoviruses. We have observed that Ad5 mutants with large deletions in E3 that infringe on the U exon display a mild growth defect, as well as an aberrant Ad E2 DNA-binding protein (DBP) intranuclear localization pattern and an apparent failure to organize replication centers during late infection. Mutants in which the U exon DNA is reconstructed have a reversed phenotype. Chow et al. (L. T. Chow et al., J. Mol. Biol. 134:265-303, 1979) described mRNAs initiating in the region of the U exon and spliced to downstream sequences in the late DBP mRNA leader and the DBP-coding region. We have cloned this mRNA (as cDNA) from Ad5 late mRNA; the predicted protein is 217 amino acids, initiating in the U exon and continuing in frame in the DBP leader and in the DBP-coding region but in a different reading frame from DBP. Polyclonal and monoclonal antibodies generated against the predicted U exon protein (UXP) showed that UXP is approximately 24K in size by immunoblot and is a late protein. At 18 to 24 h postinfection, UXP is strongly associated with nucleoli and is found throughout the nucleus; later, UXP is associated with the periphery of replication centers, suggesting a function relevant to Ad DNA replication or RNA transcription. UXP is expressed by all four species C Ads. When expressed in transient transfections, UXP complements the aberrant DBP localization pattern of UXP-negative Ad5 mutants. Our data indicate that UXP is a previously unrecognized protein derived from a novel late l-strand transcription unit.
  Journal of Virology 01/2008; 81(23):12918-26. · 5.40 Impact Factor
• Article: Preparation and titration of CsCl-banded adenovirus stocks.

  Ann E Tollefson, Mohan Kuppuswamy, Elena V Shashkova, Konstantin Doronin, William S M Wold
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  ABSTRACT: Adenovirus research often requires purified high-titer virus stocks and accurate virus titers for use in experiments. Accurate titers are important for quantitative, interpretable, and reproducible results. This is especially true when there are comparisons of different mutant viruses following infection. This chapter details the large-scale preparation of adenovirus (either replication-competent or replication-defective) in spinner cultures (e.g., KB, HeLa, or 293 cells). Protocols for harvesting cells and isolation of adenovirus by CsCl banding are presented. Methods for titering adenovirus by plaque assay are presented along with a discussion of how plaque assays can be used to determine the kinetics of cell killing and cytolysis by adenoviruses.
  Methods in molecular medicine 01/2007; 130:223-35.
• Article: 642. Toxicological Findings with Oncolytic Adenovirus Vector VRX-007, Wild-Type Ad5, and a Replication-Defective Adenovirus Vector in Syrian Hamsters and C57BL/6 Mice|[ast]|

  Karoly Toth, Jacqueline F. Spencer, Drew L. Lichtenstein, Ann E. Tollefson, Debabrata Patra, Jennifer M. Meyer, Elena Shashkova, Mohan Kuppuswamy, Konstantin Doronin, Debanjan Dhar, Maria A. Thomas, Louis A. Zumstein, William S.M. Wold
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  ABSTRACT: Molecular Therapy (2006) 13, S247|[ndash]|S247; doi: 10.1016/j.ymthe.2006.08.718 642. Toxicological Findings with Oncolytic Adenovirus Vector VRX-007, Wild-Type Ad5, and a Replication-Defective Adenovirus Vector in Syrian Hamsters and C57BL/6 Mice|[ast]| Karoly Toth1, Jacqueline F. Spencer1, Drew L. Lichtenstein2, Ann E. Tollefson1, Debabrata Patra2, Jennifer M. Meyer2, Elena Shashkova2, Mohan Kuppuswamy1, Konstantin Doronin1, Debanjan Dhar1, Maria A. Thomas1, Louis A. Zumstein3 and William S.M. Wold1,21Molecular Microbiology and Immunology, Saint Louis University School of Medicine, St. Louis, MO2VirRx, Inc., St. Louis, MO3Introgen Therapeutics, Inc., Houston, TX|[ast]|A portion of this research was funded by a biotechnology company named VirRx, Inc. KT, AET, KD, and WSMW have equity in VirRx.
  Molecular Therapy 04/2006; · 6.87 Impact Factor
• Article: 651. VRX-007, an Oncolytic Adenovirus Vector, Replicates in Syrian Hamsters but Not Mice: Comparison of Biodistribution Studies Performed in the Syrian Hamster and Mouse

  Drew L. Lichtenstein, Karoly Toth, Jaqueline F. Spencer, Jenifer Meyer, Baoling Ying, Ann E. Tollefson, Debabrata Patra, Elena Shashkova, Mohan Kuppuswamy, Konstantin Doronin, Debanjan Dhar, Louis A. Zumstein, William S.M. Wold
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  ABSTRACT: Molecular Therapy (2006) 13, S251|[ndash]|S251; doi: 10.1016/j.ymthe.2006.08.727 651. VRX-007, an Oncolytic Adenovirus Vector, Replicates in Syrian Hamsters but Not Mice: Comparison of Biodistribution Studies Performed in the Syrian Hamster and Mouse Drew L. Lichtenstein1, Karoly Toth2,|[ast]|, Jaqueline F. Spencer2, Jenifer Meyer1, Baoling Ying1, Ann E. Tollefson2,|[ast]|, Debabrata Patra1, Elena Shashkova1, Mohan Kuppuswamy2,|[ast]|, Konstantin Doronin2,|[ast]|, Debanjan Dhar2, Louis A. Zumstein3 and William S.M. Wold2,|[ast]|1Pre-clinical Vector Development, VirRx Inc., St. Louis, MO2Molecular Microbiology and Immunology, Saint Louis University, St. Louis, MO3Research Development, Introgen Therapeutics Inc., Houston, TX|[ast]|AET, KD, KT, MK, and WSMW have equity in VirRx Inc.
  Molecular Therapy 04/2006; · 6.87 Impact Factor
• Article: Syrian hamster as a permissive immunocompetent animal model for the study of oncolytic adenovirus vectors.

  Maria A Thomas, Jacqueline F Spencer, Marie C La Regina, Debanjan Dhar, Ann E Tollefson, Karoly Toth, William S M Wold
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  ABSTRACT: Oncolytic adenoviruses represent an innovative approach to cancer therapy. These vectors are typically evaluated in immunodeficient mice with human xenograft tumors. However, in addition to being immunodeficient, this model is limited because normal and cancerous mouse tissues are poorly permissive for human adenovirus replication. This prompted us to search for a model that more accurately reflects the use of oncolytic adenoviruses in cancer patients. To this end, we developed a novel Syrian hamster model that is both immunocompetent and replication-permissive. We found that human adenovirus replicates well in Syrian hamster cell lines and confirmed replication in the lungs. Oncolytic adenovirus injection showed efficacy in three different hamster tumor models. Furthermore, i.t. oncolytic adenovirus injection resulted in suppression of primary and metastatic lesions, i.t. replication and necrosis, vector entrance into the bloodstream, replication in the liver and lungs, and anti-adenovirus antibody induction. Our findings show that the Syrian hamster is a promising immunocompetent model that is permissive to human adenovirus replication in tumors as well as normal tissues. Therefore, the Syrian hamster model may become a valuable tool for the field of oncolytic adenovirus vectors in which vector safety and efficacy can be evaluated.
  Cancer Research 03/2006; 66(3):1270-6. · 7.86 Impact Factor
• Article: Ceramide regulates SR protein phosphorylation during adenoviral infection.

  Souha S Kanj, Nadine Dandashi, Aimee El-Hed, Hisham Harik, Maria Maalouf, Lina Kozhaya, Talal Mousallem, Ann E Tollefson, William S Wold, Charles E Chalfant, Ghassan S Dbaibo
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  ABSTRACT: In this study, we show that adenoviral infection induced accumulation of the sphingolipid ceramide in a dose- and time-dependent manner. This accumulation preceded cell lysis, occurred in the absence of biochemical evidence of apoptosis, and was derived from de novo synthesis of ceramide. An adenovirus mutant that lacks the adenovirus death protein (ADP) produced ceramide accumulation in the absence of cell lysis. This suggested that ceramide accumulation was either driven by adenovirus or was a cellular stress response but was unlikely a result of cell death. The use of inhibitors of ceramide synthesis resulted in a significant delay in cell lysis, suggesting that ceramide was necessary for the lytic phase of the infection. Serine/arginine-rich (SR) proteins were dephosphorylated during the late phase of the viral cycle, and inhibitors of ceramide synthesis reversed this. These findings suggest that adenovirus utilizes the ceramide pathway to regulate SR proteins during infection.
  Virology 03/2006; 345(1):280-9. · 3.35 Impact Factor
• Article: 1021. Development of a Syrian Hamster Animal Model To Study Replication Competent Adenovirus Vectors for Cancer Gene Therapy

  Maria A. Thomas, Jacqueline F. Spencer, Karoly Toth, Marie C. LaRegina, Ann E. Tollefson, Baoling Ying, Mohan Kuppuswamy, Jennifer M. Meyer, Drew L. Lichenstein, Louis A. Zumstein, William S.M. Wold
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  ABSTRACT: Molecular Therapy (2005) 11, S394|[ndash]|S395; doi: 10.1016/j.ymthe.2005.07.568 1021. Development of a Syrian Hamster Animal Model To Study Replication Competent Adenovirus Vectors for Cancer Gene Therapy Maria A. Thomas1, Jacqueline F. Spencer1, Karoly Toth1, Marie C. LaRegina2, Ann E. Tollefson1, Baoling Ying3, Mohan Kuppuswamy1, Jennifer M. Meyer3, Drew L. Lichenstein3, Louis A. Zumstein4 and William S.M. Wold1,|[ast]|1Molecular Microbiology and Immunology, Saint Louis University, St. Louis, MO2Division of Comparative Medicine, Washington University School of Medicine, St. Louis, MO3VirRx, Inc., St. Louis, MO4Introgen Therapeutics, Inc., Houston, TX|[ast]|William S. M. Wold is a founder of VirRx, Inc., and he is a shareholder in the company.
  Molecular Therapy 04/2005; · 6.87 Impact Factor
• Article: 1023. Determination of the Maximum Tolerated Dose of the Replication-Competent Adenovirus Vector VRX-007 Following Intravenous Administration in Syrian Hamsters

  Maria A. Thomas, Jacqueline F. Spencer, Jennifer M. Meyer, Marie C. LaRegina, Karoly Toth, Drew L. Lichtenstein, Ann E. Tollefson, Mohan Kuppuswamy, Baoling Ying, Louis A. Zumstein, William S.M. Wold
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  ABSTRACT: Molecular Therapy (2005) 11, S395|[ndash]|S395; doi: 10.1016/j.ymthe.2005.07.570 1023. Determination of the Maximum Tolerated Dose of the Replication-Competent Adenovirus Vector VRX-007 Following Intravenous Administration in Syrian Hamsters Maria A. Thomas1, Jacqueline F. Spencer1, Jennifer M. Meyer2, Marie C. LaRegina3, Karoly Toth1, Drew L. Lichtenstein2, Ann E. Tollefson1, Mohan Kuppuswamy1, Baoling Ying2, Louis A. Zumstein4 and William S.M. Wold1,|[ast]|1Molecular Microbiology & Immunology, Saint Louis University School of Medicine, St. Louis, MO2VirRx, Inc., St. Louis, MO3Division of Comparative Medicine, Washington University School of Medicine, St. Louis, MO4Introgen Therapeutics, Inc., Houston, TX|[ast]|William S. M. Wold is a founder of VirRx, Inc., and he is a shareholder in the company.
  Molecular Therapy 04/2005; · 6.87 Impact Factor
• Article: 1022. Single-Dose Intravenous Toxicity Study with the Oncolytic Adenovirus Vector VRX-007, Adenovirus Type 5, and a Replication-Defective Adenovirus in Syrian Hamsters

  Karoly Toth, Jacqueline F. Spencer, Drew L. Lichtenstein, Ann E. Tollefson, Jennifer M. Meyer, Maria A. Thomas, Mohan Kuppuswamy, Baoling Ying, Louis A. Zumstein, William S.M. Wold
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  ABSTRACT: Molecular Therapy (2005) 11, S395|[ndash]|S395; doi: 10.1016/j.ymthe.2005.07.569 1022. Single-Dose Intravenous Toxicity Study with the Oncolytic Adenovirus Vector VRX-007, Adenovirus Type 5, and a Replication-Defective Adenovirus in Syrian Hamsters Karoly Toth1, Jacqueline F. Spencer1, Drew L. Lichtenstein2, Ann E. Tollefson1, Jennifer M. Meyer2, Maria A. Thomas1, Mohan Kuppuswamy1, Baoling Ying2, Louis A. Zumstein3 and William S.M. Wold1,|[ast]|1Molecular Microbiology & Immunology, Saint Louis University School of Medicine, St. Louis, MO2VirRx, Inc., St. Louis, MO3Introgen Therapeutics, Inc., Houston, TX|[ast]|William S. M. Wold is a founder of VirRx, Inc., and he is a shareholder in the company.
  Molecular Therapy 04/2005; · 6.87 Impact Factor
• Article: Adenovirus immunoregulatory E3 proteins prolong transplants of human cells in immunocompetent mice.

  Karoly Toth, Konstantin Doronin, Mohan Kuppuswamy, Peter Ward, Ann E Tollefson, William S M Wold
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  ABSTRACT: The majority of proteins encoded in the early 3 (E3) region of human subgroup C adenoviruses function to modulate the host immune response. For example, gp19K, one of these E3 proteins, prevents the major histocompatibility complex type I (MHC-I) from presenting viral antigens on the surface of the infected cell. Other E3 proteins, such as the RID and 14.7K proteins, counteract the effector phase of the cellular immune response. In order to study further the effects of these proteins, we constructed an E1-/E3- adenovirus vector, Ad/E3, that contains all the E3 genes with the exception of the cytolytic adp gene, inserted into the deleted E1 region. The transcription of the E3 genes in this vector is driven by a CMV promoter in place of the native E3 promoter. Ad/E3 expressed close to wild-type adenovirus levels of all E3 proteins, and these proteins appear to function normally in cell culture. For example, in Ad/E3-infected cells, surface expression of MHC-I was down-regulated, as was cell surface display of death receptors Fas and TRAIL Receptor 1. A human cell line of lung origin (A549), which was rapidly rejected after transplantation into C57BL/6 mice, was protected for an extended time from the host immune response after infection with an Ad/E3, and went through a number of divisions in immunocompetent mice. These latter results indicate that the E3 proteins protect cells from destruction by the immune system.
  Virus Research 04/2005; 108(1-2):149-59. · 2.94 Impact Factor
• Article: Cotton rat tumor model for the evaluation of oncolytic adenoviruses.

  Karoly Toth, Jacqueline F Spencer, Ann E Tollefson, Mohan Kuppuswamy, Konstantin Doronin, Drew L Lichtenstein, Marie C La Regina, Gregory A Prince, William S M Wold
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  ABSTRACT: Oncolytic human adenovirus (Ad) vectors exert their antitumor effect by replicating in and lysing tumor cells. These vectors are commonly evaluated in immunodeficient mice bearing human tumor xenografts. However, this model suffers because the mice are immunodeficient and are not permissive for human Ads. We have developed a cotton rat model to test the selectivity, immunogenicity, and efficacy of oncolytic Ad vectors. The cotton rat is a rodent species that is semipermissive for human Ads. We show that the cotton cancer rat cell line LCRT supports the replication of human Ad in tissue culture and that the cells are destroyed on virus replication. When injected subcutaneously, LCRT cells formed tumors in immunocompetent cotton rats, and the growth of these tumors was delayed by the injection of an oncolytic Ad vector. Replication of the Ad vector in the tumor was demonstrated by sampling tumor tissue and isolating infectious virus particles at various times after intratumoral injection of the virus. We propose that the cotton rat can be used as an animal model to evaluate oncolytic Ad vectors.
  Human Gene Therapy 02/2005; 16(1):139-46. · 4.22 Impact Factor
• Article: Adenovirus E3-6.7K protein is required in conjunction with the E3-RID protein complex for the internalization and degradation of TRAIL receptor 2.

  Drew L Lichtenstein, Konstantin Doronin, Karoly Toth, Mohan Kuppuswamy, William S M Wold, Ann E Tollefson
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  ABSTRACT: Adenoviruses (Ads) encode several proteins within the early region 3 (E3) transcription unit that help protect infected cells from elimination by the immune system. Among these immunomodulatory proteins, the receptor internalization and degradation (RID) protein complex, which is composed of the RIDalpha (formerly E3-10.4K) and RIDbeta (formerly E3-14.5K) subunits, stimulates the internalization and degradation of certain members of the tumor necrosis factor (TNF) receptor superfamily, thus blocking apoptosis initiated by Fas and TNF-related apoptosis-inducing ligand (TRAIL). The experiments reported here show that TRAIL receptor 2 (TR2) is cleared from the cell surface in Ad-infected cells. Virus mutants containing deletions that span E3 were used to show that the RID and E3-6.7K proteins are both necessary for the internalization and degradation of TR2, whereas only the RID protein is required for TRAIL receptor 1 downregulation. In addition, replication-defective Ad vectors that express individual E3 proteins were used to establish that the RID and E3-6.7K proteins are sufficient to clear TR2. These data demonstrate that E3-6.7K is an important component of the antiapoptosis arsenal encoded by the E3 transcription unit of subgroup C Ads.
  Journal of Virology 12/2004; 78(22):12297-307. · 5.40 Impact Factor
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  Article: An oncolytic adenovirus vector combining enhanced cell-to-cell spreading, mediated by the ADP cytolytic protein, with selective replication in cancer cells with deregulated wnt signaling.

  Karoly Toth, Hakim Djeha, Baoling Ying, Ann E Tollefson, Mohan Kuppuswamy, Konstantin Doronin, Peter Krajcsi, Kai Lipinski, Christopher J Wrighton, William S M Wold
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  ABSTRACT: We have constructed a novel oncolytic adenovirus (Ad) vector named VRX-009 that combines enhanced cell spread with tumor-specific replication. Enhanced spread, which could significantly increase antitumor efficacy, is mediated by overexpression of the Ad cytolytic protein named ADP (also known as E3-11.6K). Replication of VRX-009 is restricted to cells with a deregulated wnt signal transduction pathway by replacement of the wild-type Ad E4 promoter with a synthetic promoter consisting of five consensus binding sites for the T-cell factor transcription factor. Tumor-selective replication is indicated by several lines of evidence. VRX-009 expresses E4ORF3, a representative Ad E4 protein, only in colon cancer cell lines. Furthermore, VRX-009 replicates preferentially in colon cancer cell lines as evidenced by virus productivity 2 orders of magnitude higher in SW480 colon cancer cells than in A549 lung cancer cells. Replication in primary human bronchial epithelial cells and human umbilical vein endothelial cells was also significantly lower than in SW480 cells. When tested in human tumor xenografts in nude mice, VRX-009 effectively suppressed the growth of SW480 colon tumors but not of A549 lung tumors. VRX-009 may provide greater level of antitumor efficacy than standard oncolytic Ad vectors in tumors in which a defect in wnt signaling increases the level of nuclear beta-catenin.
  Cancer Research 06/2004; 64(10):3638-44. · 7.86 Impact Factor
• Article: 1018. Immune-Competent Cotton Rat Animal Model for Evaluation of Oncolytic Adenoviruses

  Karoly Toth, Jacqueline F. Spencer, Ann E. Tollefson, Mohan Kuppuswamy, Konstantin Doronin, Drew L. Lichtenstein, William S. M. Wold
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  ABSTRACT: Molecular Therapy (2004) 9, S390–S390; doi: 10.1016/j.ymthe.2004.06.963 1018. Immune-Competent Cotton Rat Animal Model for Evaluation of Oncolytic Adenoviruses Karoly Toth1, Jacqueline F. Spencer1, Ann E. Tollefson1, Mohan Kuppuswamy1, Konstantin Doronin1, Drew L. Lichtenstein2 and William S. M. Wold1,21Molecular Microbiology & Immunology, Saint Louis University School of Medicine, St. Louis, MO2VirRx, Inc., St. Louis, MO
  Molecular Therapy 04/2004; · 6.87 Impact Factor
• Article: Distinct domains in the adenovirus E3 RIDalpha protein are required for degradation of Fas and the epidermal growth factor receptor.

  Tom A Zanardi, Soonpin Yei, Drew L Lichtenstein, Ann E Tollefson, William S M Wold
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  ABSTRACT: Adenovirus (Ad) types 2 and 5 encode at least five proteins within the E3 transcription unit that help the virus evade the immune system. Two such proteins, RIDalpha (formerly E3-10.4K) and RIDbeta (formerly E3-14.5K), form the RID (receptor internalization and degradation) complex (formerly E3-10.4K/14.5K). RID mediates clearance from the cell surface and lysosomal degradation of a number of important members in the tumor necrosis factor receptor (TNFR) superfamily and the receptor tyrosine kinase receptor family. Affected receptors include Fas, TRAIL (TNF-related apoptosis-inducing ligand) receptor 1 (TR1), TR2, and epidermal growth factor receptor (EGFR). Degradation of Fas and TRAIL receptors protects Ad-infected cells from apoptosis. To investigate the mechanism of action of RIDalpha, 14 mutant RIDalpha proteins, each containing a three- to five-amino-acid deletion, were constructed and then expressed from the E3 region of a replication-competent recombinant Ad in the same context as wild-type RIDalpha. Each mutant protein was characterized with regard to five physical properties associated with wild-type RIDalpha, namely, protein stability, proteolytic cleavage, insertion into the membrane, complex formation with RIDbeta, and transport to the cell surface. Additionally, the mutant proteins were tested for their ability to mediate internalization and degradation of EGFR and Fas and to protect cells from Fas-mediated apoptosis. The majority of mutant RIDalpha proteins (8 out of 14) were physically similar to wild-type RIDalpha. With regard to functional characteristics, the cytoplasmic domain of RIDalpha is largely unimportant for receptor internalization and degradation and the extracellular domain of RIDalpha is important for down-regulation of EGFR but not Fas.
  Journal of Virology 12/2003; 77(21):11685-96. · 5.40 Impact Factor
• Article: Mutations within the ADP (E3-11.6K) protein alter processing and localization of ADP and the kinetics of cell lysis of adenovirus-infected cells.

  Ann E Tollefson, Abraham Scaria, Baoling Ying, William S M Wold
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  ABSTRACT: ADP (also known as E3-11.6K protein) is synthesized abundantly in late adenovirus infection and is required for efficient lysis of infected cells and release of viral progeny at the end of the viral replication cycle. ADP is a type III bitopic N(endo)C(exo) nuclear membrane and Golgi glycoprotein that is produced at high levels in late adenovirus infection (>24 h postinfection). We show pulse-chase and other studies indicating that ADP undergoes a complex process of N- and O-linked glycosylation and proteolytic cleavage. In order to further characterize ADP, a series of 23 deletion and point mutations has been constructed in the adenovirus serotype 2 adp gene and then built into a wild-type adenovirus background. These mutants were analyzed for processing and intracellular localization of ADP. Mutation of the single predicted N glycosylation site eliminated N glycosylation. Deletion of a region in ADP rich in serine and threonine residues reduced O glycosylation. In general, mutations within the lumenal domain of ADP resulted in lower protein stability; immunofluorescence assays indicated that these ADPs were primarily present in the Golgi apparatus. Viruses with mutations within the cytoplasmic-nucleoplasmic domain of ADP showed normal glycosylation patterns and protein abundance for ADP, but the protein was often found throughout cellular membranes rather than being localized specifically to the nuclear membrane and Golgi apparatus. The ADP virus mutants were analyzed by cell viability assays to determine the kinetics of cell lysis following infection of human A549 cells. In general, viruses with mutations within the lumenal domain of ADP display greatly reduced efficiencies of cell lysis. Viruses with large deletions in the cytoplasmic-nucleoplasmic domain of ADP retain much of their ability to lyse infected cells.
  Journal of Virology 07/2003; 77(14):7764-78. · 5.40 Impact Factor