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Sample records for adenovirus e1a oncoprotein

  1. The adaptor protein DCAF7 mediates the interaction of the adenovirus E1A oncoprotein with the protein kinases DYRK1A and HIPK2

    PubMed Central

    Glenewinkel, Florian; Cohen, Michael J.; King, Cason R.; Kaspar, Sophie; Bamberg-Lemper, Simone; Mymryk, Joe S.; Becker, Walter

    2016-01-01

    DYRK1A is a constitutively active protein kinase that has a critical role in growth and development which functions by regulating cell proliferation, differentiation and survival. DCAF7 (also termed WDR68 or HAN11) is a cellular binding partner of DYRK1A and also regulates signalling by the protein kinase HIPK2. DCAF7 is an evolutionarily conserved protein with a single WD40 repeat domain and has no catalytic activity. We have defined a DCAF7 binding motif of 12 amino acids in the N-terminal domain of class 1 DYRKs that is functionally conserved in DYRK1 orthologs from Xenopus, Danio rerio and the slime mold Dictyostelium discoideum. A similar sequence was essential for DCAF7 binding to HIPK2, whereas the closely related HIPK1 family member did not bind DCAF7. Immunoprecipitation and pulldown experiments identified DCAF7 as an adaptor for the association of the adenovirus E1A protein with DYRK1A and HIPK2. Furthermore, DCAF7 was required for the hyperphosphorylation of E1A in DYRK1A or HIPK2 overexpressing cells. Our results characterize DCAF7 as a substrate recruiting subunit of DYRK1A and HIPK2 and suggest that it is required for the negative effect of DYRK1A on E1A-induced oncogenic transformation. PMID:27307198

  2. Adenovirus E1A specifically blocks SWI/SNF-dependent transcriptional activation.

    PubMed Central

    Miller, M E; Cairns, B R; Levinson, R S; Yamamoto, K R; Engel, D A; Smith, M M

    1996-01-01

    Expression of the adenovirus E1A243 oncoprotein in Saccharomyces cerevisiae produces a slow-growth phenotype with accumulation of cells in the G1 phase of the cell cycle. This effect is due to the N-terminal and CR1 domains of E1A243, which in rodent cells are involved in triggering cellular transformation and also in binding to the cellular transcriptional coactivator p300. A genetic screen was undertaken to identify genes required for the function of E1A243 in S. cerevisiae. This screen identified SNF12, a gene encoding the 73-kDa subunit of the SWI/SNF transcriptional regulatory complex. Mutation of genes encoding known members of the SWI/SNF complex also led to loss of E1A function, suggesting that the SWI/SNF complex is a target of E1A243. Moreover, expression of E1A in wild-type cells specifically blocked transcriptional activation of the INO1 and SUC2 genes, whose activation pathways are distinct but have a common requirement for the SWI/SNF complex. These data demonstrate a specific functional interaction between E1A and the SWI/SNF complex and suggest that a similar interaction takes place in rodent and human cells. PMID:8816487

  3. Functional analysis of the C-terminal region of human adenovirus E1A reveals a misidentified nuclear localization signal

    SciTech Connect

    Cohen, Michael J.; King, Cason R.; Dikeakos, Jimmy D.; Mymryk, Joe S.

    2014-11-15

    The immortalizing function of the human adenovirus 5 E1A oncoprotein requires efficient localization to the nucleus. In 1987, a consensus monopartite nuclear localization sequence (NLS) was identified at the C-terminus of E1A. Since that time, various experiments have suggested that other regions of E1A influence nuclear import. In addition, a novel bipartite NLS was recently predicted at the C-terminal region of E1A in silico. In this study, we used immunofluorescence microscopy and co-immunoprecipitation analysis with importin-α to verify that full nuclear localization of E1A requires the well characterized NLS spanning residues 285–289, as well as a second basic patch situated between residues 258 and 263 ({sup 258}RVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP{sup 289}). Thus, the originally described NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified in the existing literature as a monopartite signal, altering our understanding of one of the oldest documented NLSs. - Highlights: • Human adenovirus E1A is localized to the nucleus. • The C-terminus of E1A contains a bipartite nuclear localization signal (NLS). • This signal was previously misidentified to be a monopartite NLS. • Key basic amino acid residues within this sequence are highly conserved.

  4. Transcription factor TFIID is a direct functional target of the adenovirus E1A transcription-repression domain.

    PubMed Central

    Song, C Z; Loewenstein, P M; Toth, K; Green, M

    1995-01-01

    The 243-amino acid adenovirus E1A oncoprotein both positively and negatively modulates the expression of cellular genes involved in the regulation of cell growth. The E1A transcription repression function appears to be linked with its ability to induce cellular DNA synthesis, cell proliferation, and cell transformation, as well as to inhibit cell differentiation. The mechanism by which E1A represses the transcription of various promoters has proven enigmatic. Here we provide several lines of evidence that the "TATA-box" binding protein (TBP) component of transcription factor TFIID is a cellular target of the E1A repression function encoded within the E1A N-terminal 80 amino acids. (i) The E1A N-terminal 80 amino acids [E1A-(1-80)protein] efficiently represses basal transcription from TATA-containing core promoters in vitro. (ii) TBP reverses completely E1A repression in vitro. (iii) TBP restores transcriptional activity to E1A-(1-80) protein affinity-depleted nuclear extracts. (iv) The N-terminal repression domain of E1A interacts directly and specifically with TBP in vitro. These results may help explain how E1A represses a set of genes that lack common upstream promoter elements. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7479778

  5. Silencing E1A mRNA by RNA interference inhibits adenovirus replication.

    PubMed

    Chung, Y-S; Kim, M-K; Lee, W-J; Kang, C

    2007-01-01

    The adenovirus family contains 51 human serotypes, and most human adenoviruses cause widespread respiratory tract infections. Adenovirus infections can result in severe complications in some cases, such as in adenovirus type 11 infection in immunocompromised patients. However, effective treatment methods for adenovirus infections are currently unavailable. This prompted the search for antiviral agents effective against adenovirus infections. In the present study, adenovirus E1A was targeted by RNA interference (RNAi) using synthetic small interfering RNAs (siRNAs) in an attempt to inhibit viral replication, since adenovirus E1A proteins are known to be involved in the transcriptional activation of the viral and cellular genes necessary for controlling the cell cycle and viral replication. The results indicated that the siRNAs effectively reduced the amount of adenovirus E1A mRNA and the levels of replicative intermediates. Additionally, siRNA-mediated gene silencing inhibited adenovirus replication by suppressing the E1A mRNA. These results suggest that the RNAi-mediated targeting of adenovirus E1A may have a potentially therapeutic effect in controlling adenovirus infections.

  6. Targeting of adenovirus E1A and E4-ORF3 proteins to nuclear matrix- associated PML bodies

    PubMed Central

    1995-01-01

    The PML protein was first identified as part of a fusion product with the retinoic acid receptor alpha (RAR alpha), resulting from the t(15;17) chromosomal translocation associated with acute promyelocytic leukemia (APL). It has been previously demonstrated that PML, which is tightly bound to the nuclear matrix, concentrates in discrete subnuclear compartments that are disorganized in APL cells due to the expression of the PML-RAR alpha hybrid. Here we report that adenovirus infection causes a drastic redistribution of PML from spherical nuclear bodies into fibrous structures. The product encoded by adenovirus E4- ORF3 is shown to be responsible for this reorganization and to colocalize with PML into these fibers. In addition, we demonstrate that E1A oncoproteins concentrate in the PML domains, both in infected and transiently transfected cells, and that this association requires the conserved amino acid motif (D)LXCXE, common to all viral oncoproteins that bind pRB or the related p107 and p130 proteins. The SV-40 large T antigen, another member of this oncoprotein family is also found in close association with the PML nuclear bodies. Taken together, the present data indicate that the subnuclear domains containing PML represent a preferential target for DNA tumor viruses, and therefore suggest a more general involvement of the PML nuclear bodies in oncogenic processes. PMID:7559785

  7. Adenovirus E1A protein activates transcription of the E1A gene subsequent to transcription complex formation.

    PubMed Central

    Schaack, J; Logan, J; Vakalopoulou, E; Shenk, T

    1991-01-01

    The mechanism of transcriptional activation of the adenovirus E1A and E3 genes by E1A protein during infection was examined by using transcription-competition assays. Infection of HeLa cells with one virus led to inhibition of mRNA accumulation from a superinfecting virus. Synthesis of the E1A 289R protein by the first virus to infect reduced inhibition of transcription of the superinfecting virus, indicating that the E1A 289R protein was limiting for E1A-activated transcription. Infection with an E1A- virus, followed 6 h later by superinfection with a wild-type virus, led to preferential transcriptional activation of the E1A gene of the first virus, suggesting that a host transcription component(s) stably associated with the E1A promoter in the absence of E1A protein and that this complex was the substrate for transcriptional activation by E1A protein. The limiting host transcription component(s) bound to the E1A promoter to form a complex with a half-life greater than 24 h in the absence of E1A 289R protein, as demonstrated in a challenge assay with a large excess of superinfecting virus. In the presence of the E1A 289R protein, the E1A gene of the superinfecting virus was gradually activated with a reduction in E1A mRNA accumulation from the first virus. The kinetics of the activation suggest that this was due to an indirect effect rather than to destabilization of stable transcription complexes by the 289R protein. Images PMID:1825853

  8. Transcription activation by the adenovirus E1a protein

    NASA Astrophysics Data System (ADS)

    Lillie, James W.; Green, Michael R.

    1989-03-01

    The adenovirus Ela protein stimulates transcription of a wide variety of viral and cellular genes. It is shown here that Ela has the two functions characteristic of a typical cellular activator: one direct Ela to the promoter, perhaps by interacting with a DMA-bound protein, and the other, an activating region, enables the bound activator to stimulate transcription.

  9. The degradation sequence of adenovirus E1A consists of the amino-terminal tetrapeptide Met-Arg-His-Ile.

    PubMed Central

    Simon, R; Richter, J D

    1990-01-01

    The adenovirus E1A gene product is a potent transcriptional activator and nuclear oncoprotein. Like other regulatory proteins, E1A has a short half-life, in the range of 30 to 120 min. This short half-life, which was measured in cells synthesizing E1A, is not observed in cells injected with E1A protein made in bacteria or in vitro. In these cases, E1A is essentially refractory to degradation. In an attempt to reconcile this apparent paradox, we suggested that E1A was marked for degradation during its synthesis. Furthermore, we showed that a domain in the amino terminus of E1A was required for rapid degradation in cells translating E1A mRNA (J. M. Slavicek, N. C. Jones, and J. D. Richter, EMBO J. 7:3171-3180, 1988). In this study, we have used Xenopus laevis oocytes injected with mRNAs encoding altered E1A proteins to show that the amino-terminal tetrapeptide Met-Arg-His-Ile is required for E1A degradation. Even conservative amino acid substitutions in this degradation sequence render it nonfunctional. This degradation sequence can function as a transferable signal, since it induces instability when fused to another normally stable protein. Furthermore, the degradation sequence requires a proximity of no more than six residues from the amino terminus for activity. These data suggest that a trans-acting factor recognizes the amino terminus of E1A during the translation of its message to mark the protein for subsequent destruction. Images PMID:2146491

  10. E1A RNA transcripts amplify adenovirus-mediated tumor reduction.

    PubMed

    Dion, L D; Goldsmith, K T; Strong, T V; Bilbao, G; Curiel, D T; Garver, R I

    1996-11-01

    Previous work by this group has established that E1-defective, recombinant adenoviruses can be replication-enabled by the codelivery of a plasmid encoding the deleted E1 functions, a strategy now designated conditional replication-enablement system for adenovirus (CRESA). In the studies reported here, the original replication-enabling plasmid was replaced by two separate plasmids that encoded the necessary E1A and E1B functions, respectively. An RNA transcript encoding the requisite E1A functions was shown to substitute functionally for the E1A plasmid without significant loss of new adenovirus production in in vitro experiments. No replication competent adenovirus was detectable in the cells treated with the plasmids, or the RNA and plasmid combinations. Subcutaneous human tumor nodules containing a fraction of cells cotransduced with the replication-enabling RNA + DNA and an adenovirus containing a herpes simplex virus thymidine kinase (HSVtk) expression cassette were reduced to a greater extent than control nodules containing the same fraction of cells cotransduced with the virus and an irrelevant plasmid. These experiments show that an E1-defective adenovirus can be conditionally replication-enabled by an RNA transcript encoding the required E1 functions, and that the replication-enablement is sufficient to produce an augmentation of an adenovirus-mediated therapeutic effect in vivo.

  11. Transcription control region within the protein-coding portion of adenovirus E1A genes.

    PubMed Central

    Osborne, T F; Arvidson, D N; Tyau, E S; Dunsworth-Browne, M; Berk, A J

    1984-01-01

    A single-base deletion within the protein-coding region of the adenovirus type 5 early region 1A (E1A) genes, 399 bases downstream from the transcription start site, depresses transcription to 2% of the wild-type rate. Complementation studies demonstrated that this was due to two effects of the mutation: first, inactivation of an E1A protein, causing a reduction by a factor of 5; second, a defect which acts in cis to depress E1A mRNA and nuclear RNA concentrations by a factor of 10. A larger deletion within the protein-coding region of E1A which overlaps the single-base deletion produces the same phenotype. In contrast, a linker insertion which results in a similar truncated E1A protein does not produce the cis-acting defect in E1A transcription. These results demonstrate that a critical cis-acting transcription control region occurs within the protein coding sequence in adenovirus type 5 E1A. The single-base deletion occurs in a sequence which shows extensive homology with a sequence from the enhancer regions of simian virus 40 and polyomavirus. This region is not required for E1A transcription during the late phase of infection. Images PMID:6334230

  12. Limited temperature-sensitive transactivation by mutant adenovirus type 2 E1a proteins.

    PubMed Central

    Fahnestock, M L; Lewis, J B

    1989-01-01

    A series of linker-scanning and deletion mutations was generated in the transactivating domain of the larger, 289-amino-acid-residue E1a protein of adenovirus type 2. Mutant genes were recombined into virus to assay the ability of the variant E1a proteins to activate expression of an E1a-dependent viral gene during infection. Results of assays performed at 32, 37, and 40 degrees C indicated that at least 2 of the 10 mutants tested showed limited temperature sensitivity for transactivation. Images PMID:2523001

  13. Identification and characterization of multiple conserved nuclear localization signals within adenovirus E1A

    SciTech Connect

    Marshall, Kris S.; Cohen, Michael J.; Fonseca, Greg J.; Todorovic, Biljana; King, Cason R.; Yousef, Ahmed F.; Zhang, Zhiying; Mymryk, Joe S.

    2014-04-15

    The human adenovirus 5 (HAdV-5) E1A protein has a well defined canonical nuclear localization signal (NLS) located at its C-terminus. We used a genetic assay in the yeast Saccharomyces cerevisiae to demonstrate that the canonical NLS is present and functional in the E1A proteins of each of the six HAdV species. This assay also detects a previously described non-canonical NLS within conserved region 3 and a novel active NLS within the N-terminal/conserved region 1 portion of HAdV-5 E1A. These activities were also present in the E1A proteins of each of the other five HAdV species. These results demonstrate that, despite substantial differences in primary sequence, HAdV E1A proteins are remarkably consistent in that they contain one canonical and two non-canonical NLSs. By utilizing independent mechanisms, these multiple NLSs ensure nuclear localization of E1A in the infected cell. - Highlights: • HAdV E1A uses multiple mechanisms for nuclear import. • We identified an additional non-canonical NLS in the N-terminal/CR1 portion of E1A. • The new NLS does not contact importin-alpha directly. • All NLSs are functionally conserved in the E1A proteins of all 6 HAdV species.

  14. Adenovirus E1A/E1B Transformed Amniotic Fluid Cells Support Human Cytomegalovirus Replication

    PubMed Central

    Krömmelbein, Natascha; Wiebusch, Lüder; Schiedner, Gudrun; Büscher, Nicole; Sauer, Caroline; Florin, Luise; Sehn, Elisabeth; Wolfrum, Uwe; Plachter, Bodo

    2016-01-01

    The human cytomegalovirus (HCMV) replicates to high titers in primary human fibroblast cell cultures. A variety of primary human cells and some tumor-derived cell lines do also support permissive HCMV replication, yet at low levels. Cell lines established by transfection of the transforming functions of adenoviruses have been notoriously resistant to HCMV replication and progeny production. Here, we provide first-time evidence that a permanent cell line immortalized by adenovirus type 5 E1A and E1B (CAP) is supporting the full HCMV replication cycle and is releasing infectious progeny. The CAP cell line had previously been established from amniotic fluid cells which were likely derived from membranes of the developing fetus. These cells can be grown under serum-free conditions. HCMV efficiently penetrated CAP cells, expressed its immediate-early proteins and dispersed restrictive PML-bodies. Viral DNA replication was initiated and viral progeny became detectable by electron microscopy in CAP cells. Furthermore, infectious virus was released from CAP cells, yet to lower levels compared to fibroblasts. Subviral dense bodies were also secreted from CAP cells. The results show that E1A/E1B expression in transformed cells is not generally repressive to HCMV replication and that CAP cells may be a good substrate for dense body based vaccine production. PMID:26848680

  15. The regions of the retinoblastoma protein needed for binding to adenovirus E1A or SV40 large T antigen are common sites for mutations.

    PubMed Central

    Hu, Q J; Dyson, N; Harlow, E

    1990-01-01

    The protein product of the retinoblastoma (RB) gene is thought to function in a pathway that restricts cell proliferation. Recently, transforming proteins from three different classes of DNA tumor viruses have been shown to form complexes with the RB protein. Genetic studies suggest that these interactions with the RB protein are important steps in transformation by these viruses. In order to understand better the function of the RB-viral oncoprotein complexes, we have mapped the regions of the RB protein that are necessary for these associations. Two non-contiguous regions of RB were found to be essential for complex formation with adenovirus E1A or SV40 large T antigen. These two regions are found between amino acids 393 and 572 and 646 and 772. Interestingly, these binding sites on RB overlap with the positions of naturally occurring, inactivating mutations of the RB gene. These results strongly suggest that these viral oncoproteins are targeting a protein domain that is an important site in the normal function of the RB protein. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 9. PMID:2138977

  16. The adenovirus E1A N-terminal repression domain represses transcription from a chromatin template in vitro

    PubMed Central

    Loewenstein, Paul M.; Wu, Shwu-Yuan; Chiang, Cheng-Ming

    2013-01-01

    The adenovirus repression domain of E1A 243R at the E1A N-terminus (E1A 1–80) transcriptionally represses genes involved in differentiation and cell cycle progression. E1A 1–80 represses transcription in vitro from naked DNA templates through its interaction with p300 and TFIID. E1A 1–80 can also interact with several chromatin remodeling factors and associates with chromatin in vivo. We show here that E1A 243R and E1A 1–80 can repress transcription from a reconstituted chromatin template in vitro. Temporal analysis reveals strong repression by E1A 1–80 when added at pre-activation, activation and early transcription stages. Interestingly, E1A 1–80 can greatly enhance transcription from chromatin templates, but not from naked DNA, when added at pre-initiation complex (PIC) formation and transcription-initiation stages. These data reveal a new dimension for E1A 1–80's interface with chromatin and may reflect its interaction with key players in PIC formation, p300 and TFIID, and/or possibly a role in chromatin remodeling. PMID:22521914

  17. The adenovirus E1A N-terminal repression domain represses transcription from a chromatin template in vitro.

    PubMed

    Loewenstein, Paul M; Wu, Shwu-Yuan; Chiang, Cheng-Ming; Green, Maurice

    2012-06-20

    The adenovirus repression domain of E1A 243R at the E1A N-terminus (E1A 1-80) transcriptionally represses genes involved in differentiation and cell cycle progression. E1A 1-80 represses transcription in vitro from naked DNA templates through its interaction with p300 and TFIID. E1A 1-80 can also interact with several chromatin remodeling factors and associates with chromatin in vivo. We show here that E1A 243R and E1A 1-80 can repress transcription from a reconstituted chromatin template in vitro. Temporal analysis reveals strong repression by E1A 1-80 when added at pre-activation, activation and early transcription stages. Interestingly, E1A 1-80 can greatly enhance transcription from chromatin templates, but not from naked DNA, when added at pre-initiation complex (PIC) formation and transcription-initiation stages. These data reveal a new dimension for E1A 1-80's interface with chromatin and may reflect its interaction with key players in PIC formation, p300 and TFIID, and/or possibly a role in chromatin remodeling.

  18. Preclinical safety assessment of Ad[I/PPT-E1A], a novel oncolytic adenovirus for prostate cancer.

    PubMed

    Schenk, Ellen; Essand, Magnus; Kraaij, Robert; Adamson, Rachel; Maitland, Norman J; Bangma, Chris H

    2014-03-01

    Prostate cancer is the most common malignancy in the Western world. Patients can be cured only when the tumor has not metastasized outside the prostate. However, treatment with curative intent fails in a significant number of men, often resulting in untreatable progressive disease with a fatal outcome. Oncolytic adenovirus therapy may be a promising adjuvant treatment to reduce local failure or the outgrowth of micrometastatic disease. Within the European gene therapy consortium GIANT, we have developed a novel prostate-specific oncolytic adenovirus: Ad[I/PPT-E1A]. This adenovirus specifically kills prostate cells via prostate-specific replication. This article describes the clinical development of Ad[I/PPT-E1A] with particular reference to the preclinical safety assessment of this novel virus. The preclinical safety assessment involved an efficacy study in a human orthotopic xenograft mouse model, a specificity study in human primary cells, and a toxicity study in normal mice. These studies confirmed that Ad[I/PPT-E1A] efficiently kills prostate tumor cells in vivo, is not harmful to other organs, and is well tolerated in mice after systemic delivery. The safety, as well as the immunological effects of Ad[I/PPT-E1A] as a local adjuvant therapy, will now be studied in a phase I dose-escalating trial in patients with localized prostate cancer who are scheduled for curative radical prostatectomy and can be used as an updated paradigm for similar therapeutic viruses.

  19. A simple method for the simultaneous detection of E1A and E1B in adenovirus stocks.

    PubMed

    Suzuki, Erika; Murata, Takehide; Watanabe, Sanae; Kujime, Yukari; Hirose, Megumi; Pan, Jianzhi; Yamazaki, Takahito; Ugai, Hideyo; Yokoyama, Kazunari K

    2004-01-01

    Recombinant adenoviral vectors have been developed for use as therapeutic agents and for the introduction of exogenous genes into living cells. However, the occurrence of replication-competent adenoviruses (RCA) in adenovirus stocks produced in 293 cells remains a major problem in terms of the safe use of such vectors. To overcome the problems associated with the occurrence of RCA, we have established a simple method for the simultaneous detection of amplified E1A and E1B from RCA that might contaminate adenoviral stocks. The products amplified by polymerase chain reaction (PCR) were fractionated by regular electrophoresis on agarose gels and visualized by staining with ethidium bromide. This method is rapid and inexpensive for detection of RCA in the preparation of adenoviruses.

  20. Role of an adenovirus E2 promoter binding factor in E1A-mediated coordinate gene control.

    PubMed Central

    Kovesdi, I; Reichel, R; Nevins, J R

    1987-01-01

    A product of the adenovirus gene E1A is responsible for the stimulation of transcription from six viral promoters as well as at least two cellular promoters. We have detected a HeLa cell factor, termed E2 promoter binding factor (E2F), that appears to mediate the transcriptional stimulation of the viral E2 promoter. Competition experiments revealed that E2F did not recognize and bind to the E1B, E3, E4, or major late promoter sequences. Furthermore, three additional promoters stimulated by E1A, heat shock protein 70, beta-globin, and early simian virus 40, do not bind E2F. In contrast, the factor does recognize sequences in the E1A enhancer, and within the E1A enhancer are duplicated binding sites for E2F. Finally, a single E2F binding site from the E1A enhancer can confer increased transcription to a mouse beta-globin promoter, dependent on the action of the E1A gene product. This stimulation requires binding of E2F since methylation of the binding site, which blocks binding in vitro, reduces transcription stimulation in vivo. We, therefore, conclude that E2F is likely to be responsible for the E1A-mediated stimulation of the E1A gene as well as the E2 gene but is not involved in the activation of the other E1A-inducible promoters. Images PMID:2951737

  1. Suppression of mutations in two Saccharomyces cerevisiae genes by the adenovirus E1A protein.

    PubMed Central

    Zieler, H A; Walberg, M; Berg, P

    1995-01-01

    The protein products of the adenoviral E1A gene are implicated in a variety of transcriptional and cell cycle events, involving interactions with several proteins present in human cells, including parts of the transcriptional machinery and negative regulators of cell division such as the Rb gene product and p107. To determine if there are functional homologs of E1A in Saccharomyces cerevisiae, we have developed a genetic screen for mutants that depend on E1A for growth. The screen is based on a colony color sectoring assay which allows the identification of mutants dependent on the maintenance and expression of an E1A-containing plasmid. Using this screen, we have isolated five mutants that depend on expression of the 12S or 13S cDNA of E1A for growth. A plasmid shuffle assay confirms that the plasmid-dependent phenotype is due to the presence of either the 12S or the 13S E1A cDNA and that both forms of E1A rescue growth of all mutants equally well. The five mutants fall into two classes that were named web1 and web2 (for "wants E1A badly"). Plasmid shuffle assays with mutant forms of E1A show that conserved region 1 (CR1) is required for rescue of the growth of the web1 and web2 E1A-dependent yeast mutants, while the N-terminal 22 amino acids are only partially required; conserved region 2 (CR2) and the C terminus are dispensable. The phenotypes of mutants in both the web1 and the web2 groups are due to a single gene defect, and the yeast genes that fully complement the mutant phenotypes of both groups were cloned. The WEB1 gene sequence encodes a 1,273-amino-acid protein that is identical to SEC31, a protein involved in the budding of transport vesicles from the endoplasmic reticulum. The WEB2 gene encodes a 1,522-amino-acid protein with homology to nucleic acid-dependent ATPases. Deletion of either WEB1 or WEB2 is lethal. Expression of E1A is not able to rescue the lethality of either the web1 or the web2 null allele, implying allele-specific mutations that lead

  2. Inhibition of adenovirus replication by the E1A antisense transcript initiated from hsp70 and VA-1 promoters.

    PubMed

    Miroshnichenko, O I; Borisenko, A S; Ponomareva, T I; Tikhonenko, T I

    1990-03-01

    The E1A region of the adenoviral genome, important for initiation of virus infection and activation of other viral genes, was chosen as a target for engineering antisense RNA (asRNA) to inhibit adenovirus 5 (Ad5) replication in COS-1 cell culture in vitro. The hsp70 promoter, taken from the appropriate heat-shock-protein gene of Drosophila melanogaster, and the VA-1 RNA promoter, derived from the Ad5 gene coding for low-molecular-mass VA-1 RNA and recognized by RNA polymerase III were used as regulatory elements of transcription. The two types of recombinant constructs contained E1A fragments of 710 bp (hsp70 constructs) or 380 or 740 bp (VA-1 RNA constructs) in reverse orientation relative to the promoter position, as well as a transcription termination signal, the SV40 ori, and the gene controlling Geneticin (antibiotic G418) resistance (G418R). After selection of transfected COS-1 cells in the presence of G418, a number of stable G418R cell lines were raised which expressed engineered asRNAs. Plating of Ad5 suspensions of known titre on monolayers of transfected COS-1 cells clearly showed strong inhibition of adenovirus replication by asRNAs: 75% with the hsp70 promoter and 90% with the VA-1 RNA promoter.

  3. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Enhances E1A Functional Activity

    PubMed Central

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G. Eric; Dobner, Thomas; Branton, Philip E.

    2015-01-01

    ABSTRACT Human adenovirus (Ad) E1A proteins have long been known as the central regulators of virus infection as well as the major source of adenovirus oncogenic potential. Not only do they activate expression of other early viral genes, they make viral replication possible in terminally differentiated cells, at least in part, by binding to the retinoblastoma (Rb) tumor suppressor family of proteins to activate E2F transcription factors and thus viral and cellular DNA synthesis. We demonstrate in an accompanying article (F. Dallaire et al., mSphere 1:00014-15, 2016) that the human adenovirus E3 ubiquitin ligase complex formed by the E4orf6 and E1B55K proteins is able to mimic E1A activation of E2F transactivation factors. Acting alone in the absence of E1A, the Ad5 E4orf6 protein in complex with E1B55K was shown to bind E2F, disrupt E2F/Rb complexes, and induce hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis, as well as stimulation of early and late viral gene expression and production of viral progeny. While these activities were significantly lower than those exhibited by E1A, we report here that this ligase complex appeared to enhance E1A activity in two ways. First, the E4orf6/E1B55K complex was shown to stabilize E1A proteins, leading to higher levels in infected cells. Second, the complex was demonstrated to enhance the activation of E2F by E1A products. These findings indicated a new role of the E4orf6/E1B55K ligase complex in promoting adenovirus replication. IMPORTANCE Following our demonstration that adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins is able to mimic the activation of E2F by E1A, we conducted a series of studies to determine if this complex might also promote the ability of E1A to do so. We found that the complex both significantly stabilizes E1A proteins and also enhances their ability to activate E2F. This finding is of significance because it represents an entirely new

  4. Posttranslational modification at the N terminus of the human adenovirus type 12 E1A 235R tumor antigen.

    PubMed Central

    Lucher, L A; Brackmann, K H; Symington, J S; Green, M

    1986-01-01

    The adenovirus E1A transforming region, which encodes immortalization, partial cell transformation, and gene activation functions, expresses two early mRNAs, 13S and 12S. Multiple-T antigen species with different electrophoretic mobilities are formed from each mRNA, presumably by unknown posttranslational modifications. The adenovirus type 12 (Ad12) 13S and 12S mRNAs encode E1A T antigens of 266 and 235 amino acid residues (266R and 235R), respectively. To study possible posttranslational processing at the N and C termini and to distinguish between the Ad12 266R and 235R T antigens, we prepared antibodies targeted to synthetic peptides encoded at the common C (peptide 204) and N (peptide 202) termini of the 266R and 235R T antigens and at the unique internal domain of the 266R T antigen (peptide 206). The specificity of each anti-peptide antibody was confirmed by immunoprecipitation of the 266R and 235R T antigens produced in Escherichia coli. Immunoprecipitation analysis of the E1A T antigens synthesized in Ad12-infected KB cells revealed the following. Antibody to the common C terminus recognized three T antigens with apparent Mrs of 43,000, 42,000, and 39,000 (43K, 42K, and 39K). All three forms were phosphorylated and were present in both the nucleus and the cytoplasm. The 43K and 42K T antigens were rapidly synthesized during a 10-min pulse with [35S]methionine in Ad12-infected cells. The 43K T antigen had a half-life of 20 min, the 42K T antigen had a longer half-life of about 40 min, and the 39K T antigen became the predominant E1A T antigen. Antibodies to the unique region immunoprecipitated the 43K T antigen but not the 42K and 39K T antigens. Antibody to the N terminus immunoprecipitated the 43K and 42K T antigens but not the 39K T antigen, suggesting that the 39K T antigen possessed a modified N terminus. Partial N-terminal amino acid sequence analysis showed that the 43K and 42K T antigens contain methionine at residues 1 and 5, as predicted from the

  5. Interaction of CtBP with adenovirus E1A suppresses immortalization of primary epithelial cells and enhances virus replication during productive infection

    SciTech Connect

    Subramanian, T.; Zhao, Ling-jun; Chinnadurai, G.

    2013-09-01

    Adenovirus E1A induces cell proliferation, oncogenic transformation and promotes viral replication through interaction with p300/CBP, TRRAP/p400 multi-protein complex and the retinoblastoma (pRb) family proteins through distinct domains in the E1A N-terminal region. The C-terminal region of E1A suppresses E1A/Ras co-transformation and interacts with FOXK1/K2, DYRK1A/1B/HAN11 and CtBP1/2 (CtBP) protein complexes. To specifically dissect the role of CtBP interaction with E1A, we engineered a mutation (DL→AS) within the CtBP-binding motif, PLDLS, and investigated the effect of the mutation on immortalization and Ras cooperative transformation of primary cells and viral replication. Our results suggest that CtBP–E1A interaction suppresses immortalization and Ras co-operative transformation of primary rodent epithelial cells without significantly influencing the tumorigenic activities of transformed cells in immunodeficient and immunocompetent animals. During productive infection, CtBP–E1A interaction enhances viral replication in human cells. Between the two CtBP family proteins, CtBP2 appears to restrict viral replication more than CtBP1 in human cells. - Highlights: • Adenovirus E1A C-terminal region suppresses E1A/Ras co-transformation. • This E1A region binds with FOXK, DYRK1/HAN11 and CtBP cellular protein complexes. • We found that E1A–CtBP interaction suppresses immortalization and transformation. • The interaction enhances viral replication in human cells.

  6. Adenovirus Small E1A Employs the Lysine Acetylases p300/CBP and Tumor Suppressor Rb to Repress Select Host Genes and Promote Productive Virus Infection

    PubMed Central

    Ferrari, Roberto; Gou, Dawei; Jawdekar, Gauri; Johnson, Sarah A.; Nava, Miguel; Su, Trent; Yousef, Ahmed F.; Zemke, Nathan R.; Pellegrini, Matteo; Kurdistani, Siavash K.; Berk, Arnold J.

    2015-01-01

    SUMMARY Oncogenic transformation by adenovirus small e1a depends on simultaneous interactions with the host lysine acetylases p300/CBP and the tumor suppressor RB. How these interactions influence cellular gene expression remains unclear. We find that e1a displaces RBs from E2F transcription factors and promotes p300 acetylation of RB1 K873/K874 to lock it into a repressing conformation that interacts with repressive chromatin-modifying enzymes. These repressing p300-e1a-RB1 complexes specifically interact with host genes that have unusually high p300 association within the gene body. The TGFβ-, TNF-, and interleukin-signaling pathway components are enriched among such p300-targeted genes. The p300-e1a-RB1 complex condenses chromatin in a manner dependent on HDAC activity, p300 lysine acetylase activity, the p300 bromodomain, and RB K873/K874 and e1a K239 acetylation to repress host genes that would otherwise inhibit productive virus infection. Thus, adenovirus employs e1a to repress host genes that interfere with viral replication. PMID:25525796

  7. The amino-terminal portion of CD1 of the adenovirus E1A proteins is required to induce susceptibility to tumor necrosis factor cytolysis in adenovirus-infected mouse cells.

    PubMed Central

    Duerksen-Hughes, P J; Hermiston, T W; Wold, W S; Gooding, L R

    1991-01-01

    Previous work by our laboratory and others has shown that mouse cells normally resistant to tumor necrosis factor can be made sensitive to the cytokine by the expression of adenovirus E1A. The E1A gene can be introduced by either infection or transfection, and either of the two major E1A proteins, 289R or 243R, can induce this sensitivity. The E1A proteins are multifunctional and modular, with specific domains associated with specific functions. Here, we report that the CD1 domain of E1A is required to induce susceptibility to tumor necrosis factor cytolysis in adenovirus-infected mouse C3HA fibroblasts. Amino acids C terminal to residue 60 and N terminal to residue 36 are not necessary for this function. This conclusion is based on 51Cr-release assays for cytolysis in cells infected with adenovirus mutants with deletions in various portions of E1A. These E1A mutants are all in an H5dl309 background and therefore they lack the tumor necrosis factor protection function provided by the 14.7-kilodalton (14.7K) protein encoded by region E3. Western blot (immunoblot) analysis indicated that most of the mutant E1A proteins were stable in infected C3HA cells, although with certain large deletions the E1A proteins were unstable. The region between residues 36 and 60 is included within but does not precisely correlate with domains in E1A that have been implicated in nuclear localization, enhancer repression, cellular immortalization, cell transformation in cooperation with ras, induction of cellular DNA synthesis and proliferation, induction of DNA degradation, and binding to the 300K protein and the 105K retinoblastoma protein. Images PMID:1825340

  8. E1A-engineered human umbilical cord mesenchymal stem cells as carriers and amplifiers for adenovirus suppress hepatocarcinoma in mice

    PubMed Central

    Li, Zhenzhen; Ye, Zhou; Zhang, Xiaolong; Zhang, Qing; Fan, Dongmei; Zhang, Yanjun; Luo, Hongbo R.; Yuan, Xiangfei; Li, Zongfang; Xiong, Dongsheng

    2016-01-01

    Gene therapy is an attractive approach for hepatocellular carcinoma (HCC) patients. Nevertheless, efficient transgene delivery remains a challenge. In this study, we explored a new targeted system based on human umbilical cord-derived mesenchymal stem cells (HUMSCs), which were engineered to deliver adenovirus to tumor sites, and to replicate and assemble into new adenovirus against HCC. Our results showed that HUMSCs infected by Ad-hTERTp-IL24 followed by LentiR.E1A infection could specifically migrate to HepG2 tumor cells and support adenoviral replication in vitro and in vivo 36 h after LentiR.E1A infection. Ad-hTERTp-IL24 specifically inhibited HepG2 cells growth, and this inhibitory effect was enhanced by low doses of 5-fluorouracil (5-Fu), because the expression levels of coxsackie adenovirus receptor (CAR) and integrin ανβ3 on tumor cells were significantly increased, causing higher viral uptake. Compared with the no treatment groups, Ad-hTERTp-IL24 and LentiR.E1A co-loaded HUMSCs exhibited significant anti-tumor activity in vivo, particularly in combination with low doses of 5-Fu. In summary, this study provides a promising targeted gene therapeutic strategy dependent on the tumor tropism of HUMSCs, to improve the outcome of virotherapy for tumor patients especially those with metastatic diseases. PMID:27322080

  9. Identification of functional domains of adenovirus tumor-specific transplantation antigen in types 5 and 12 by viable viruses carrying chimeric E1A genes.

    PubMed

    Sawada, Y; Rasková, J; Fujinaga, K; Raska, K

    1994-05-15

    The adenovirus (Ad) E1A gene induces in immunized animals a strong tumor transplantation (TSTA) immunity against Ad tumors. Such immunity with group-A and group-C viruses is highly group-specific and no cross-protection is detected between serotypes 5 and 12. This fact was used to map the domains of the Ad5 and Ad12 E1A gene products, respectively, which control the TSTA. We constructed a library of 8 recombinant viruses (H5sub1101 through H5sub1108) which carry chimeric Ad5/Ad12 E1A genes in the background of Ad5. The chimeric genes are functional and these viruses are viable. Some of these constructs induce strong and highly specific tumor syngraft immunity in immunized rats. The viruses carrying the 5' terminus of the first E1A exon derived from Ad12 (viruses H5sub1101, H5sub1102 and H5sub1103) induce strong protection against Ad12 tumors irrespective of the rest of their E1A sequence. The viruses which carry the second exon of the Ad5 E1A gene (viruses H5sub1101, H5sub1102 and H5sub1106) protect against group-C tumors, regardless of the origin of the rest of their E1A gene. The 2 viruses that carry the 5' E1A terminus of the first exon of Ad12 and the second exon of Ad5 (H5sub1101 and H5sub1102) are thus effective in inducing immunity against Ad12 tumors as well as against Ad2 tumors. The viruses which carry the 5' terminus of the first exon derived from Ad5 and the second exon of Ad12 (H5sub1107 and H5sub 1108) fail to induce immunity against either tumor. Expression of only the truncated 5' terminus of the Ad12 E1A gene (viruses H5sub1104 and H5sub1105) is sufficient for induction of Ad12 TSTA. Our results provide direct and unequivocal in vivo evidence that TSTA activities of adenovirus groups A and C are controlled by different domains of their respective E1A genes. The Ad12 TSTA is a function of the 5' terminus of the first E1A exon, while the Ad5 TSTA is coded for by the 3' exon of its E1A gene.

  10. Adenovirus type 5 early region 1B 55-kDa oncoprotein can promote cell transformation by a mechanism independent from blocking p53-activated transcription.

    PubMed

    Härtl, B; Zeller, T; Blanchette, P; Kremmer, E; Dobner, T

    2008-06-12

    Inhibition of p53-activated transcription is an integral part of the mechanism by which early region 1B 55K oncoprotein (E1B-55K) from adenovirus type 5 (Ad5) contributes to complete cell transformation in combination with Ad E1A. In addition, more recent data suggest that the mode of action of the Ad protein during transformation may involve additional functions and other protein interactions. In the present study, we performed a comprehensive mutational analysis to assign further transforming functions of Ad5 E1B-55K to distinct domains within the viral polypeptide. Results from these studies show that the functions required for transformation are encoded within several patches of the 55K primary sequence, including several clustered cysteine and histidine residues, some of which match the consensus for zinc fingers. In addition, two amino-acid substitutions (C454S/C456S) created a 55K mutant protein, which had substantially reduced transforming activity. Interestingly, the same mutations neither affected binding to p53 nor inhibition of p53-mediated transactivation. Therefore, an activity necessary for efficient transformation of primary rat cells can be separated from functions required for inhibition of p53-stimulated transcription. Our data indicate that this activity is linked to the ability of the Ad5 protein to bind to components of the Mre11/Rad50/NBS1 DNA double-strand break repair complex, and/or its ability to assemble multiprotein aggregates in the cytoplasm and nucleus of transformed rat cells. These results introduce a new function for Ad5 E1B-55K and suggest that the viral protein contributes to cell transformation through p53 transcription-dependent and -independent pathways.

  11. Preclinical pharmacology and toxicology study of Ad-hTERT-E1a-Apoptin, a novel dual cancer-specific oncolytic adenovirus

    SciTech Connect

    Qi, Yanxin; Guo, Huanhuan; Hu, Ningning; He, Dongyun; Zhang, Shi; Chu, Yunjie; Huang, Yubin; Li, Xiao; Sun, LiLi; Jin, Ningyi

    2014-10-15

    Clinical studies have demonstrated that conditionally replicating adenovirus is safe. We constructed an oncolytic adenovirus, Ad-hTERT-E1a-Apoptin, using a cancer-specific promoter (human telomerase reverse transcriptase promoter, hTERTp) and a cancer cell-selective apoptosis-inducing gene (Apoptin). Ad-hTERT-E1a-Apoptin was proven effective both in vitro and in vivo in our previous study. In this study, the preclinical safety profiles of Ad-hTERT-E1a-Apoptin in animal models were investigated. At doses of 5.0 × 10{sup 8}, 2.5 × 10{sup 9}, and 1.25 × 10{sup 10} viral particles (VP)/kg, Ad-hTERT-E1a-Apoptin had no adverse effects on mouse behavior, muscle cooperation, sedative effect, digestive system, and nervous systems, or on beagle cardiovascular and respiratory systems at 5.0 × 10{sup 8}, 2.5 × 10{sup 9}, and 1.25 × 10{sup 10} VP/kg doses. In acute toxicity tests in mice, the maximum tolerated dose > 5 × 10{sup 10} VP/kg. There was no inflammation or ulceration at the injection sites within two weeks. In repeat-dose toxicological studies, the no observable adverse effect levels of Ad-hTERT-E1a-Apoptin in rats (1.25 × 10{sup 10} VP/kg) and beagles (2.5 × 10{sup 9} VP/kg) were 62.5- and 12.5-fold of the proposed clinical dose, respectively. The anti-virus antibody was produced in animal sera. Bone marrow examination revealed no histopathological changes. Guinea pigs sensitized by three repeated intraperitoneal injections of 1.35 × 10{sup 10} VP/mL Ad-hTERT-E1a-Apoptin each and challenged by one intravenous injection of 1.67 × 10{sup 8} VP/kg Ad-hTERT-E1a-Apoptin did not exhibit any sign of systemic anaphylaxis. Our data from different animal models suggest that Ad-hTERT-E1a-Apoptin is a safe anti-tumor therapeutic agent. - Highlights: • We use the rodents and non-rodents animal models to evaluation Ad-hTERT-E1a-Apoptin. • Ad-hTERT-E1a-Apoptin is a safe anti-tumor therapeutic agent. • Demonstrate the safety and feasibility dose of injected Ad-hTERT-E

  12. E1B and E4 oncoproteins of adenovirus antagonize the effect of apoptosis inducing factor

    SciTech Connect

    Turner, Roberta L.; Wilkinson, John C.; Ornelles, David A.

    2014-05-15

    Adenovirus inundates the productively infected cell with linear, double-stranded DNA and an abundance of single-stranded DNA. The cellular response to this stimulus is antagonized by the adenoviral E1B and E4 early genes. A mutant group C adenovirus that fails to express the E1B-55K and E4ORF3 genes is unable to suppress the DNA-damage response. Cells infected with this double-mutant virus display significant morphological heterogeneity at late times of infection and frequently contain fragmented nuclei. Nuclear fragmentation was due to the translocation of apoptosis inducing factor (AIF) from the mitochondria into the nucleus. The release of AIF was dependent on active poly(ADP-ribose) polymerase-1 (PARP-1), which appeared to be activated by viral DNA replication. Nuclear fragmentation did not occur in AIF-deficient cells or in cells treated with a PARP-1 inhibitor. The E1B-55K or E4ORF3 proteins independently prevented nuclear fragmentation subsequent to PARP-1 activation, possibly by altering the intracellular distribution of PAR-modified proteins. - Highlights: • E1B-55K or E4orf3 prevents nuclear fragmentation. • Nuclear fragmentation requires AIF and PARP-1 activity. • Adenovirus DNA replication activates PARP-1. • E1B-55K or E4orf3 proteins alter the distribution of PAR.

  13. Induction of sensitivity to the cytotoxic action of tumor necrosis factor alpha by adenovirus E1A is independent of transformation and transcriptional activation.

    PubMed Central

    Ames, R S; Holskin, B; Mitcho, M; Shalloway, D; Chen, M J

    1990-01-01

    We have previously shown that expression of the adenovirus E1A 12S or 13S products in NIH 3T3 fibroblasts induces susceptibility to the cytotoxic actions of tumor necrosis factor alpha (TNF alpha). A large number of studies have mapped the multiple biological functions of the 12S and 13S products to three highly conserved regions (CR) within the E1A sequence. Here we used plasmids coding for E1A deletion and point mutants in these regions to generate target cell lines for TNF alpha cytotoxicity assays to determine which regions and functions are necessary for the induction of TNF alpha sensitivity. Expression of CR1 was required for the induction of TNF alpha sensitivity. This finding did not reflect a requirement for transforming or transcriptional repression activity, since some mutants that were defective in both of these properties were able to induce TNF alpha sensitivity. CR2 transformation-defective point mutants, but not a CR2/3 region deletion mutant, were also able to induce sensitivity. In addition, NIH 3T3 cells expressing the retroviral transcription activators tat from human immunodeficiency virus type 1 and tax from human T-lymphotropic virus type I were not sensitive to TNF alpha. However, the possibility that E1A-mediated transcriptional activation can augment the induction of TNF alpha sensitivity is not excluded. Comparison of data from previous biological studies with the TNF alpha cytotoxicity assays presented here suggested that the mechanism by which E1A induces sensitivity to TNF alpha in NIH 3T3 cells is independent of many of the known E1A biological functions, including transformation in cooperation with ras, immortalization, induction of DNA synthesis in quiescent cells, and transcriptional repression. A novel E1A-mediated effect may be involved, although our data do not exclude the possibility that sensitization to TNF alpha is mediated through E1A binding to cellular proteins. Images PMID:2143540

  14. A bidirectional Tet-dependent promotor construct regulating the expression of E1A for tight control of oncolytic adenovirus replication.

    PubMed

    Fechner, Henry; Wang, Xiaomin; Picó, Almudena Hurtado; Wildner, Judith; Suckau, Lennart; Pinkert, Sandra; Sipo, Isaac; Weger, Stefan; Poller, Wolfgang

    2007-01-20

    Tight regulation of oncolytic adenoviruses (oAdV) represents an important requirement for their safe application. Here we describe a new doxycycline (Dox)-dependent oAdV with a bidirectional expression cassette, which drives the expression of the reverse tetracycline-controlled transactivator (rtTA(s)-M2) from a lung tumor-specific promoter and, in the opposite direction, the expression of the adenoviral E1A gene from a second generation TetO(7) sequence linked to an isolated TATA box. In H441 lung cancer cells, this oAdV showed a strictly Dox-dependent E1A expression, adenoviral replication, cell killing activity and a 450-fold induction of progeny virus production. The virus could be shut off again by withdrawal of Dox and, in contrast to a control oAdV expressing E1A directly from the SP-B promoter, did not replicate in non-target cells. However, the absolute values of virus production and the cell killing activity in the presence of the inducer were still reduced as compared to the control oAdV. The results demonstrate, for the first time, Dox-dependent oAdV replication from a single adenoviral vector genome. Future improvement of the Dox-dependent E1A regulation cassette should lead to the generation of an oAdV well suited to meet the demands for a highly regulated and efficient oncolytic virus for in vivo applications.

  15. Identification of specific adenovirus E1A N-terminal residues critical to the binding of cellular proteins and to the control of cell growth.

    PubMed Central

    Wang, H G; Rikitake, Y; Carter, M C; Yaciuk, P; Abraham, S E; Zerler, B; Moran, E

    1993-01-01

    Adenovirus early region 1A (E1A) oncogene-encoded sequences essential for transformation- and cell growth-regulating activities are localized at the N terminus and in regions of highly conserved amino acid sequence designated conserved regions 1 and 2. These regions interact to form the binding sites for two classes of cellular proteins: those, such as the retinoblastoma gene product, whose association with the E1A products is specifically dependent on region 2, and another class which so far is known to include only a large cellular DNA-binding protein, p300, whose association with the E1A products is specifically dependent on the N-terminal region. Association between the E1A products and either class of cellular proteins can be disrupted by mutations in conserved region 1. While region 2 has been studied intensively, very little is known so far concerning the nature of the essential residues in the N-terminal region, or about the manner in which conserved region 1 participates in the binding of two distinct sets of cellular proteins. A combination of site-directed point mutagenesis and monoclonal antibody competition experiments reported here suggests that p300 binding is dependent on specific, conserved residues in the N terminus, including positively charged residues at positions 2 and 3 of the E1A proteins, and that p300 and pRB bind to distinct, nonoverlapping subregions within conserved region 1. The availability of precise point mutations disrupting p300 binding supports previous data linking p300 with cell cycle control and enhancer function. Images PMID:8416379

  16. Tamoxifen-regulated adenoviral E1A chimeras for the control of tumor selective oncolytic adenovirus replication in vitro and in vivo.

    PubMed

    Sipo, I; Wang, X; Hurtado Picó, A; Suckau, L; Weger, S; Poller, W; Fechner, H

    2006-01-01

    Pharmacological control is a desirable safety feature of oncolytic adenoviruses (oAdV). It has recently been shown that oAdV replication may be controlled by drug-dependent transcriptional regulation of E1A expression. Here, we present a novel concept that relies on tamoxifen-dependent regulation of E1A activity through functional linkage to the mutated hormone-binding domain of the murine estrogen receptor (Mer). Four different E1A-Mer chimeras (ME, EM, E(DeltaNLS)M, MEM) were constructed and inserted into the adenoviral genome under control of a lung-specific surfactant protein B promoter. The highest degree of regulation in vitro was seen for the corresponding oAdVs Ad.E(DeltaNLS)M and Ad.MEM, which exhibited an up to 100-fold higher oAdV replication in the presence as compared with the absence of 4-OH-tamoxifen. Moreover, destruction of nontarget cells was six- and 13-fold reduced for Ad.E(DeltaNLS)M and Ad.MEM, respectively, as compared with Ad.E. Further investigations supported tamoxifen-dependent regulation of Ad.E(DeltaNLS)M and Ad.MEM in vivo. Induction of Ad.E(DeltaNLS)M inhibited growth of H441 lung tumors as efficient as a control oAdV expressing E1A. E(DeltaNLS)M and the MEM chimeras can be easily inserted into a single vector genome, which extends their application to existing oAdVs and strongly facilitates in vivo application.

  17. The human papillomavirus E7 oncoprotein

    SciTech Connect

    McLaughlin-Drubin, Margaret E. Muenger, Karl

    2009-02-20

    The human papillomavirus (HPV) E7 oncoprotein shares functional similarities with such proteins as adenovirus E1A and SV40 large tumor antigen. As one of only two viral proteins always expressed in HPV-associated cancers, E7 plays a central role in both the viral life cycle and carcinogenic transformation. In the HPV viral life cycle, E7 disrupts the intimate association between cellular differentiation and proliferation in normal epithelium, allowing for viral replication in cells that would no longer be in the dividing population. This function is directly reflected in the transforming activities of E7, including tumor initiation and induction of genomic instability.

  18. Characterization of Murine Thymic Stromal-Cell Lines Immortalized by Temperature-Sensitive Simian Virus 40 Large T or Adenovirus 5 E1a

    PubMed Central

    Larsson, Lena; Timms, Emma; Blight, Kenneth; Restall, Deborah E.; Jat, Parmjit S.; Fisher, Amanda G.

    1991-01-01

    The heterogeneity of thymic stromal cells is probably related to their role in providing different microenvironments where T cells can develop. We have immortalized thymic stromal elements using recombinant retroviral constructs containing a temperature-sensitive simian virus 40 (SV40tsA58) large-T antigen gene or the adenovirus 5 E1a region linked to the gene coding for resistance to G418. Cell lines containing the thermolabile large T antigen encoded by SV40 proliferate at the permissive temperature of 33°C and arrest growth when transferred to the nonpermissive temperature of 39°C. At the nonpermissive temperature, ts-derived cell lines are shown to alter their phenotype but remain metabolically active, as indicated by the inducible expression of class I and class II MHC antigens. Here we describe the generation of a total of 84 thymic stromal-cell lines, many of which show distinct morphologic, phenotypic, and functional properties consistent with fibroblastoid, epithelial, or monocytoid origins. Several E1a and SV40tsA58-derived cell lines generated exhibit the epithelial characteristic of desmosome formation and, in addition, two of these lines (15.5 and 15.18) form multicellular complexes (rosettes) when incubated with unfractionated thymocytes from syngeneic mice. A single line (14.5) displays very strong nonspecific esterase activity, suggesting it may represent a macrophagelike cell type. We describe the generation of stromal cell lines with different properties, which is consistent with the heterogeneity found in the thymic microenvironment. In addition to documenting this diversity, these cell lines may be useful tools for studying T-cell development in vitro and give access to model systems in which stromal-thymocyte interactions can be examined. PMID:1668372

  19. The Human Adenovirus Type 5 E4orf6/E1B55K E3 Ubiquitin Ligase Complex Can Mimic E1A Effects on E2F

    PubMed Central

    Dallaire, Frédéric; Schreiner, Sabrina; Blair, G. Eric; Dobner, Thomas; Branton, Philip E.

    2015-01-01

    ABSTRACT The human adenovirus E4orf6/E1B55K E3 ubiquitin ligase is well known to promote viral replication by degrading an increasing number of cellular proteins that inhibit the efficient production of viral progeny. We report here a new function of the adenovirus 5 (Ad5) viral ligase complex that, although at lower levels, mimics effects of E1A products on E2F transcription factors. When expressed in the absence of E1A, the E4orf6 protein in complex with E1B55K binds E2F, disrupts E2F/retinoblastoma protein (Rb) complexes, and induces hyperphosphorylation of Rb, leading to induction of viral and cellular DNA synthesis as well as stimulation of early and late viral gene expression and production of viral progeny of E1/E3-defective adenovirus vectors. These new and previously undescribed functions of the E4orf6/E1B55K E3 ubiquitin ligase could play an important role in promoting the replication of wild-type viruses. IMPORTANCE During the course of work on the adenovirus E3 ubiquitin ligase formed by the viral E4orf6 and E1B55K proteins, we found, very surprisingly, that expression of these species was sufficient to permit low levels of replication of an adenovirus vector lacking E1A, the central regulator of infection. E1A products uncouple E2F transcription factors from Rb repression complexes, thus stimulating viral gene expression and cell and viral DNA synthesis. We found that the E4orf6/E1B55K ligase mimics these functions. This finding is of significance because it represents an entirely new function for the ligase in regulating adenovirus replication. PMID:27303679

  20. Interaction of Adenovirus E1A with the HHV8 Promoter of Latent Genes: E1A Proteins are Able to Activate the HHV-8 LANAp in MV3 Reporter Cells

    PubMed Central

    Koehler-Hansner, Karin; Flore, Ornella; Opalka, Bertram; Hengge, Ulrich R

    2008-01-01

    Human herpesvirus 8 (HHV-8) is associated with Kaposi’s sarcoma, body cavity-based lymphoma, and Castleman’s disease. Adenoviral (Ad) E1A proteins regulate the activity of cellular and viral promoters/enhancers and transcription factors and can suppress tumorigenicity of human cancers. As (i) HHV-8 and Ad may co-exist in immunocompromised patients and (ii) E1A might be considered as therapeutic transgene for HHV-8-associated neoplasms we investigated whether the promoter of the latency-associated nuclear antigen (LANAp) controlling expression of vCyclin, vFLIP, and LANA proteins required for latent type infection is regulated by E1A. Transfection experiments in MV3 melanoma cells revealed activation of the LANAp by Ad5 E1A constructs containing an intact N terminus (aa 1-119). In particular, an Ad12 E1A mutant, Spm2, lacking six consecutive alanine residues in the “spacer” region activated the HHV-8 promoter about 15-fold compared to vector controls. In summary, we report the activation of the LANAp by E1A as a novel interaction of E1A with a viral promoter. These data may have relevance for the management of viral infections in immunocompromised patients. A role for E1A as a therapeutic in this context remains to be defined. PMID:19440465

  1. Interaction of Adenovirus E1A with the HHV8 Promoter of Latent Genes: E1A Proteins are Able to Activate the HHV-8 LANAp in MV3 Reporter Cells.

    PubMed

    Koehler-Hansner, Karin; Flore, Ornella; Opalka, Bertram; Hengge, Ulrich R

    2008-01-01

    Human herpesvirus 8 (HHV-8) is associated with Kaposi's sarcoma, body cavity-based lymphoma, and Castleman's disease. Adenoviral (Ad) E1A proteins regulate the activity of cellular and viral promoters/enhancers and transcription factors and can suppress tumorigenicity of human cancers. As (i) HHV-8 and Ad may co-exist in immunocompromised patients and (ii) E1A might be considered as therapeutic transgene for HHV-8-associated neoplasms we investigated whether the promoter of the latency-associated nuclear antigen (LANAp) controlling expression of vCyclin, vFLIP, and LANA proteins required for latent type infection is regulated by E1A. Transfection experiments in MV3 melanoma cells revealed activation of the LANAp by Ad5 E1A constructs containing an intact N terminus (aa 1-119). In particular, an Ad12 E1A mutant, Spm2, lacking six consecutive alanine residues in the "spacer" region activated the HHV-8 promoter about 15-fold compared to vector controls. In summary, we report the activation of the LANAp by E1A as a novel interaction of E1A with a viral promoter. These data may have relevance for the management of viral infections in immunocompromised patients. A role for E1A as a therapeutic in this context remains to be defined.

  2. Human papillomavirus E6E7-mediated adenovirus cell killing: selectivity of mutant adenovirus replication in organotypic cultures of human keratinocytes.

    PubMed

    Balagué, C; Noya, F; Alemany, R; Chow, L T; Curiel, D T

    2001-08-01

    Replication-competent adenoviruses are being investigated as potential anticancer agents. Exclusive virus replication in cancer cells has been proposed as a safety trait to be considered in the design of oncolytic adenoviruses. From this perspective, we have investigated several adenovirus mutants for their potential to conditionally replicate and promote the killing of cells expressing human papillomavirus (HPV) E6 and E7 oncoproteins, which are present in a high percentage of anogenital cancers. For this purpose, we have employed an organotypic model of human stratified squamous epithelium derived from primary keratinocytes that have been engineered to express HPV-18 oncoproteins stably. We show that, whereas wild-type adenovirus promotes a widespread cytopathic effect in all infected cells, E1A- and E1A/E1B-deleted adenoviruses cause no deleterious effect regardless of the coexpression of HPV18 E6E7. An adenovirus deleted in the CR2 domain of E1A, necessary for binding to the pRB family of pocket proteins, shows no selectivity of replication as it efficiently kills all normal and E6E7-expressing keratinocytes. Finally, an adenovirus mutant deleted in the CR1 and CR2 domains of E1A exhibits preferential replication and cell killing in HPV E6E7-expressing cultures. We conclude that the organotypic keratinocyte culture represents a distinct model to evaluate adenovirus selectivity and that, based on this model, further modifications of the adenovirus genome are required to restrict adenovirus replication to tumor cells.

  3. Human Papillomavirus E6E7-Mediated Adenovirus Cell Killing: Selectivity of Mutant Adenovirus Replication in Organotypic Cultures of Human Keratinocytes

    PubMed Central

    Balagué, Cristina; Noya, Francisco; Alemany, Ramon; Chow, Louise T.; Curiel, David T.

    2001-01-01

    Replication-competent adenoviruses are being investigated as potential anticancer agents. Exclusive virus replication in cancer cells has been proposed as a safety trait to be considered in the design of oncolytic adenoviruses. From this perspective, we have investigated several adenovirus mutants for their potential to conditionally replicate and promote the killing of cells expressing human papillomavirus (HPV) E6 and E7 oncoproteins, which are present in a high percentage of anogenital cancers. For this purpose, we have employed an organotypic model of human stratified squamous epithelium derived from primary keratinocytes that have been engineered to express HPV-18 oncoproteins stably. We show that, whereas wild-type adenovirus promotes a widespread cytopathic effect in all infected cells, E1A- and E1A/E1B-deleted adenoviruses cause no deleterious effect regardless of the coexpression of HPV18 E6E7. An adenovirus deleted in the CR2 domain of E1A, necessary for binding to the pRB family of pocket proteins, shows no selectivity of replication as it efficiently kills all normal and E6E7-expressing keratinocytes. Finally, an adenovirus mutant deleted in the CR1 and CR2 domains of E1A exhibits preferential replication and cell killing in HPV E6E7-expressing cultures. We conclude that the organotypic keratinocyte culture represents a distinct model to evaluate adenovirus selectivity and that, based on this model, further modifications of the adenovirus genome are required to restrict adenovirus replication to tumor cells. PMID:11462032

  4. Ad5/35E1aPSESE4: A novel approach to marking circulating prostate tumor cells with a replication competent adenovirus controlled by PSA/PSMA transcription regulatory elements.

    PubMed

    Hwang, Ji-Eun; Joung, Jae Young; Shin, Seung-Phil; Choi, Moon-Kyung; Kim, Jeong Eun; Kim, Yon Hui; Park, Weon Seo; Lee, Sang-Jin; Lee, Kang Hyun

    2016-03-01

    Circulating tumor cells serve as useful biomarkers with which to identify disease status associated with survival, metastasis and drug sensitivity. Here, we established a novel application for detecting PSA/PSMA-positive prostate cancer cells circulating in peripheral blood employing an adenovirus called Ad5/35E1aPSESE4. Ad5/35E1aPSESE4 utilized PSES, a chimeric enhancer derived from PSA/PSMA promoters that is highly active with and without androgen. A fluorescence signal mediated by GFP expression upon Ad5/35E1aPSESE4 infection was selectively amplified in PSA/PSMA-positive prostate cancer cells in vitro and ex vivo. Furthermore, for the in vivo model, blood drawn from TRAMP was tested for CTCs with Ad5/35E1aPSESE4 infection and was positive for CTCs at week 16. Validation was performed on patient blood at various clinical stages and found out 1-100 CTCs expressing GFP upon Ad5/35E1aPSESE4 infection. Interestingly, CTC from one patient was confirmed to be sensitive to docetaxel chemotherapeutic reagent and to abundantly express metastasis-related genes like MMP9, Cofilin1, and FCER1G through RNA-seq. Our study established that the usage of Ad5/35E1aPSESE4 is effective in marking PSA/PSMA-positive prostate cancer cells in patient blood to improve the efficacy of utilizing CTCs as a biomarker.

  5. Functional and Structural Mimicry of Cellular Protein Kinase A Anchoring Proteins by a Viral Oncoprotein

    PubMed Central

    King, Cason R.; Cohen, Michael J.; Fonseca, Gregory J.; Dirk, Brennan S.; Dikeakos, Jimmy D.; Mymryk, Joe S.

    2016-01-01

    The oncoproteins of the small DNA tumor viruses interact with a plethora of cellular regulators to commandeer control of the infected cell. During infection, adenovirus E1A deregulates cAMP signalling and repurposes it for activation of viral gene expression. We show that E1A structurally and functionally mimics a cellular A-kinase anchoring protein (AKAP). E1A interacts with and relocalizes protein kinase A (PKA) to the nucleus, likely to virus replication centres, via an interaction with the regulatory subunits of PKA. Binding to PKA requires the N-terminus of E1A, which bears striking similarity to the amphipathic α-helical domain present in cellular AKAPs. E1A also targets the same docking-dimerization domain of PKA normally bound by cellular AKAPs. In addition, the AKAP like motif within E1A could restore PKA interaction to a cellular AKAP in which its normal interaction motif was deleted. During infection, E1A successfully competes with endogenous cellular AKAPs for PKA interaction. E1A’s role as a viral AKAP contributes to viral transcription, protein expression and progeny production. These data establish HAdV E1A as the first known viral AKAP. This represents a unique example of viral subversion of a crucial cellular regulatory pathway via structural mimicry of the PKA interaction domain of cellular AKAPs. PMID:27137912

  6. “Hit-and-Run” Transformation by Adenovirus Oncogenes

    PubMed Central

    Nevels, Michael; Täuber, Birgitt; Spruss, Thilo; Wolf, Hans; Dobner, Thomas

    2001-01-01

    According to classical concepts of viral oncogenesis, the persistence of virus-specific oncogenes is required to maintain the transformed cellular phenotype. In contrast, the “hit-and-run” hypothesis claims that viruses can mediate cellular transformation through an initial “hit,” while maintenance of the transformed state is compatible with the loss (“run”) of viral molecules. It is well established that the adenovirus E1A and E1B gene products can cooperatively transform primary human and rodent cells to a tumorigenic phenotype and that these cells permanently express the viral oncogenes. Additionally, recent studies have shown that the adenovirus E4 region encodes two novel oncoproteins, the products of E4orf6 and E4orf3, which cooperate with the viral E1A proteins to transform primary rat cells in an E1B-like fashion. Unexpectedly, however, cells transformed by E1A and either E4orf6 or E4orf3 fail to express the viral E4 gene products, and only a subset contain E1A proteins. In fact, the majority of these cells lack E4- and E1A-specific DNA sequences, indicating that transformation occurred through a hit-and-run mechanism. We provide evidence that the unusual transforming activities of the adenoviral oncoproteins may be due to their mutagenic potential. Our results strongly support the possibility that even tumors that lack any detectable virus-specific molecules can be of viral origin, which could have a significant impact on the use of adenoviral vectors for gene therapy. PMID:11238835

  7. Innate Immunity to Adenovirus

    PubMed Central

    Hendrickx, Rodinde; Stichling, Nicole; Koelen, Jorien; Kuryk, Lukasz; Lipiec, Agnieszka

    2014-01-01

    Abstract Human adenoviruses are the most widely used vectors in gene medicine, with applications ranging from oncolytic therapies to vaccinations, but adenovirus vectors are not without side effects. In addition, natural adenoviruses pose severe risks for immunocompromised people, yet infections are usually mild and self-limiting in immunocompetent individuals. Here we describe how adenoviruses are recognized by the host innate defense system during entry and replication in immune and nonimmune cells. Innate defense protects the host and represents a major barrier to using adenoviruses as therapeutic interventions in humans. Innate response against adenoviruses involves intrinsic factors present at constant levels, and innate factors mounted by the host cell upon viral challenge. These factors exert antiviral effects by directly binding to viruses or viral components, or shield the virus, for example, soluble factors, such as blood clotting components, the complement system, preexisting immunoglobulins, or defensins. In addition, Toll-like receptors and lectins in the plasma membrane and endosomes are intrinsic factors against adenoviruses. Important innate factors restricting adenovirus in the cytosol are tripartite motif-containing proteins, nucleotide-binding oligomerization domain-like inflammatory receptors, and DNA sensors triggering interferon, such as DEAD (Asp-Glu-Ala-Asp) box polypeptide 41 and cyclic guanosine monophosphate–adenosine monophosphate synthase. Adenovirus tunes the function of antiviral autophagy, and counters innate defense by virtue of its early proteins E1A, E1B, E3, and E4 and two virus-associated noncoding RNAs VA-I and VA-II. We conclude by discussing strategies to engineer adenovirus vectors with attenuated innate responses and enhanced delivery features. PMID:24512150

  8. Novel mechanism of JNK pathway activation by adenoviral E1A.

    PubMed

    Romanov, Vasily S; Brichkina, Anna I; Morrison, Helen; Pospelova, Tatiana V; Pospelov, Valery A; Herrlich, Peter

    2014-04-30

    The adenoviral oncoprotein E1A influences cellular regulation by interacting with a number of cellular proteins. In collaboration with complementary oncogenes, E1A fully transforms primary cells. As part of this action, E1A inhibits transcription of c-Jun:Fos target genes while promoting that of c-Jun:ATF2-dependent genes including jun. Both c-Jun and ATF2 are hyperphosphorylated in response to E1A. In the current study, E1A was fused with the ligand binding domain of the estrogen receptor (E1A-ER) to monitor the immediate effect of E1A activation. With this approach we now show that E1A activates c-Jun N-terminal kinase (JNK), the upstream kinases MKK4 and MKK7, as well as the small GTPase Rac1. Activation of the JNK pathway requires the N-terminal domain of E1A, and, importantly, is independent of transcription. In addition, it requires the presence of ERM proteins. Downregulation of signaling components upstream of JNK inhibits E1A-dependent JNK/c-Jun activation. Taking these findings together, we show that E1A activates the JNK/c-Jun signaling pathway upstream of Rac1 in a transcription-independent manner, demonstrating a novel mechanism of E1A action.

  9. Waterborne adenovirus.

    PubMed

    Mena, Kristina D; Gerba, Charles P

    2009-01-01

    Adenoviruses are associated with numerous disease outbreaks, particularly those involving d-cares, schools, children's camps, hospitals and other health care centers, and military settings. In addition, adenoviruses have been responsible for many recreational water outbreaks, including a great number of swimming pool outbreaks than any other waterborne virus (Gerba and Enriquez 1997). Two drinking water outbreaks have been documented for adenovirus (Divizia et al. 2004; Kukkula et al. 1997) but none for food. Of the 51 known adenovirus serotypes, one third are associated with human disease, while other infections are asymptomatic. Human disease associated with adenovirus infections include gastroenteritis, respiratory infections, eye infections, acute hemorrhagic cystitis, and meningoencephalitis (Table 2). Children and the immunocompromised are more severely impacted by adenovirus infections. Subsequently, adenovirus is included in the EPA's Drinking Water Contaminant Candidate List (CCL), which is a list of unregulated contaminants found in public water systems that may pose a risk to public health (National Research Council 1999). Adenoviruses have been detected in various waters worldwide including wastewater, river water, oceans, and swimming pools (Hurst et al. 1988; Irving and Smith 1981; Pina et al. 1998). Adenoviruses typically outnumber the enteroviruses, when both are detected in surface waters. Chapron et al. (2000) found that 38% of 29 surface water samples were positive for infectious Ad40 and Ad41. Data are lacking regarding the occurrence of adenovirus in water in the US, particularly for groundwater and drinking water. Studies have shown, however, that adenoviruses survive longer in water than enteroviruses and hepatitis A virus (Enriquez et al. 1995), which may be due to their double-stranded DNA. Risk assessments have been conducted on waterborne adenovirus (Crabtree et al. 1997; van Heerden et al. 2005c). Using dose-response data for inhalation

  10. E1a induces the expression of epithelial characteristics

    PubMed Central

    1994-01-01

    Cells closely resembling epithelia constitute the first specific cell type in a mammalian embryo. Many other cell types emerge via epithelial- mesenchymal differentiation. The transcription factors and signal transduction pathways involved in this differentiation are being elucidated. I have previously reported (Frisch, 1991) that adenovirus E1a is a tumor suppressor gene in certain human cell lines. In the present report, I demonstrate that E1a expression caused diverse human tumor cells (rhabdomyosarcoma, fibrosarcoma, melanoma, osteosarcoma) and fibroblasts to assume at least two of the following epithelial characteristics: (a) epithelioid morphology; (b) epithelial-type intercellular adhesion proteins localized to newly formed junctional complexes; (c) keratin-containing intermediate filaments; and (d) down- regulation of non-epithelial genes. E1a thus appeared to partially convert diverse human tumor cells into an epithelial phenotype. This provides a new system for molecular analysis of epithelial-mesenchymal interconversions. This effect may also contribute to E1a's tumor suppression activity, possibly through sensitization to anoikis (Frisch, S.M., and H. Francis, 1994. J. Cell Biol. 124:619-626). PMID:7525602

  11. E1A dependent up-regulation of c-jun/AP-1 activity.

    PubMed Central

    Kitabayashi, I; Chiu, R; Gachelin, G; Yokoyama, K

    1991-01-01

    E1A, the early region 1A transcription unit of human adenovirus, exhibits multiple functions that regulate the expression of some cellular genes and promote cell growth and division. We found that E1A stimulated c-jun gene expression at least fifty-fold in rat 3Y1 cells in a serum-independent manner, concomitantly with E1A down-regulation of jun B expression. The E1A-dependent induction of c-jun transcription resulted in increase amount of cJun/AP1. This induction was mediated by the enhancement of the binding activity of the transcription factor cJun/AP1 to an AP1 binding site in the c-jun promoter. Additionally, this induction can be repressed by introducing junB into the cells. Taken collectively, these results suggest that the differential expression of two closely related proteins greatly expands their cellular regulation. Induction of c-jun expression by E1A as well as c-jun autoregulation may amplify the action of E1A during adenovirus infection. Therefore, some of the biological effects of E1A may include mediating the constitutive activation of c-jun, which is important in transcriptional regulation and oncogenic transformation. Images PMID:1826351

  12. Regulation of Human Adenovirus Replication by RNA Interference

    PubMed Central

    Nikitenko, N. A.; Speiseder, T.; Lam, E.; Rubtsov, P. M.; Tonaeva, Kh. D.; Borzenok, S. A.; Dobner, T.; Prassolov, V. S.

    2015-01-01

    Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to be promising targets. Here, we propose an effective approach to downregulate the replication of human species D adenoviruses by means of RNA interference. We generated E1A expressing model cell lines enabling fast evaluation of the RNA interference potential. Small interfering RNAs complementary to the E1A mRNA sequences of human species D adenoviruses mediate significant suppression of the E1A expression in model cells. Furthermore, we observed a strong downregulation of replication of human adenoviruses type D8 and D37 by small hairpin RNAs complementary to the E1A or E2B mRNA sequences in primary human limbal cells. We believe that our results will contribute to the development of efficient anti-adenoviral therapy. PMID:26483965

  13. Regulation of Human Adenovirus Replication by RNA Interference.

    PubMed

    Nikitenko, N A; Speiseder, T; Lam, E; Rubtsov, P M; Tonaeva, Kh D; Borzenok, S A; Dobner, T; Prassolov, V S

    2015-01-01

    Adenoviruses cause a wide variety of human infectious diseases. Adenoviral conjunctivitis and epidemic keratoconjunctivitis are commonly associated with human species D adenoviruses. Currently, there is no sufficient or appropriate treatment to counteract these adenovirus infections. Thus, there is an urgent need for new etiology-directed therapies with selective activity against human adenoviruses. To address this problem, the adenoviral early genes E1A and E2B (viral DNA polymerase) seem to be promising targets. Here, we propose an effective approach to downregulate the replication of human species D adenoviruses by means of RNA interference. We generated E1A expressing model cell lines enabling fast evaluation of the RNA interference potential. Small interfering RNAs complementary to the E1A mRNA sequences of human species D adenoviruses mediate significant suppression of the E1A expression in model cells. Furthermore, we observed a strong downregulation of replication of human adenoviruses type D8 and D37 by small hairpin RNAs complementary to the E1A or E2B mRNA sequences in primary human limbal cells. We believe that our results will contribute to the development of efficient anti-adenoviral therapy.

  14. Human papillomavirus oncoproteins and apoptosis (Review)

    PubMed Central

    JIANG, PEIYUE; YUE, YING

    2014-01-01

    The aim of this study was to review the literature and identify the association between human papillomavirus (HPV) oncoproteins and apoptosis. HPV-associated apoptosis may be primarily blocked by a number of oncoproteins, including E5, E6 and E7. E5 protein protects cells from tumor necrosis factor-associated apoptosis; the oncoprotein E6 predominantly inhibits apoptosis through the p53 pathway; and oncoprotein E7 is involved in apoptosis activation and inhibition. In addition, HPV oncoproteins are involved in activating or repressing the transcription of E6/E7. In conclusion, HPV oncoproteins, including E5, E6 and E7 protein, may interfere with apoptosis via certain regulatory principles. PMID:24348754

  15. The C-terminal region of E1A: a molecular tool for cellular cartography.

    PubMed

    Yousef, Ahmed F; Fonseca, Gregory J; Cohen, Michael J; Mymryk, Joe S

    2012-04-01

    The adenovirus E1A proteins function via protein-protein interactions. By making many connections with the cellular protein network, individual modules of this virally encoded hub reprogram numerous aspects of cell function and behavior. Although many of these interactions have been thoroughly studied, those mediated by the C-terminal region of E1A are less well understood. This review focuses on how this region of E1A affects cell cycle progression, apoptosis, senescence, transformation, and conversion of cells to an epithelial state through interactions with CTBP1/2, DYRK1A/B, FOXK1/2, and importin-α. Furthermore, novel potential pathways that the C-terminus of E1A influences through these connections with the cellular interaction network are discussed.

  16. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  17. Activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  18. Adenovirus structure.

    PubMed

    Rux, John J; Burnett, Roger M

    2004-12-01

    Structural studies continue to play an essential role as the focus of adenovirus research shifts in emphasis from basic biology to adenovirus-based vector technologies. A crucial step in developing novel therapeutics for gene replacement, cancer, and vaccines is often to modify the virion. Such engineered changes are designed to retarget the virus, or to reduce the immunological responses to infection. These efforts are far more effective when they are based on detailed structural knowledge. This minireview provides a brief summary of the wealth of information that has been obtained from the combined application of X-ray crystallography and electron microscopy. This knowledge now includes a good working model for the architectural organization of the virion, and atomic resolution molecular structures for all the major capsid proteins, hexon, penton, and fiber. We highlight new developments, which include the structure of the penton base and the discovery that adenovirus has several relatives. We sketch how the structural information can be used to engineer novel virions and conclude with the prospects for future progress.

  19. Suppression of Adenovirus Replication by Cardiotonic Steroids.

    PubMed

    Grosso, Filomena; Stoilov, Peter; Lingwood, Clifford; Brown, Martha; Cochrane, Alan

    2017-02-01

    The dependence of adenovirus on the host pre-RNA splicing machinery for expression of its complete genome potentially makes it vulnerable to modulators of RNA splicing, such as digoxin and digitoxin. Both drugs reduced the yields of four human adenoviruses (HAdV-A31, -B35, and -C5 and a species D conjunctivitis isolate) by at least 2 to 3 logs by affecting one or more steps needed for genome replication. Immediate early E1A protein levels are unaffected by the drugs, but synthesis of the delayed protein E4orf6 and the major late capsid protein hexon is compromised. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that both drugs altered E1A RNA splicing (favoring the production of 13S over 12S RNA) early in infection and partially blocked the transition from 12S and 13S to 9S RNA at late stages of virus replication. Expression of multiple late viral protein mRNAs was lost in the presence of either drug, consistent with the observed block in viral DNA replication. The antiviral effect was dependent on the continued presence of the drug and was rapidly reversible. RIDK34, a derivative of convallotoxin, although having more potent antiviral activity, did not show an improved selectivity index. All three drugs reduced metabolic activity to some degree without evidence of cell death. By blocking adenovirus replication at one or more steps beyond the onset of E1A expression and prior to genome replication, digoxin and digitoxin show potential as antiviral agents for treatment of serious adenovirus infections. Furthermore, understanding the mechanism(s) by which digoxin and digitoxin inhibit adenovirus replication will guide the development of novel antiviral therapies.

  20. DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1

    PubMed Central

    1994-01-01

    The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells. PMID:8175885

  1. Inhibitory effect of interferon-gamma on adenovirus replication and late transcription.

    PubMed

    Mistchenko, A S; Diez, R A; Falcoff, R

    1989-06-15

    We have previously shown that human interferon-gamma inhibited adenovirus multiplication in vitro in a dose-dependent fashion. This action was previous to capsid proteins synthesis and did not involve virus adsorption nor penetration. In this report we have analysed viral mRNA levels at early (7 hr post infection (p.i.)) or late (20 hr p.i.) times, as well as DNA replication in Wish cells pretreated with interferon-gamma and infected with adenovirus 5. Controls included untreated cells as well as cells treated with interferon-alpha, to which adenovirus are reported to be resistant. Transcription of adenovirus regions E1, E4, L1 and L2 has been analysed by Northern blot. Adenovirus DNA replication was determined by DNA-DNA hybridization with total adenovirus 2 DNA. We have also searched for adenovirus E1A proteins by immunoblot with a specific monoclonal antibody. Although pretreatment of cells with either interferon-alpha or interferon-gamma resulted in reduced amounts of E1 and E4 mRNA in the early phase of infection (7 hr p.i.), the near complete inhibition of viral DNA and late transcription was only achieved by interferon-gamma. Immunoblot has shown the absence of the 48-kD E1A protein in cells pretreated with interferon-gamma. The lack of this regulatory adenovirus protein may be involved in the inhibitory mechanism of interferon-gamma on adenovirus.

  2. Dissecting the roles of E1A and E1B in adenoviral replication and RCAd-enhanced RDAd transduction efficacy on tumor cells

    PubMed Central

    Wei, Fang; Wang, Huiping; Chen, Xiafang; Li, Chuanyuan; Huang, Qian

    2014-01-01

    Oncolytic viruses have recently received widespread attention for their potential in innovative cancer therapy. Many telomerase promoter-regulated oncolytic adenoviral vectors retain E1A and E1B. However, the functions of E1A and E1B proteins in the oncolytic role of replication-competent adenovirus (RCAd) and RCAd enhanced transduction of replication defective adenoviruses (RDAd) have not been addressed well. In this study, we constructed viruses expressing E1A alone, E1A plus E1B-19 kDa, and E1A plus E1B-19 kDa/55 kDa. We then tested their roles in oncolysis and replication of RCAd as well as their roles in RCAd enhanced transfection rate and transgene expression of RDAd in various cancer cells in vitro and in xenografted human NCI-H460 tumors in nude mice. We demonstrated that RCAds expressing E1A alone and plus E1B-19 kDa exhibited an obvious ability in replication and oncolytic effects as well as enhanced RDAd replication and transgene expression, with the former showed more effective oncolysis, while the latter exhibited superior viral replication and transgene promotion activity. However, RCAd expressing both E1A and E1B-19 kDa/55 kDa was clearly worst in all these abilities. The effects of E1A and E1B observed through using RCAd were further validated by using plasmids expressing E1A alone, E1A plus E1B-19 kDa, and E1A plus E1B-19 kDa/55 kDa proteins. Our study provided evidence that E1A was essential for inducing replication and oncolytic effects of RCAd as well as RCAd enhanced RDAd transduction, and expression of E1B-19 kDa other than E1B-55 kDa could promote these effects. E1B-55 kDa is not necessary for the oncolytic effects of adenoviruses and somehow inhibits RCAd-mediated RDAd replication and transgene expression. PMID:25019940

  3. Adenovirus (For Parents)

    MedlinePlus

    ... common in late winter, spring, and early summer conjunctivitis (pinkeye) and pharyngoconjunctival fever caused by adenovirus tend to ... cystitis usually resolves on its own. Eye infections: Pinkeye (conjunctivitis) is a mild inflammation of the conjunctiva ( ...

  4. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, C.W.; Mangel, W.F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying the peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described. 29 figs.

  5. Co-factor activated recombinant adenovirus proteinases

    DOEpatents

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  6. Recombinant soluble adenovirus receptor

    DOEpatents

    Freimuth, Paul I.

    2002-01-01

    Disclosed are isolated polypeptides from human CAR (coxsackievirus and adenovirus receptor) protein which bind adenovirus. Specifically disclosed are amino acid sequences which corresponds to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2. In other aspects, the disclosure relates to nucleic acid sequences encoding these domains as well as expression vectors which encode the domains and bacterial cells containing such vectors. Also disclosed is an isolated fusion protein comprised of the D1 polypeptide sequence fused to a polypeptide sequence which facilitates folding of D1 into a functional, soluble domain when expressed in bacteria. The functional D1 domain finds application for example in a therapeutic method for treating a patient infected with a virus which binds to D1, and also in a method for identifying an antiviral compound which interferes with viral attachment. Also included is a method for specifically targeting a cell for infection by a virus which binds to D1.

  7. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... and antisera used in serological tests to identify antibodies to adenovirus in serum. Additionally... identify adenoviruses directly from clinical specimens. The identification aids in the diagnosis of disease caused by adenoviruses and provides epidemiological information on these diseases. Adenovirus...

  8. Use of macrophages to target therapeutic adenovirus to human prostate tumors.

    PubMed

    Muthana, Munitta; Giannoudis, Athina; Scott, Simon D; Fang, Hsin-Yu; Coffelt, Seth B; Morrow, Fiona J; Murdoch, Craig; Burton, Julian; Cross, Neil; Burke, Bernard; Mistry, Roshna; Hamdy, Freddie; Brown, Nicola J; Georgopoulos, Lindsay; Hoskin, Peter; Essand, Magnus; Lewis, Claire E; Maitland, Norman J

    2011-03-01

    New therapies are required to target hypoxic areas of tumors as these sites are highly resistant to conventional cancer therapies. Monocytes continuously extravasate from the bloodstream into tumors where they differentiate into macrophages and accumulate in hypoxic areas, thereby opening up the possibility of using these cells as vehicles to deliver gene therapy to these otherwise inaccessible sites. We describe a new cell-based method that selectively targets an oncolytic adenovirus to hypoxic areas of prostate tumors. In this approach, macrophages were cotransduced with a hypoxia-regulated E1A/B construct and an E1A-dependent oncolytic adenovirus, whose proliferation is restricted to prostate tumor cells using prostate-specific promoter elements from the TARP, PSA, and PMSA genes. When such cotransduced cells reach an area of extreme hypoxia, the E1A/B proteins are expressed, thereby activating replication of the adenovirus. The virus is subsequently released by the host macrophage and infects neighboring tumor cells. Following systemic injection into mice bearing subcutaneous or orthotopic prostate tumors, cotransduced macrophages migrated into hypoxic tumor areas, upregulated E1A protein, and released multiple copies of adenovirus. The virus then infected neighboring cells but only proliferated and was cytotoxic in prostate tumor cells, resulting in the marked inhibition of tumor growth and reduction of pulmonary metastases. This novel delivery system employs 3 levels of tumor specificity: the natural "homing" of macrophages to hypoxic tumor areas, hypoxia-induced proliferation of the therapeutic adenovirus in host macrophages, and targeted replication of oncolytic virus in prostate tumor cells.

  9. Adenovirus infection elevates levels of cellular topoisomerase I.

    PubMed Central

    Chow, K C; Pearson, G D

    1985-01-01

    We have developed a specific, sensitive, and quantitative assay for topoisomerase I, which is based on the formation of a covalent enzyme-DNA intermediate. Our assay measures the quantitative transfer of 32P radioactivity from 32P-labeled DNA to topoisomerase I. Since 32P-labeled topoisomerase molecules are resolved by NaDodSO4/PAGE, HeLa topoisomerase I (100 kDa) and calf thymus topoisomerase I (82 kDa) can be quantitatively assayed in the same reaction mixture. The assay can detect at least 0.3 ng (3 fmol) of topoisomerase I. We have used our assay to measure the levels of topoisomerase I activity in crude extracts of nuclei prepared from uninfected, adenovirus-infected, and adenovirus-transformed human cells. The evidence suggests that an adenovirus early gene product, presumably a protein encoded in early region 1A (E1A), increases cellular topoisomerase I activity at least 10-fold. Immunoblotting analysis with antiserum against calf thymus topoisomerase I shows that the increase in activity is due to an increase in the amount of enzyme. Images PMID:2986107

  10. New Approach to Identify Novel Regulators of Myc Oncoprotein Stability

    DTIC Science & Technology

    2012-09-01

    Regulators of Myc Oncoprotein Stability PRINCIPAL INVESTIGATOR: Linda Z. Penn, Ph D CONTRACTING ORGANIZATION : University Health Network...uhnres.utoronto.ca 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBER...research. 6 KEY RESEARCH ACCOMPLISHMENTS: Constructed new Myc-FP chimeras . Showed that Myc half-life and Myc-FP fluorescence are

  11. Targeting of the MUC1-C Oncoprotein in Colitis-Associated Colorectal Cancer

    DTIC Science & Technology

    2013-09-01

    expression of the mucin 1 (MUC1) oncoprotein by chronic intestinal inflammation contributes to the development of colorectal cancers that are in turn...regulation. Oncogene, 2010. 29:5663-6. 3. Kufe, D, MUC1-C oncoprotein as a target in breast cancer : activation of signaling pathways and therapeutic...of the MUC1 oncoprotein blocks survival and tumorigenicity of human breast carcinoma cells. Cancer Res, 2009. 69:5133–41. 5. Raina, D, Kosugi M

  12. Canine adenovirus based rabies vaccines.

    PubMed

    Tordo, N; Foumier, A; Jallet, C; Szelechowski, M; Klonjkowski, B; Eloit, M

    2008-01-01

    Adenovirus based vectors are very attractive candidates for vaccination purposes as they induce in mammalian hosts potent humoral, mucosal and cellular immune responses to antigens encoded by the inserted genes. We have generated E1-deleted and replication-competent recombinant canine type-2 adenoviruses expressing the rabies virus glycoprotein (G). The effectiveness of both vectors to express a native G protein has been characterized in vitro in permissive cell lines. We compared the humoral and cellular immune responses induced in mice by intramuscular injection of the recombinant canine adenovirus vectors with those induced by a human (Ad5) E1-deleted virus expressing the same rabies G protein. Humoral responses specific to the adenoviruses or the rabies glycoprotein antigens were studied. The influence of the mouse strain was observed using replication-competent canine adenovirus. A high level of rabies neutralizing antibody was observed upon i.m. inoculation, and 100% of mice survived lethal challenge. These results are very promising in the perspective of oral vaccine for dog rabies control.

  13. The adenoviral E1A N-terminal domain represses MYC transcription in human cancer cells by targeting both p300 and TRRAP and inhibiting MYC promoter acetylation of H3K18 and H4K16

    PubMed Central

    Zhao, Ling-Jun; Loewenstein, Paul M.; Green, Maurice

    2016-01-01

    Human cancers frequently arise from increased expression of proto-oncogenes, such as MYC and HER2. Understanding the cellular pathways regulating the transcription and expression of proto-oncogenes is important for targeted therapies for cancer treatment. Adenoviral (Ad) E1A 243R (243 aa residues) is a viral oncoprotein that interacts with key regulators of gene transcription and cell proliferation. We have shown previously that the 80 amino acid N-terminal transcriptional repression domain of E1A 243R (E1A 1-80) can target the histone acetyltransferase (HAT) p300 and repress HER2 in the HER2-overexpressing human breast cancer cell line SKBR3. Expression of E1A 1-80 induces death of SKBR3 and other cancer cell lines. In this study, we performed total cell RNA sequence analysis and identified MYC as the regulatory gene for cellular proliferation most strongly repressed by E1A 1-80. By RT-quantitative PCR analysis we show that repression of MYC in SKBR3 cells occurs early after expression of E1A 1-80, suggesting that MYC may be an early responder of E1A 1-80-mediated transcriptional repression. Of interest, while E1A 1-80 repression of MYC occurs in all eight human cancer cell lines examined, repression of HER2 is cell-type dependent. We demonstrate by ChIP analysis that MYC transcriptional repression by E1A 1-80 is associated with inhibition of acetylation of H3K18 and H4K16 on the MYC promoter, as well as inhibition of RNA Pol II binding to the MYC promoter. Deletion mutant analysis of E1A 1-80 suggests that both p300/CBP and TRRAP are involved in E1A 1-80 repression of MYC transcription. Further, E1A 1-80 interaction with p300/CBP and TRRAP is correlated with inhibition of H3K18 and H4K16 acetylation on the MYC promoter, respectively. Our results indicate that E1A 1-80 may target two important pathways for histone modification to repress transcription in human cancer cells. PMID:27382434

  14. Structure-function analysis of the human papillomavirus type 16 E7 oncoprotein.

    PubMed Central

    Phelps, W C; Münger, K; Yee, C L; Barnes, J A; Howley, P M

    1992-01-01

    The E7 gene of human papillomavirus type 16 encodes a multifunctional nuclear phosphoprotein that is functionally and structurally similar to the adenovirus (Ad) E1A proteins and the T antigens of other papovaviruses. E7 can cooperate with an activated ras oncogene to transform primary rodent cells, trans activate the Ad E2 promoter, and abrogate transforming growth factor beta-mediated repression of c-myc. Recent studies suggest that these functions may in part be a consequence of the ability of E7 to associate with the product of the retinoblastoma tumor suppressor gene (pRB). In this study, a series of site-specific mutations of the human papillomavirus type 16 E7 gene product were constructed and assessed for their effects on intracellular protein stability, ras cooperativity, transcriptional trans activation, pRB association, and phosphorylation. The results of these studies indicate that the transforming and trans-activating domains extensively overlap within a region of the protein analogous to conserved region 2 of Ad E1A, suggesting that pRB binding is necessary for both activities. Deletion of sequences in conserved region 1 abrogates cellular transformation but has only a marginal effect on trans activation. These data suggest that E7 trans activation and cellular transformation are interrelated but separable functions. Images PMID:1312637

  15. Effect of exposure to UV-C irradiation and monochloramine on adenovirus serotype 2 early protein expression and DNA replication.

    PubMed

    Sirikanchana, Kwanrawee; Shisler, Joanna L; Mariñas, Benito J

    2008-06-01

    The mechanisms of adenovirus serotype 2 inactivation with either UV light (with a narrow emission spectrum centered at 254 nm) or monochloramine were investigated by assessing the potential inhibition of two key steps of the adenovirus life cycle, namely, E1A protein synthesis and viral genomic replication. E1A early protein synthesis was assayed by using immunoblotting, while the replication of viral DNA was analyzed by using slot blotting. Disinfection experiments were performed in phosphate buffer solutions at pH 8 and room temperature (UV) or 20 degrees C (monochloramine). Experimental results revealed that normalized E1A levels at 12 h postinfection (p.i.) were statistically the same as the corresponding decrease in survival ratio for both UV and monochloramine disinfection. Normalized DNA levels at 24 h p.i. were also found to be statistically the same as the corresponding decrease in survival ratio for monochloramine disinfection. In contrast, for UV disinfection, genomic DNA levels were much lower than E1A or survival ratios, possibly as a result of a delay in DNA replication for UV-treated virions compared to that for controls. Future efforts will determine the pre-E1A synthesis step in the adenovirus life cycle affected by exposure to UV and monochloramine, with the goal of identifying the viral molecular target of these two disinfectants.

  16. Adenoviruses in the immunocompromised host.

    PubMed Central

    Hierholzer, J C

    1992-01-01

    Adenoviruses are among the many pathogens and opportunistic agents that cause serious infection in the congenitally immunocompromised, in patients undergoing immunosuppressive treatment for organ and tissue transplants and for cancers, and in human immunodeficiency virus-infected patients. Adenovirus infections in these patients tend to become disseminated and severe, and the serotypes involved are clustered according to the age of the patient and the nature of the immunosuppression. Over 300 adenovirus infections in immunocompromised patients, with an overall case fatality rate of 48%, are reviewed in this paper. Children with severe combined immunodeficiency syndrome and other primary immunodeficiencies are exposed to the serotypes of subgroups B and C that commonly infect young children, and thus their infections are due to types 1 to 7 and 31 of subgenus A. Children with bone marrow and liver transplants often have lung and liver adenovirus infections that are due to an expanded set of subgenus A, B, C, and E serotypes. Adults with kidney transplants have viruses of subgenus B, mostly types 11, 34, and 35, which cause cystitis. This review indicates that 11% of transplant recipients become infected with adenoviruses, with case fatality rates from 60% for bone marrow transplant patients to 18% for renal transplant patients. Patients with AIDS become infected with a diversity of serotypes of all subgenera because their adult age and life-style expose them to many adenoviruses, possibly resulting in antigenically intermediate strains that are not found elsewhere. Interestingly, isolates from the urine of AIDS patients are generally of subgenus B and comprise types 11, 21, 34, 35, and intermediate strains of these types, whereas isolates from stool are of subgenus D and comprise many rare, new, and intermediate strains that are untypeable for practical purposes. It has been estimated that adenoviruses cause active infection in 12% of AIDS patients and that 45% of

  17. Adenovirus Early Proteins and Host Sumoylation

    PubMed Central

    Sohn, Sook-Young

    2016-01-01

    ABSTRACT The human adenovirus genome is transported into the nucleus, where viral gene transcription, viral DNA replication, and virion assembly take place. Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) are implicated in the regulation of diverse cellular processes, particularly nuclear events. It is not surprising, therefore, that adenovirus modulates and utilizes the host sumoylation system. Adenovirus early proteins play an important role in establishing optimal host environments for virus replication within infected cells by stimulating the cell cycle and counteracting host antiviral defenses. Here, we review findings on the mechanisms and functional consequences of the interplay between human adenovirus early proteins and the host sumoylation system. PMID:27651358

  18. Combination therapy with conditionally replicating adenovirus and replication defective adenovirus.

    PubMed

    Lee, Choon-Taek; Park, Kyung-Ho; Yanagisawa, Kiyoshi; Adachi, Yasushi; Ohm, Joyce E; Nadaf, Sorena; Dikov, Mikhail M; Curiel, David T; Carbone, David P

    2004-09-15

    Low gene transfer rate is the most substantial hurdle in the practical application of gene therapy. One strategy to improve transfer efficiency is the use of a conditionally replicating adenovirus (CRAD) that can selectively replicate in tumor cells. We hypothesized that conventional E1-deleted adenoviruses (ad) can become replication-competent when cotransduced with a CRAD to selectively supply E1 in trans in tumors. The resulting selective production of large numbers of the E1-deleted ad within the tumor mass will increase the transduction efficiency. We used a CRAD (Delta24RGD) that produces a mutant E1 without the ability to bind retinoblastoma but retaining viral replication competence in cancer cells with a defective pRb/p16. Ad-lacZ, adenovirus-luciferase (ad-luc), and adenovirus insulin-like growth factor-1R/dominant-negative (ad-IGF-1R/dn; 482, 950) are E1-deleted replication-defective adenoviruses. The combination of CRAD and ad-lacZ increased the transduction efficiency of lacZ to 100% from 15% observed with ad-lacZ alone. Transfer of media of CRAD and ad-lacZ cotransduced cells induced the transfer of lacZ (media transferable bystander effect). Combination of CRAD and ad-IGF-1R/dn increased the production of truncated IGF-1R or soluble IGF-1R > 10 times compared with transduction with ad-IGF-1R/dn alone. Combined intratumoral injection of CRAD and ad-luc increased the luciferase expression about 70 times compared with ad-luc alone without substantial systemic spread. Combined intratumoral injection of CRAD and ad-IGF-1R/482 induced stronger growth suppression of established lung cancer xenografts than single injections. The combination of CRAD and E1-deleted ad induced tumor-specific replication of CRAD and E1-deleted ad and increased the transduction rate and therapeutic efficacy of these viruses in model tumors.

  19. Bak and Bax Function To Limit Adenovirus Replication through Apoptosis Induction

    PubMed Central

    Cuconati, Andrea; Degenhardt, Kurt; Sundararajan, Ramya; Anschel, Alan; White, Eileen

    2002-01-01

    Adenovirus infection and expression of E1A induces both proliferation and apoptosis, the latter of which is blocked by the adenovirus Bcl-2 homologue E1B 19K. The mechanism of apoptosis induction and the role that it plays in productive infection are not known. Unlike apoptosis mediated by death receptors, infection with proapoptotic E1B 19K mutant viruses did not induce cleavage of Bid but nonetheless induced changes in Bak and Bax conformation, Bak-Bax interaction, caspase 9 and 3 activation, and apoptosis. In wild-type-adenovirus-infected cells, in which E1B 19K inhibits apoptosis, E1B 19K was bound to Bak, precluding Bak-Bax interaction and changes in Bax conformation. Infection with E1B 19K mutant viruses induced apoptosis in wild-type and Bax- or Bak-deficient baby mouse kidney cells but not in those deficient for both Bax and Bak. Furthermore, Bax and Bak deficiency dramatically increased E1A expression and virus replication. Thus, Bax- and Bak-mediated apoptosis severely limits adenoviral replication, demonstrating that Bax and Bak function as an antiviral response at the cellular level. PMID:11932420

  20. Bak and Bax function to limit adenovirus replication through apoptosis induction.

    PubMed

    Cuconati, Andrea; Degenhardt, Kurt; Sundararajan, Ramya; Anschel, Alan; White, Eileen

    2002-05-01

    Adenovirus infection and expression of E1A induces both proliferation and apoptosis, the latter of which is blocked by the adenovirus Bcl-2 homologue E1B 19K. The mechanism of apoptosis induction and the role that it plays in productive infection are not known. Unlike apoptosis mediated by death receptors, infection with proapoptotic E1B 19K mutant viruses did not induce cleavage of Bid but nonetheless induced changes in Bak and Bax conformation, Bak-Bax interaction, caspase 9 and 3 activation, and apoptosis. In wild-type-adenovirus-infected cells, in which E1B 19K inhibits apoptosis, E1B 19K was bound to Bak, precluding Bak-Bax interaction and changes in Bax conformation. Infection with E1B 19K mutant viruses induced apoptosis in wild-type and Bax- or Bak-deficient baby mouse kidney cells but not in those deficient for both Bax and Bak. Furthermore, Bax and Bak deficiency dramatically increased E1A expression and virus replication. Thus, Bax- and Bak-mediated apoptosis severely limits adenoviral replication, demonstrating that Bax and Bak function as an antiviral response at the cellular level.

  1. A double-regulated oncolytic adenovirus with improved safety for adenocarcinoma therapy

    SciTech Connect

    Wei, Na; Fan, Jun Kai; Gu, Jin Fa; He, Ling Feng; Tang, Wen Hao; Cao, Xin; Liu, Xin Yuan

    2009-10-16

    Safety and efficiency are equally important to be considered in developing oncolytic adenovirus. Previously, we have reported that ZD55, an oncolytic adenovirus with the deletion of E1B-55K gene, exhibited potent antitumor activity. In this study, to improve the safety of ZD55, we utilized MUC1 promoter to replace the native promoter of E1A on the basis of ZD55, and generated a double-regulated adenovirus, named MUD55. Our data demonstrated that the expression of early and late genes of MUD55 was both reduced in MUC1-negative cells, resulting in its stricter glandular-tumor selective progeny production. The cytopathic effect of MUD55 was about 10-fold lower than mono-regulated adenovirus ZD55 or Ad.MUC1 in normal cells and not obviously attenuated in glandular tumor cells. Moreover, MUD55 showed the least liver toxicity when administrated by intravenous injection in nude mice. These results indicate that MUD55 could be a promising candidate for the treatment of adenocarcinoma.

  2. Construction and Evaluation of Novel Rhesus Monkey Adenovirus Vaccine Vectors

    DOE PAGES

    Abbink, Peter; Maxfield, Lori F.; Ng'ang'a, David; ...

    2014-11-19

    Adenovirus vectors are widely used as vaccine candidates for a variety of pathogens, including HIV-1. To date, human and chimpanzee adenoviruses have been explored in detail as vaccine vectors. Furthermore, the phylogeny of human and chimpanzee adenoviruses is overlapping, and preexisting humoral and cellular immunity to both are exhibited in human populations worldwide. More distantly related adenoviruses may therefore offer advantages as vaccine vectors. We describe the primary isolation and vectorization of three novel adenoviruses from rhesus monkeys. The seroprevalence of these novel rhesus monkey adenovirus vectors was extremely low in sub-Saharan Africa human populations, and these vectors proved tomore » have immunogenicity comparable to that of human and chimpanzee adenovirus vaccine vectors in mice. These rhesus monkey adenoviruses phylogenetically clustered with the poorly described adenovirus species G and robustly stimulated innate immune responses. These novel adenoviruses represent a new class of candidate vaccine vectors.« less

  3. Construction and Evaluation of Novel Rhesus Monkey Adenovirus Vaccine Vectors

    SciTech Connect

    Abbink, Peter; Maxfield, Lori F.; Ng'ang'a, David; Borducchi, Erica N.; Iampietro, M. Justin; Bricault, Christine A.; Teigler, Jeffrey E.; Blackmore, Stephen; Parenteau, Lily; Wagh, Kshitij; Handley, Scott A.; Zhao, Guoyan; Virgin, Herbert W.; Korber, Bette; Barouch, Dan H.

    2014-11-19

    Adenovirus vectors are widely used as vaccine candidates for a variety of pathogens, including HIV-1. To date, human and chimpanzee adenoviruses have been explored in detail as vaccine vectors. Furthermore, the phylogeny of human and chimpanzee adenoviruses is overlapping, and preexisting humoral and cellular immunity to both are exhibited in human populations worldwide. More distantly related adenoviruses may therefore offer advantages as vaccine vectors. We describe the primary isolation and vectorization of three novel adenoviruses from rhesus monkeys. The seroprevalence of these novel rhesus monkey adenovirus vectors was extremely low in sub-Saharan Africa human populations, and these vectors proved to have immunogenicity comparable to that of human and chimpanzee adenovirus vaccine vectors in mice. These rhesus monkey adenoviruses phylogenetically clustered with the poorly described adenovirus species G and robustly stimulated innate immune responses. These novel adenoviruses represent a new class of candidate vaccine vectors.

  4. Phylogenetic analysis of adenovirus sequences.

    PubMed

    Harrach, Balázs; Benko, Mária

    2007-01-01

    Members of the family Adenoviridae have been isolated from a large variety of hosts, including representatives from every major vertebrate class from fish to mammals. The high prevalence, together with the fairly conserved organization of the central part of their genomes, make the adenoviruses one of (if not the) best models for studying viral evolution on a larger time scale. Phylogenetic calculation can infer the evolutionary distance among adenovirus strains on serotype, species, and genus levels, thus helping the establishment of a correct taxonomy on the one hand, and speeding up the process of typing new isolates on the other. Initially, four major lineages corresponding to four genera were recognized. Later, the demarcation criteria of lower taxon levels, such as species or types, could also be defined with phylogenetic calculations. A limited number of possible host switches have been hypothesized and convincingly supported. Application of the web-based BLAST and MultAlin programs and the freely available PHYLIP package, along with the TreeView program, enables everyone to make correct calculations. In addition to step-by-step instruction on how to perform phylogenetic analysis, critical points where typical mistakes or misinterpretation of the results might occur will be identified and hints for their avoidance will be provided.

  5. Small-Molecule Inhibitors of the Myc Oncoprotein

    PubMed Central

    Fletcher, Steven; Prochownik, Edward V.

    2014-01-01

    The c-Myc (Myc) oncoprotein is among the most attractive of cancer targets given that is deregulated in the majority of tumors and that its inhibition profoundly affects their growth and/or survival. However, its role as a seldom-mutated transcription factor, its lack of enzymatic activity for which suitable pharmaceutical inhibitors could be crafted and its expression by normal cells have largely been responsible for its being viewed as “undruggable”. Work over the past several years, however, has begun to reverse this idea by allowing us to view Myc within the larger context of global gene regulatory control. Thus, Myc and its obligate heterodimeric partner, Max, are integral to the coordinated recruitment and post-translational modification of components of the core transcriptional machinery. Moreover, Myc over-expression re-programs numerous critical cellular functions and alters the cell’s susceptibility to their inhibition. This new knowledge has therefore served as a framework upon which to develop new pharmaceutical approaches. These include the continuing development of small molecules which act directly to inhibit the critical Myc-Max interaction, those which act indirectly to prevent Myc-directed post-translational modifications necessary to initiate productive transcription and those which inhibit vital pathways upon which the Myc-transformed cell is particularly reliant. PMID:24657798

  6. Adenovirus Replaces Mitotic Checkpoint Controls

    PubMed Central

    Turner, Roberta L.; Groitl, Peter; Dobner, Thomas

    2015-01-01

    ABSTRACT Infection with adenovirus triggers the cellular DNA damage response, elements of which include cell death and cell cycle arrest. Early adenoviral proteins, including the E1B-55K and E4orf3 proteins, inhibit signaling in response to DNA damage. A fraction of cells infected with an adenovirus mutant unable to express the E1B-55K and E4orf3 genes appeared to arrest in a mitotic-like state. Cells infected early in G1 of the cell cycle were predisposed to arrest in this state at late times of infection. This arrested state, which displays hallmarks of mitotic catastrophe, was prevented by expression of either the E1B-55K or the E4orf3 genes. However, E1B-55K mutant virus-infected cells became trapped in a mitotic-like state in the presence of the microtubule poison colcemid, suggesting that the two viral proteins restrict entry into mitosis or facilitate exit from mitosis in order to prevent infected cells from arresting in mitosis. The E1B-55K protein appeared to prevent inappropriate entry into mitosis through its interaction with the cellular tumor suppressor protein p53. The E4orf3 protein facilitated exit from mitosis by possibly mislocalizing and functionally inactivating cyclin B1. When expressed in noninfected cells, E4orf3 overcame the mitotic arrest caused by the degradation-resistant R42A cyclin B1 variant. IMPORTANCE Cells that are infected with adenovirus type 5 early in G1 of the cell cycle are predisposed to arrest in a mitotic-like state in a p53-dependent manner. The adenoviral E1B-55K protein prevents entry into mitosis. This newly described activity for the E1B-55K protein appears to depend on the interaction between the E1B-55K protein and the tumor suppressor p53. The adenoviral E4orf3 protein facilitates exit from mitosis, possibly by altering the intracellular distribution of cyclin B1. By preventing entry into mitosis and by promoting exit from mitosis, these adenoviral proteins act to prevent the infected cell from arresting in a

  7. Monoclonal antibodies specific for adenovirus early region 1A proteins: extensive heterogeneity in early region 1A products.

    PubMed Central

    Harlow, E; Franza, B R; Schley, C

    1985-01-01

    Hybridomas secreting monoclonal antibodies specific for the adenovirus early region 1A (E1A) proteins were prepared from BALB/c mice immunized with a bacterial trpE-E1A fusion protein. This protein is encoded by a hybrid gene that joins a portion of the Escherichia coli trpE gene and a cDNA copy of the E1A 13S mRNA (Spindler et al., J. Virol. 49:132-141, 1984). Eighty-three hybridomas that secrete antibodies which recognize the immunogen were isolated and single cell cloned. Twenty-nine of these antibodies are specific for the E1A portion of the fusion protein. Only 12 of the monoclonal antibodies can efficiently immunoprecipitate E1A polypeptides from detergent lysates of infected cells. E1A polypeptides were analyzed on one-dimensional, sodium dodecyl sulfate-polyacrylamide gels and two-dimensional, isoelectric focusing polyacrylamide gels. The E1A proteins that are specifically immunoprecipitated by the monoclonal antibodies are heterogeneous in size and charge and can be resolved into approximately 60 polypeptide species. This heterogeneity is due not only to synthesis from multiple E1A mRNAs, but also at least in part to post-translational modification. Several of the monoclonal antibodies divide the E1A polypeptides into immunological subclasses based on the ability of the antibodies to bind to the antigen. In particular, two of the monoclonal antibodies bind to the polypeptides synthesized from the 13S E1A mRNA, but not to other E1A proteins. Images PMID:3894685

  8. Adenovirus sensing by the immune system.

    PubMed

    Atasheva, Svetlana; Shayakhmetov, Dmitry M

    2016-12-01

    The host immune system developed multiple ways for recognition of viral pathogens. Upon disseminated adenovirus infection, the immune system senses adenovirus invasion from the moment it enters the bloodstream. The soluble blood factors, FX, antibodies, and complement, can bind and activate plethora of host-protective immune responses. Adenovirus binding to the cellular β3 integrin and endosomal membrane rupture trigger activation of IL-1α/IL-1R1 proinflammatory cascade leading to attraction of cytotoxic immune cells to the site of infection. Upon cell entry, adenovirus exposes its DNA genome in the cytoplasm and triggers DNA sensors signaling. Even when inside the nucleus, the specialized cellular machinery that recognizes the double-strand DNA breaks become activated and triggers viral DNA replication arrest. Thus, the host employs very diverse mechanisms to prevent viral dissemination.

  9. Core labeling of adenovirus with EGFP

    SciTech Connect

    Le, Long P.; Le, Helen N.; Nelson, Amy R.; Matthews, David A.; Yamamoto, Masato; Curiel, David T. . E-mail: curiel@uab.edu

    2006-08-01

    The study of adenovirus could greatly benefit from diverse methods of virus detection. Recently, it has been demonstrated that carboxy-terminal EGFP fusions of adenovirus core proteins Mu, V, and VII properly localize to the nucleus and display novel function in the cell. Based on these observations, we hypothesized that the core proteins may serve as targets for labeling the adenovirus core with fluorescent proteins. To this end, we constructed various chimeric expression vectors with fusion core genes (Mu-EGFP, V-EGFP, preVII-EGFP, and matVII-EGFP) while maintaining expression of the native proteins. Expression of the fusion core proteins was suboptimal using E1 expression vectors with both conventional CMV and modified (with adenovirus tripartite leader sequence) CMV5 promoters, resulting in non-labeled viral particles. However, robust expression equivalent to the native protein was observed when the fusion genes were placed in the deleted E3 region. The efficient Ad-wt-E3-V-EGFP and Ad-wt-E3-preVII-EGFP expression vectors were labeled allowing visualization of purified virus and tracking of the viral core during early infection. The vectors maintained their viral function, including viral DNA replication, viral DNA encapsidation, cytopathic effect, and thermostability. Core labeling offers a means to track the adenovirus core in vector targeting studies as well as basic adenovirus virology.

  10. Molecular Analysis of Adenovirus Isolates from Previously Vaccinated Young Adults

    DTIC Science & Technology

    2004-04-01

    NAVAL HEALTH RESEARCH CENTER MOLECULAR ANALYSIS OF ADENOVIRUS ISOLATES FROM PREVIOUSLY VACCINATED YOUNG ADULTS D. A. Blasiole...Molecular Analysis of Adenovirus Isolates From Previously Vaccinated Young Adults . 6. AUTHORS Daniel A Blasiole, David Metzgar, Luke T Daum, Margaret AK

  11. The Dual Nature of Nek9 in Adenovirus Replication

    PubMed Central

    Jung, Richard; Radko, Sandi

    2015-01-01

    ABSTRACT To successfully replicate in an infected host cell, a virus must overcome sophisticated host defense mechanisms. Viruses, therefore, have evolved a multitude of devices designed to circumvent cellular defenses that would lead to abortive infection. Previous studies have identified Nek9, a cellular kinase, as a binding partner of adenovirus E1A, but the biology behind this association remains a mystery. Here we show that Nek9 is a transcriptional repressor that functions together with E1A to silence the expression of p53-inducible GADD45A gene in the infected cell. Depletion of Nek9 in infected cells reduces virus growth but unexpectedly enhances viral gene expression from the E2 transcription unit, whereas the opposite occurs when Nek9 is overexpressed. Nek9 localizes with viral replication centers, and its depletion reduces viral genome replication, while overexpression enhances viral genome numbers in infected cells. Additionally, Nek9 was found to colocalize with the viral E4 orf3 protein, a repressor of cellular stress response. Significantly, Nek9 was also shown to associate with viral and cellular promoters and appears to function as a transcriptional repressor, representing the first instance of Nek9 playing a role in gene regulation. Overall, these results highlight the complexity of virus-host interactions and identify a new role for the cellular protein Nek9 during infection, suggesting a role for Nek9 in regulating p53 target gene expression. IMPORTANCE In the arms race that exists between a pathogen and its host, each has continually evolved mechanisms to either promote or prevent infection. In order to successfully replicate and spread, a virus must overcome every mechanism that a cell can assemble to block infection. On the other hand, to counter viral spread, cells must have multiple mechanisms to stifle viral replication. In the present study, we add to our understanding of how the human adenovirus is able to circumvent cellular roadblocks

  12. Analysis of the viral replication cycle of adenovirus serotype 2 after inactivation by free chlorine.

    PubMed

    Gall, Aimee M; Shisler, Joanna L; Mariñas, Benito J

    2015-04-07

    Free chlorine is effective at inactivating a wide range of waterborne viral pathogens including human adenovirus (HAdV), but the mechanisms by which free chlorine inactivates HAdV and other human viruses remain to be elucidated. Such advances in fundamental knowledge are key for development of new disinfection technologies and novel sensors to detect infectious viruses in drinking water. We developed and tested a quantitative assay to analyze several steps in the HAdV replication cycle upon increasing free chlorine exposure. We used quantitative polymerase chain reaction (qPCR) to detect HAdV genomic DNA as a means to quantify attachment and genome replication of untreated and treated virions. Also, we used quantitative reverse-transcription PCR (RT-qPCR) to quantify the transcription of E1A (first early protein) and hexon mRNA. We compared these replication cycle events to virus inactivation kinetics to determine what stage of the virus replication cycle was inhibited as a function of free chlorine exposure. We observed that adenovirus inactivated at levels up to 99.99% by free chlorine still attached to host cells; however, viral DNA synthesis and early E1A and late hexon gene transcription were inhibited. We conclude that free chlorine exposure interferes with a replication cycle event occurring postbinding but prior to early viral protein synthesis.

  13. Anti-Tumor Activity of a miR-199-dependent Oncolytic Adenovirus

    PubMed Central

    Callegari, Elisa; Elamin, Bahaeldin K.; D’Abundo, Lucilla; Falzoni, Simonetta; Donvito, Giovanna; Moshiri, Farzaneh; Milazzo, Maddalena; Altavilla, Giuseppe; Giacomelli, Luciano; Fornari, Francesca; Hemminki, Akseli; Di Virgilio, Francesco; Gramantieri, Laura; Negrini, Massimo; Sabbioni, Silvia

    2013-01-01

    The down-regulation of miR-199 occurs in nearly all primary hepatocellular carcinomas (HCCs) and HCC cell lines in comparison with normal liver. We exploited this miR-199 differential expression to develop a conditionally replication-competent oncolytic adenovirus, Ad-199T, and achieve tumor-specific viral expression and replication. To this aim, we introduced four copies of miR-199 target sites within the 3’ UTR of E1A gene, essential for viral replication. As consequence, E1A expression from Ad-199T virus was tightly regulated both at RNA and protein levels in HCC derived cell lines, and replication controlled by the level of miR-199 expression. Various approaches were used to asses in vivo properties of Ad-199T. Ad-199T replication was inhibited in normal, miR-199 positive, liver parenchyma, thus resulting in reduced hepatotoxicity. Conversely, the intrahepatic delivery of Ad-199T in newborn mice led to virus replication and fast removal of implanted HepG2 liver cancer cells. The ability of Ad-199T to control tumor growth was also shown in a subcutaneous xenograft model in nude mice and in HCCs arising in immune-competent mice. In summary, we developed a novel oncolytic adenovirus, Ad-199T, which could demonstrate a therapeutic potential against liver cancer without causing significant hepatotoxicity. PMID:24069256

  14. Adenovirus Serotype 14 Infection, New Brunswick, Canada, 2011

    PubMed Central

    Garceau, Richard; Thibault, Louise; Oussedik, Youcef; Bastien, Nathalie; Li, Yan

    2013-01-01

    We describe 3 culture-proven cases of adenovirus serotype 14 infection in New Brunswick, Canada, during the summer of 2011. Strains isolated from severely ill patients were closely related to strains of a genomic variant, adenovirus 14p1, circulating in the United States and Ireland. Physicians in Canada should be aware of this emerging adenovirus. PMID:23260201

  15. Elasticity and Binding of Adenovirus

    NASA Astrophysics Data System (ADS)

    Matthews, Garrett; Negishi, Atsuko; Seeger, Adam; McCarty, Doug; Taylor, Russell; Samulshi, Jude; Superfine, Richard

    1999-11-01

    Adenovirus was the first human virus found to cause the transformation of cells and is one of the more common vectors being used for the development of gene therapy. As such, much is known about the viral structure and genome; however, the events of the early infection cycle, such as binding of the virus to the cell membrane and the release of genetic material from the capsid, for this and other nonenveloped viruses, are not fully understood. With the atomic force microscope (AFM) we are able to image the virus in both air and liquids, allowing us to change the surrounding environment, varying such physiologically relevant parameters as osmolality or pH. We additionally have the ability to do manipulations on single virus particles in these environments using the nanoManipulator. The nanoManipulator is an advanced interface for AFM that allows real time three dimensional rendering of the topographical data, allows the sample surface to be non-destructively felt using a hand held stylus that responds to the information being sensed at the tip, and allows controlled modification of the surface. Using this tool we have translated single virions over various surfaces, allowing us to measure the adhesion between the capsid and these surfaces. Additionally, we are able to place the tip directly atop individual viruses and measure their elasticity under a compressive load being supplied by that tip. We can explore how this property changes as a function of the properties of the surrounding liquid.

  16. Components of Adenovirus Genome Packaging

    PubMed Central

    Ahi, Yadvinder S.; Mittal, Suresh K.

    2016-01-01

    Adenoviruses (AdVs) are icosahedral viruses with double-stranded DNA (dsDNA) genomes. Genome packaging in AdV is thought to be similar to that seen in dsDNA containing icosahedral bacteriophages and herpesviruses. Specific recognition of the AdV genome is mediated by a packaging domain located close to the left end of the viral genome and is mediated by the viral packaging machinery. Our understanding of the role of various components of the viral packaging machinery in AdV genome packaging has greatly advanced in recent years. Characterization of empty capsids assembled in the absence of one or more components involved in packaging, identification of the unique vertex, and demonstration of the role of IVa2, the putative packaging ATPase, in genome packaging have provided compelling evidence that AdVs follow a sequential assembly pathway. This review provides a detailed discussion on the functions of the various viral and cellular factors involved in AdV genome packaging. We conclude by briefly discussing the roles of the empty capsids, assembly intermediates, scaffolding proteins, portal vertex and DNA encapsidating enzymes in AdV assembly and packaging. PMID:27721809

  17. MicroRNA-Mediated Suppression of Oncolytic Adenovirus Replication in Human Liver

    PubMed Central

    Ylösmäki, Erkko; Lavilla-Alonso, Sergio; Jäämaa, Sari; Vähä-Koskela, Markus; af Hällström, Taija; Hemminki, Akseli; Arola, Johanna; Mäkisalo, Heikki; Saksela, Kalle

    2013-01-01

    MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3′ untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5) in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver. PMID:23349911

  18. MicroRNA-mediated suppression of oncolytic adenovirus replication in human liver.

    PubMed

    Ylösmäki, Erkko; Lavilla-Alonso, Sergio; Jäämaa, Sari; Vähä-Koskela, Markus; af Hällström, Taija; Hemminki, Akseli; Arola, Johanna; Mäkisalo, Heikki; Saksela, Kalle

    2013-01-01

    MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression that can suppress their target genes by translational inhibition as well as mRNA destruction. Cell type-specific miRNA expression patterns have been successfully exploited for targeting the expression of experimental and therapeutic gene constructs, for example to reduce pathogenic effects of cancer virotherapy in normal tissues. In order to avoid liver damage associated with systemic or intrahepatic delivery of oncolytic adenoviruses we have introduced the concept of suppressing adenovirus replication in hepatic cells by inserting target elements for the liver-specific miR122 into the viral genome. Here we show using ex vivo cultured tissue specimens that six perfectly complementary miR122 target sites in the 3' untranslated region of the viral E1A gene are sufficient in the absence of any other genetic modifications to prevent productive replication of serotype 5 adenovirus (Ad5) in normal human liver. This modification did not compromise the replicative capacity of the modified virus in cancer tissue derived from a colon carcinoma liver metastasis or its oncolytic potency in a human lung cancer xenograft mouse model. Unlike wild-type Ad5, the modified virus did not result in increased serum levels of liver enzymes in infected mice. These results provide a strong preclinical proof of concept for the use of miR122 target sites for reducing the risk of liver damage caused by oncolytic adenoviruses, and suggest that ectopic miR122 target elements should be considered as an additional safety measure included in any therapeutic virus or viral vector posing potential hazard to the liver.

  19. Late-phase miRNA-controlled oncolytic adenovirus for selective killing of cancer cells

    PubMed Central

    Fillat, Cristina

    2015-01-01

    Tissue-specific detargeting by miRNAs has been demonstrated to be a potent strategy to restrict adenoviral replication to cancer cells. These studies have generated adenoviruses with miRNA target sites placed in the 3′UTR of early gene products. In this work, we have studied the feasibility of providing tissue-specific selectivity to replication-competent adenoviruses through the regulation of the late structural protein fiber (L5 gene). We have engineered a 3′UTR containing eight miR-148a binding sites downstream the L5 coding sequence (Ad-L5-8miR148aT). We present in vitro and in vivo evidences of Ad-L5-8miR148aT miRNA-dependent regulation. In vitro data show that at 72 hours post-infection miR-148a-regulation impaired fiber expression leading to a 70% reduction of viral release. The application of seven consecutive rounds of infection in miR-148a cells resulted in 10.000-fold reduction of viral genomes released. In vivo, liver production of infective viral particles was highly impaired, similarly to that triggered by an adenovirus with miRNA target sites regulating the early E1A gene. Noticeably, mice treated with Ad-L5-8miR148aT showed an attenuation of adenoviral-induced hepatotoxicity but retained full lytic activity in cancer cells and exhibited robust antitumoral responses in patient-derived xenografts. Thus, miRNA-control of late proteins constitutes a novel strategy to provide selectivity to adenoviruses. PMID:25714032

  20. Expression of Marek's disease virus oncoprotein Meq during infection in the natural host

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marek's disease virus (MDV, Gallid herpesvirus 2) causes a lymphoproliferative disease known as Marek's disease (MD), which is unique among alphaherpesviruses as the viral genome encodes an oncoprotein, Meq. Previous studies, using cultured fibroblasts and MDV-transformed lymphoblastoid cell lines, ...

  1. Problem-Solving Test: The Mechanism of Action of a Human Papilloma Virus Oncoprotein

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2009-01-01

    Terms to be familiar with before you start to solve the test: human papilloma virus; cervical cancer; oncoproteins; malignant transformation; retinoblastoma protein; cell cycle; quiescent and cycling cells; cyclin/cyclin-dependent kinase (Cdk) complexes; E2F; S-phase genes; enhancer element; proto-oncogenes; tumor suppressor genes; radioactive…

  2. Immunologic and Genetic Selection of Adenovirus Vaccine Strains: Synthesis and Characterization of Adenovirus Antigens.

    DTIC Science & Technology

    1984-08-01

    exhibited strikingly different chromatographic characteristics. 2. Effect of proflavine on the synthesis of adenovirus, type 5, and associated soluble...antigens. The synthesis of type 5 adenovirus in HeLa cells was suppressed to a considerable extent by low concentrations of proflavine , an acridine dye...chemical. Addition of proflavine to infected cells at different times during the virus growth cycle revealed that the processes leading to the synthesis

  3. Structure and Uncoating of Immature Adenovirus

    SciTech Connect

    Perez-Berna, A.J.; Mangel, W.; Marabini, R.; Scheres, S. H. W., Menendez-Conejero, R.; Dmitriev, I. P.; Curiel, D. T.; Flint, S. J.; San Martin, C.

    2009-09-18

    Maturation via proteolytic processing is a common trait in the viral world and is often accompanied by large conformational changes and rearrangements in the capsid. The adenovirus protease has been shown to play a dual role in the viral infectious cycle: (a) in maturation, as viral assembly starts with precursors to several of the structural proteins but ends with proteolytically processed versions in the mature virion, and (b) in entry, because protease-impaired viruses have difficulties in endosome escape and uncoating. Indeed, viruses that have not undergone proteolytic processing are not infectious. We studied the three-dimensional structure of immature adenovirus particles as represented by the adenovirus type 2 thermosensitive mutant ts1 grown under non-permissive conditions and compared it with the mature capsid. Our three-dimensional electron microscopy maps at subnanometer resolution indicate that adenovirus maturation does not involve large-scale conformational changes in the capsid. Difference maps reveal the locations of unprocessed peptides pIIIa and pVI and help define their role in capsid assembly and maturation. An intriguing difference appears in the core, indicating a more compact organization and increased stability of the immature cores. We have further investigated these properties by in vitro disassembly assays. Fluorescence and electron microscopy experiments reveal differences in the stability and uncoating of immature viruses, both at the capsid and core levels, as well as disassembly intermediates not previously imaged.

  4. Chimpanzee Adenovirus Vector Ebola Vaccine.

    PubMed

    Ledgerwood, Julie E; DeZure, Adam D; Stanley, Daphne A; Coates, Emily E; Novik, Laura; Enama, Mary E; Berkowitz, Nina M; Hu, Zonghui; Joshi, Gyan; Ploquin, Aurélie; Sitar, Sandra; Gordon, Ingelise J; Plummer, Sarah A; Holman, LaSonji A; Hendel, Cynthia S; Yamshchikov, Galina; Roman, Francois; Nicosia, Alfredo; Colloca, Stefano; Cortese, Riccardo; Bailer, Robert T; Schwartz, Richard M; Roederer, Mario; Mascola, John R; Koup, Richard A; Sullivan, Nancy J; Graham, Barney S

    2017-03-09

    Background The unprecedented 2014 epidemic of Ebola virus disease (EVD) prompted an international response to accelerate the availability of a preventive vaccine. A replication-defective recombinant chimpanzee adenovirus type 3-vectored ebolavirus vaccine (cAd3-EBO), encoding the glycoprotein from Zaire and Sudan species, that offers protection in the nonhuman primate model, was rapidly advanced into phase 1 clinical evaluation. Methods We conducted a phase 1, dose-escalation, open-label trial of cAd3-EBO. Twenty healthy adults, in sequentially enrolled groups of 10 each, received vaccination intramuscularly in doses of 2×10(10) particle units or 2×10(11) particle units. Primary and secondary end points related to safety and immunogenicity were assessed throughout the first 8 weeks after vaccination; in addition, longer-term vaccine durability was assessed at 48 weeks after vaccination. Results In this small study, no safety concerns were identified; however, transient fever developed within 1 day after vaccination in two participants who had received the 2×10(11) particle-unit dose. Glycoprotein-specific antibodies were induced in all 20 participants; the titers were of greater magnitude in the group that received the 2×10(11) particle-unit dose than in the group that received the 2×10(10) particle-unit dose (geometric mean titer against the Zaire antigen at week 4, 2037 vs. 331; P=0.001). Glycoprotein-specific T-cell responses were more frequent among those who received the 2×10(11) particle-unit dose than among those who received the 2×10(10) particle-unit dose, with a CD4 response in 10 of 10 participants versus 3 of 10 participants (P=0.004) and a CD8 response in 7 of 10 participants versus 2 of 10 participants (P=0.07) at week 4. Assessment of the durability of the antibody response showed that titers remained high at week 48, with the highest titers in those who received the 2×10(11) particle-unit dose. Conclusions Reactogenicity and immune responses

  5. The drug-induced degradation of oncoproteins: an unexpected Achilles' heel of cancer cells?

    PubMed

    Ablain, Julien; Nasr, Rihab; Bazarbachi, Ali; de Thé, Hugues

    2011-07-01

    Many targeted therapies against cancer are aimed at inhibiting the enzymatic activity of kinases. Thus far, this approach has undoubtedly yielded significant clinical improvements, but has only rarely achieved cures. Other drugs, which selectively elicit proteasome-dependent degradation of oncoproteins, induce the loss of cancer cell self-renewal and promote cell differentiation and/or apoptosis. In acute promyelocytic leukemia, the cooperative degradation of PML/RARA by arsenic and retinoic acid cures most patients. In this condition and others, drug-induced proteolysis of oncoproteins is feasible and underlies improved clinical outcome. Several transcription factors, nuclear receptors, or fusion proteins driving cancer growth could be candidates for proteolysis-based drug-discovery programs.

  6. The human papillomavirus (HPV) E6 oncoproteins promotes nuclear localization of active caspase 8

    SciTech Connect

    Manzo-Merino, Joaquin; Lizano, Marcela

    2014-02-15

    The HPV-16 E6 and E6{sup ⁎} proteins have been shown previously to be capable of regulating caspase 8 activity. We now show that the capacity of E6 to interact with caspase 8 is common to diverse HPV types, being also seen with HPV-11 E6, HPV-18 E6 and HPV-18 E6{sup ⁎}. Unlike most E6-interacting partners, caspase 8 does not appear to be a major proteasomal target of E6, but instead E6 appears able to stimulate caspase 8 activation, without affecting the overall apoptotic activity. This would appear to be mediated in part by the ability of the HPV E6 oncoproteins to recruit active caspase 8 to the nucleus. - Highlights: • Multiple HPV E6 oncoproteins interact with the caspase 8 DED domain. • HPV E6 stimulates activation of caspase 8. • HPV E6 promotes nuclear accumulation of caspase 8.

  7. Identification of cytotoxic agents disrupting synovial sarcoma oncoprotein interactions by proximity ligation assay

    PubMed Central

    Laporte, Aimée N.; Ji, Jennifer X.; Ma, Limin; Nielsen, Torsten O.; Brodin, Bertha A.

    2016-01-01

    Conventional cytotoxic therapies for synovial sarcoma provide limited benefit. Drugs specifically targeting the product of its driver translocation are currently unavailable, in part because the SS18-SSX oncoprotein functions via aberrant interactions within multiprotein complexes. Proximity ligation assay is a recently-developed method that assesses protein-protein interactions in situ. Here we report use of the proximity ligation assay to confirm the oncogenic association of SS18-SSX with its co-factor TLE1 in multiple human synovial sarcoma cell lines and in surgically-excised human tumor tissue. SS18-SSX/TLE1 interactions are disrupted by class I HDAC inhibitors and novel small molecule inhibitors. This assay can be applied in a high-throughput format for drug discovery in fusion-oncoprotein associated cancers where key effector partners are known. PMID:27120803

  8. The 19-kilodalton adenovirus E1B transforming protein inhibits programmed cell death and prevents cytolysis by tumor necrosis factor alpha.

    PubMed Central

    White, E; Sabbatini, P; Debbas, M; Wold, W S; Kusher, D I; Gooding, L R

    1992-01-01

    The adenovirus E1A and E1B proteins are required for transformation of primary rodent cells. When expressed in the absence of the 19,000-dalton (19K) E1B protein, however, the E1A proteins are acutely cytotoxic and induce host cell chromosomal DNA fragmentation and cytolysis, analogous to cells undergoing programmed cell death (apoptosis). E1A alone can efficiently initiate the formation of foci which subsequently undergo abortive transformation whereby stimulation of cell growth is counteracted by continual cell death. Cell lines with an immortalized growth potential eventually arise with low frequency. Coexpression of the E1B 19K protein with E1A is sufficient to overcome abortive transformation to produce high-frequency transformation. Like E1A, the tumoricidal cytokine tumor necrosis factor alpha (TNF-alpha) evokes a programmed cell death response in many tumor cell lines by inducing DNA fragmentation and cytolysis. Expression of the E1B 19K protein by viral infection, by transient expression, or in transformed cells completely and specifically blocks this TNF-alpha-induced DNA fragmentation and cell death. Cosegregation of 19K protein transforming activity with protection from TNF-alpha-mediated cytolysis demonstrates that both activities are likely the consequence of the same function of the protein. Therefore, we propose that by suppressing an intrinsic cell death mechanism activated by TNF-alpha or E1A, the E1B 19K protein enhances the transforming activity of E1A and enables adenovirus to evade TNF-alpha-dependent immune surveillance. Images PMID:1317006

  9. Molecular characterization of adenoviruses among finnish military conscripts.

    PubMed

    Mölsä, Markos; Hemmilä, Heidi; Rönkkö, Esa; Virkki, Maria; Nikkari, Simo; Ziegler, Thedi

    2016-04-01

    Although adenoviruses were identified as important respiratory pathogens many years ago, little information is available concerning the prevalence of different adenovirus serotypes, which are circulating and causing epidemics in Finnish military training centers. Over a period of five years from 2008 to 2012, 3577 respiratory specimens were collected from military conscripts presenting with symptoms compatible with acute respiratory tract infection. Upon initial testing for certain respiratory viruses by real-time PCR, 837 of these specimens were identified as adenovirus-positive. For 672 of these specimens, the serotype of the adenovirus responsible was successfully determined by DNA sequencing. Serotypes 1, 2, 3, and 4 were detected in 1, 3, 181, and 487 samples, respectively. Adenovirus epidemics were observed during each year of this study. Based on these findings, adenovirus vaccination should be considered for military conscripts in the Finnish Defence Forces.

  10. Adenovirus Type 5 E4 Open Reading Frame 4 Protein Induces Apoptosis in Transformed Cells

    PubMed Central

    Shtrichman, Ronit; Kleinberger, Tamar

    1998-01-01

    Adenovirus type 5 E4 open reading frame 4 (E4orf4) protein has been previously shown to counteract transactivation of the junB and c-fos genes by cyclic AMP plus E1A protein and to interact with protein phosphatase 2A (PP2A). Here, we show that the wild-type E4orf4 protein induces apoptosis in the E1A-expressing 293 cells, in NIH 3T3 cells transformed with v-Ras, and in the lung carcinoma cell line H1299. The induction of apoptosis is not accompanied by enhanced levels of p53 in 293 cells and occurs in the absence of p53 in H1299 cells, indicating involvement of a p53-independent pathway. A mutant E4orf4 protein that had lost the ability to induce apoptosis also lost its ability to bind PP2A. We suggest that E4orf4 antagonizes continuous signals to proliferate, like those given by E1A or v-Ras, and that the conflicting signals lead to the induction of cell death. PMID:9525619

  11. Gene expression profile regulated by the HPV16 E7 oncoprotein and estradiol in cervical tissue

    PubMed Central

    Cortés-Malagón, Enoc M.; Bonilla-Delgado, José; Díaz-Chávez, José; Hidalgo-Miranda, Alfredo; Romero-Cordoba, Sandra; Üren, Aykut; Çelik, Haydar; McCormick, Matthew; Munguía-Moreno, José A.; Ibarra-Sierra, Eloisa; Escobar-Herrera, Jaime; Lambert, Paul F.; Mendoza-Villanueva, Daniel; Bermudez-Cruz, Rosa M.; Gariglio, Patricio

    2014-01-01

    The HPV16 E7 oncoprotein and 17β-estradiol are important factors for the induction of premalignant lesions and cervical cancer. The study of these factors is crucial for a better understanding of cervical tumorigenesis. Here, we assessed the global gene expression profiles induced by the HPV16 E7 oncoprotein and/or 17β-estradiol in cervical tissue of FvB and K14E7 transgenic mice. We found that the most dramatic changes in gene expression occurred in K14E7 and FvB groups treated with 17β-estradiol. A large number of differentially expressed genes involved in the immune response were observed in 17β-estradiol treated groups. The E7 oncoprotein mainly affected the expression of genes involved in cellular metabolism. Our microarray data also identified differentially expressed genes that have not previously been reported in cervical cancer. The identification of genes regulated by E7 and 17β-estradiol, provides the basis for further studies on their role in cervical carcinogenesis. PMID:24210110

  12. Adenovirus dodecahedron, a new vector for human gene transfer.

    PubMed

    Fender, P; Ruigrok, R W; Gout, E; Buffet, S; Chroboczek, J

    1997-01-01

    Recombinant adenovirus is one of most efficient delivery vehicles for gene therapy. However, the initial enthusiasm for the use of recombinant adenovirus for gene therapy has been tempered by strong immune responses that develop to the virus and virus-infected cells. Even though recombinant adenoviruses are replication-defective, they introduce into the recipient cell, together with the gene of interest, viral genetes that might lead to fortuitous recombination if the recipient is infected by wild-type adenovirus. We propose the use of a dodecahedron made of adenovirus pentons or penton bases as an alternative vector for human gene therapy. The penton is a complex of two oligomeric proteins, a penton base and fiber, involved in the cell attachment, internalization, and liberation of virus into the cytoplasm. The dodecahedron retains many of the advantages of adenovirus for gene transfer such as efficiency of entry, efficient release of DNA from endosomes, and wide range of cell and tissue targets. Because it consists of only one or two adenovirus proteins instead of the 11 contained in an adenovirus virion and it does not contain the viral genome, it is potentially a safer alternative to recombinant adenovirus.

  13. Adenovirus type 5 exerts genome-wide control over cellular programs governing proliferation, quiescence, and survival

    PubMed Central

    Miller, Daniel L; Myers, Chad L; Rickards, Brenden; Coller, Hilary A; Flint, S Jane

    2007-01-01

    Background Human adenoviruses, such as serotype 5 (Ad5), encode several proteins that can perturb cellular mechanisms that regulate cell cycle progression and apoptosis, as well as those that mediate mRNA production and translation. However, a global view of the effects of Ad5 infection on such programs in normal human cells is not available, despite widespread efforts to develop adenoviruses for therapeutic applications. Results We used two-color hybridization and oligonucleotide microarrays to monitor changes in cellular RNA concentrations as a function of time after Ad5 infection of quiescent, normal human fibroblasts. We observed that the expression of some 2,000 genes, about 10% of those examined, increased or decreased by a factor of two or greater following Ad5 infection, but were not altered in mock-infected cells. Consensus k-means clustering established that the temporal patterns of these changes were unexpectedly complex. Gene Ontology terms associated with cell proliferation were significantly over-represented in several clusters. The results of comparative analyses demonstrate that Ad5 infection induces reversal of the quiescence program and recapitulation of the core serum response, and that only a small subset of the observed changes in cellular gene expression can be ascribed to well characterized functions of the viral E1A and E1B proteins. Conclusion These findings establish that the impact of adenovirus infection on host cell programs is far greater than appreciated hitherto. Furthermore, they provide a new framework for investigating the molecular functions of viral early proteins and information relevant to the design of conditionally replicating adenoviral vectors. PMID:17430596

  14. Structure, function and dynamics in adenovirus maturation.

    PubMed

    Mangel, Walter F; San Martín, Carmen

    2014-11-21

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a "molecular sled" to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core is more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. Finally, possible roles for maturation of the terminal protein are discussed.

  15. Structure, function and dynamics in adenovirus maturation

    DOE PAGES

    Mangel, Walter F.; San Martín, Carmen

    2014-11-21

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core ismore » more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. In conclusion, possible roles for maturation of the terminal protein are discussed.« less

  16. Structure and uncoating of immature adenovirus

    PubMed Central

    Pérez-Berná, Ana J.; Marabini, Roberto; Scheres, Sjors H. W.; Menéndez-Conejero, Rosa; Dmitriev, Igor P.; Curiel, David T.; Mangel, Walter F.; Flint, S. Jane; Martín, Carmen San

    2009-01-01

    Summary Maturation via proteolytical processing is a common trait in the viral world, and is often accompanied by large conformational changes and rearrangements in the capsid. The adenovirus protease has been shown to play a dual role in the viral infectious cycle: (a) in maturation, as viral assembly starts with precursors to several of the structural proteins, but ends with proteolytically processed versions in the mature virion; and (b) in entry, because protease-impaired viruses have difficulties in endosome escape and uncoating. Indeed, viruses that have not undergone proteolytical processing are not infectious. We present the 3D structure of immature adenovirus particles, as represented by the thermosensitive mutant Ad2 ts1 grown under non-permissive conditions, and compare it with the mature capsid. Our 3DEM maps at subnanometer resolution indicate that adenovirus maturation does not involve large scale conformational changes in the capsid. Difference maps reveal the location of unprocessed peptides pIIIa and pVI and help to define their role in capsid assembly and maturation. An intriguing difference appears in the core, indicating a more compact organization and increased stability of the immature cores. We have further investigated these properties by in vitro disassembly assays. Fluorescence and electron microscopy experiments reveal differences in the stability and uncoating of immature viruses, both at the capsid and core levels, as well as disassembly intermediates not previously imaged. PMID:19563809

  17. Structure, Function and Dynamics in Adenovirus Maturation

    PubMed Central

    Mangel, Walter F.; San Martín, Carmen

    2014-01-01

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core is more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. Finally, possible roles for maturation of the terminal protein are discussed. PMID:25421887

  18. Structure, function and dynamics in adenovirus maturation

    SciTech Connect

    Mangel, Walter F.; San Martín, Carmen

    2014-11-21

    Here we review the current knowledge on maturation of adenovirus, a non-enveloped icosahedral eukaryotic virus. The adenovirus dsDNA genome fills the capsid in complex with a large amount of histone-like viral proteins, forming the core. Maturation involves proteolytic cleavage of several capsid and core precursor proteins by the viral protease (AVP). AVP uses a peptide cleaved from one of its targets as a “molecular sled” to slide on the viral genome and reach its substrates, in a remarkable example of one-dimensional chemistry. Immature adenovirus containing the precursor proteins lacks infectivity because of its inability to uncoat. The immature core is more compact and stable than the mature one, due to the condensing action of unprocessed core polypeptides; shell precursors underpin the vertex region and the connections between capsid and core. Maturation makes the virion metastable, priming it for stepwise uncoating by facilitating vertex release and loosening the condensed genome and its attachment to the icosahedral shell. The packaging scaffold protein L1 52/55k is also a substrate for AVP. Proteolytic processing of L1 52/55k disrupts its interactions with other virion components, providing a mechanism for its removal during maturation. In conclusion, possible roles for maturation of the terminal protein are discussed.

  19. ADENOVIRUS INTERACTION WITH ITS CELLULAR RECEPTOR CAR.

    SciTech Connect

    HOWITT,J.; ANDERSON,C.W.; FREIMUTH,P.

    2001-08-01

    The mechanism of adenovirus attachment to the host cell plasma membrane has been revealed in detail by research over the past 10 years. It has long been known that receptor binding activity is associated with the viral fibers, trimeric spike proteins that protrude radially from the vertices of the icosahedral capsid (Philipson et al. 1968). In some adenovirus serotypes, fiber and other virus structural proteins are synthesized in excess and accumulate in the cell nucleus during late stages of infection. Fiber protein can be readily purified from lysates of cells infected with subgroup C viruses, for example Ad2 and Ad5 (Boulanger and Puvion 1973). Addition of purified fiber protein to virus suspensions during adsorption strongly inhibits infection, indicating that fiber and intact virus particles compete for binding sites on host cells (Philipson et al. 1968; Hautala et al. 1998). Cell binding studies using purified radiolabeled fiber demonstrated that fiber binds specifically and with high affinity to the cell plasma membrane, and that cell lines typically used for laboratory propagation of adenovirus have approximately 10{sup 4} high-affinity receptor sites per cell (Persson et al. 1985; Freimuth 1996). Similar numbers of high-affinity binding sites for radiolabeled intact virus particles also were observed (Seth et al. 1994).

  20. Adenovirus DNA polymerase is a phosphoprotein.

    PubMed

    Ramachandra, M; Nakano, R; Mohan, P M; Rawitch, A B; Padmanabhan, R

    1993-01-05

    Biological activities of many of the eukaryotic DNA replication proteins are modulated by protein phosphorylation. Investigations of the phosphorylation of adenovirus DNA polymerase (AdPol) have been difficult mainly because of its low level of synthesis in adenovirus-infected HeLa cells. However, when AdPol was overproduced using the recombinant vaccinia virus (RV-AdPol) and the baculovirus expression systems, or by a large scale metabolic labeling of adenovirus 2-infected HeLa cells (native AdPol), in vivo phosphorylation of AdPol could be demonstrated. Phosphoamino acid analysis of [32P]AdPol indicated the presence of phosphoserine independent of the source of AdPol. Comparison of tryptic peptide maps of native AdPol and RV-AdPol revealed that the majority of phosphopeptides were common. Fractionation by high performance liquid chromatography and sequencing of one of the major phosphopeptides revealed serine 67 as a site of phosphorylation. Interestingly, this site is located close to the nuclear localization signal of AdPol and has a consensus substrate recognition sequence for histone H1 (cdc2-related) kinases and mitogen-activated protein kinases. Dephosphorylation of AdPol with calf intestinal alkaline phosphatase resulted in significant decrease in its activity in the in vitro DNA replication initiation assay, suggesting that phosphorylation is important for its biological activity.

  1. A novel application of E1A in combination therapy with EGFR-TKI treatment in breast cancer

    PubMed Central

    Su, Chih-Ming; Chang, Ting-Yu; Hsu, Hui-Ping; Lai, Hui-Huang; Li, Jie-Ning; Lyu, Yu-Jhen; Kuo, Kuang-Tai; Huang, Ming-Te; Su, Jen-Liang; Chen, Pai-Sheng

    2016-01-01

    Epidermal growth factor receptor (EGFR) is commonly overexpressed in breast cancer and is associated with poor clinical outcomes; however, an increasing number of patients have shown a poor effective response to EGFR tyrosine kinase inhibitors (EGFR-TKI). Here, we found that AXL expression was positively correlated with poor progression in breast cancer patients. Suppression of AXL by an anti-tumor protein, E1A, enhanced EGFR-TKI (gefitinib, erlotinib and lapatinib) sensitization, resulting in significant inhibition of tumor growth in breast cancer cells. Additionally, AXL overexpression dramatically impaired E1A-mediated EGFR-TKI sensitization. These findings show that downregulation of AXL expression by E1A contributes to sensitization to EGFR-TKI in breast cancer, suggesting that combinatorial therapy of AXL inhibitors or E1A gene therapy with EGFR-TKI may be a potential therapeutic strategy for treatment of breast cancer patients. PMID:27590506

  2. E1A enhances cellular sensitivity to DNA-damage-induced apoptosis through PIDD-dependent caspase-2 activation

    PubMed Central

    Radke, Jay R; Siddiqui, Zeba K; Figueroa, Iris; Cook, James L

    2016-01-01

    Expression of the adenoviral protein, E1A, sensitizes mammalian cells to a wide variety of apoptosis-inducing agents through multiple cellular pathways. For example, E1A sensitizes cells to apoptosis induced by TNF-superfamily members by inhibiting NF-kappa B (NF-κB)-dependent gene expression. In contrast, E1A sensitization to nitric oxide, an inducer of the intrinsic apoptotic pathway, is not dependent upon repression of NF-κB-dependent transcription but rather is dependent upon caspase-2 activation. The latter observation suggested that E1A-induced enhancement of caspase-2 activation might be a critical factor in cellular sensitization to other intrinsic apoptosis pathway-inducing agents. Etoposide and gemcitabine are two DNA damaging agents that induce intrinsic apoptosis. Here we report that E1A-induced sensitization to both of these agents, like NO, is independent of NF-κB activation but dependent on caspase-2 activation. The results show that caspase-2 is a key mitochondrial-injuring caspase during etoposide and gemcitabine-induced apoptosis of E1A-positive cells, and that caspase-2 is required for induction of caspase-3 activity by both chemotherapeutic agents. Expression of PIDD was required for caspase-2 activation, mitochondrial injury and enhanced apoptotic cell death. Furthermore, E1A-enhanced sensitivity to injury-induced apoptosis required PIDD cleavage to PIDD-CC. These results define the PIDD/caspase-2 pathway as a key apical, mitochondrial-injuring mechanism in E1A-induced sensitivity of mammalian cells to chemotherapeutic agents. PMID:27833761

  3. E1A enhances cellular sensitivity to DNA-damage-induced apoptosis through PIDD-dependent caspase-2 activation.

    PubMed

    Radke, Jay R; Siddiqui, Zeba K; Figueroa, Iris; Cook, James L

    2016-01-01

    Expression of the adenoviral protein, E1A, sensitizes mammalian cells to a wide variety of apoptosis-inducing agents through multiple cellular pathways. For example, E1A sensitizes cells to apoptosis induced by TNF-superfamily members by inhibiting NF-kappa B (NF-κB)-dependent gene expression. In contrast, E1A sensitization to nitric oxide, an inducer of the intrinsic apoptotic pathway, is not dependent upon repression of NF-κB-dependent transcription but rather is dependent upon caspase-2 activation. The latter observation suggested that E1A-induced enhancement of caspase-2 activation might be a critical factor in cellular sensitization to other intrinsic apoptosis pathway-inducing agents. Etoposide and gemcitabine are two DNA damaging agents that induce intrinsic apoptosis. Here we report that E1A-induced sensitization to both of these agents, like NO, is independent of NF-κB activation but dependent on caspase-2 activation. The results show that caspase-2 is a key mitochondrial-injuring caspase during etoposide and gemcitabine-induced apoptosis of E1A-positive cells, and that caspase-2 is required for induction of caspase-3 activity by both chemotherapeutic agents. Expression of PIDD was required for caspase-2 activation, mitochondrial injury and enhanced apoptotic cell death. Furthermore, E1A-enhanced sensitivity to injury-induced apoptosis required PIDD cleavage to PIDD-CC. These results define the PIDD/caspase-2 pathway as a key apical, mitochondrial-injuring mechanism in E1A-induced sensitivity of mammalian cells to chemotherapeutic agents.

  4. 11 CFR 300.61 - Federal elections (2 U.S.C. 441i(e)(1)(A)).

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 11 Federal Elections 1 2010-01-01 2010-01-01 false Federal elections (2 U.S.C. 441i(e)(1)(A)). 300.61 Section 300.61 Federal Elections FEDERAL ELECTION COMMISSION BIPARTISAN CAMPAIGN REFORM ACT OF... elections (2 U.S.C. 441i(e)(1)(A)). No person described in 11 CFR 300.60 shall solicit, receive,...

  5. Enhanced inactivation of adenovirus under polychromatic UV lamps

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Adenovirus is recognized as the most UV-resistant waterborne pathogen of concern to public health microbiologists. The US EPA has stipulated that a UV fluence (dose) of 186 mJ cm-2 is required for 4-log inactivation credit in water treatment. However, all adenovirus inactivation data to date publi...

  6. Capturing and concentrating adenovirus using magnetic anionic nanobeads

    PubMed Central

    Sakudo, Akikazu; Baba, Koichi; Ikuta, Kazuyoshi

    2016-01-01

    We recently demonstrated how various enveloped viruses can be efficiently concentrated using magnetic beads coated with an anionic polymer, poly(methyl vinyl ether-maleic anhydrate). However, the exact mechanism of interaction between the virus particles and anionic beads remains unclear. To further investigate whether these magnetic anionic beads specifically bind to the viral envelope, we examined their potential interaction with a nonenveloped virus (adenovirus). The beads were incubated with either adenovirus-infected cell culture medium or nasal aspirates from adenovirus-infected individuals and then separated from the supernatant by applying a magnetic field. After thoroughly washing the beads, adsorption of adenovirus was confirmed by a variety of techniques, including immunochromatography, polymerase chain reaction, Western blotting, and cell culture infection assays. These detection methods positively identified the hexon and penton capsid proteins of adenovirus along with the viral genome on the magnetic beads. Furthermore, various types of adenovirus including Types 5, 6, 11, 19, and 41 were captured using the magnetic bead procedure. Our bead capture method was also found to increase the sensitivity of viral detection. Adenovirus below the detectable limit for immunochromatography was efficiently concentrated using the magnetic bead procedure, allowing the virus to be successfully detected using this methodology. Moreover, these findings clearly demonstrate that a viral envelope is not required for binding to the anionic magnetic beads. Taken together, our results show that this capture procedure increases the sensitivity of detection of adenovirus and would, therefore, be a valuable tool for analyzing both clinical and experimental samples. PMID:27274228

  7. The search for adenovirus 14 in children in Houston, Texas.

    PubMed

    Laham, Federico R; Jewell, Alan M; Schoonover, Shauna L; Demmler, Gail J; Piedra, Pedro A

    2008-07-01

    Adenovirus (Ad)14 has recently emerged in the United States causing outbreaks of severe respiratory disease. To determine if Ad14 circulated in Houston, Texas, during the same time as an outbreak in military recruits in nearby San Antonio, 215 pediatric adenovirus isolates were serotyped using microneutralization. None were Ad14; Ad1, Ad2, and Ad3 were the most common identified serotypes.

  8. A novel adenovirus in Chinstrap penguins (Pygoscelis antarctica) in Antarctica.

    PubMed

    Lee, Sook-Young; Kim, Jeong-Hoon; Park, Yon Mi; Shin, Ok Sarah; Kim, Hankyeom; Choi, Han-Gu; Song, Jin-Won

    2014-05-07

    Adenoviruses (family Adenoviridae) infect various organ systems and cause diseases in a wide range of host species. In this study, we examined multiple tissues from Chinstrap penguins (Pygoscelis antarctica), collected in Antarctica during 2009 and 2010, for the presence of novel adenoviruses by PCR. Analysis of a 855-bp region of the hexon gene of a newly identified adenovirus, designated Chinstrap penguin adenovirus 1 (CSPAdV-1), showed nucleotide (amino acid) sequence identity of 71.8% (65.5%) with South Polar skua 1 (SPSAdV-1), 71% (70%) with raptor adenovirus 1 (RAdV-1), 71.4% (67.6%) with turkey adenovirus 3 (TAdV-3) and 61% (61.6%) with frog adenovirus 1 (FrAdV-1). Based on the genetic and phylogenetic analyses, CSPAdV-1 was classified as a member of the genus, Siadenovirus. Virus isolation attempts from kidney homogenates in the MDTC-RP19 (ATCC® CRL-8135™) cell line were unsuccessful. In conclusion, this study provides the first evidence of new adenovirus species in Antarctic penguins.

  9. Adenovirus dodecahedron allows large multimeric protein transduction in human cells.

    PubMed

    Fender, P; Schoehn, G; Foucaud-Gamen, J; Gout, E; Garcel, A; Drouet, E; Chroboczek, J

    2003-04-01

    Adenovirus dodecahedron is a virus-like particle composed of only two viral proteins of human adenovirus serotype 3 that are responsible for virus attachment and internalization. We show here that this dodecameric particle, devoid of genetic information, efficiently penetrates human cells and can deliver large multimeric proteins such as immunoglobulins.

  10. Enhanced suppression of adenovirus replication by triple combination of anti-adenoviral siRNAs, soluble adenovirus receptor trap sCAR-Fc and cidofovir.

    PubMed

    Pozzuto, Tanja; Röger, Carsten; Kurreck, Jens; Fechner, Henry

    2015-08-01

    Adenoviruses (Ad) generally induce mild self-limiting respiratory or intestinal infections but can also cause serious disease with fatal outcomes in immunosuppressed patients. Antiviral drug therapy is an important treatment for adenoviral infections but its efficiency is limited. Recently, we have shown that gene silencing by RNA interference (RNAi) is a promising new approach to inhibit adenoviral infection. In the present in vitro study, we examined whether the efficiency of an RNAi-based anti-adenoviral therapy can be further increased by combination with a virus receptor trap sCAR-Fc and with the antiviral drug cidofovir. Initially, three siRNAs, siE1A_4, siIVa2_2 and Pol-si2, targeting the adenoviral E1A, IVa2 and DNA polymerase mRNAs, respectively, were used for gene silencing. Replication of the Ad was inhibited in a dose dependent manner by each siRNA, but the efficiency of inhibition differed (Pol-si2>siIVa2_2>siE1A_4). Double or triple combinations of the siRNAs compared with single siRNAs did not result in a measurably higher suppression of Ad replication. Combination of the siRNAs (alone or mixes of two or three siRNAs) with sCAR-Fc markedly increased the suppression of adenoviral replication compared to the same siRNA treatment without sCAR-Fc. Moreover, the triple combination of a mix of all three siRNAs, sCAR-Fc and cidofovir was about 23-fold more efficient than the combination of siRNAs mix/sCAR-Fc and about 95-fold more efficient than the siRNA mix alone. These data demonstrate that co-treatment of cells with sCAR-Fc and cidofovir is suitable to increase the efficiency of anti-adenoviral siRNAs.

  11. Synthesis in Escherichia coli of human adenovirus type 12 transforming proteins encoded by early region 1A 13S mRNA and 12S mRNA.

    PubMed Central

    Kimelman, D; Lucher, L A; Brackmann, K H; Symington, J S; Ptashne, M; Green, M

    1984-01-01

    Human adenovirus (Ad)-encoded early region 1A (E1A) tumor (T) antigens have been implicated in the positive regulation of viral early genes, the positive and negative regulation of some cellular genes, and cell immortalization and transformation. To further study the Ad E1A T antigens and to facilitate their purification, we have cloned cDNA copies of the Ad12 E1A 13S mRNA and 12S mRNA downstream of a hybrid Escherichia coli trp-lac (tac) promoter. Up to 8% of the protein synthesized in E. coli cells transformed by each of the two different Ad12 E1A cDNA constructs were immunoprecipitated as a Mr 47,000 protein by antibody to a synthetic peptide encoded in the Ad12 E1A DNA sequence. Both proteins produced in E. coli appear to be authentic and complete Ad12 E1A T antigens because they possess (i) the Ad12 E1A NH2-terminal amino acid sequence predicted from the DNA sequence; (ii) the Ad12 E1A COOH-terminal sequence, as shown by immunoprecipitation with anti-peptide antibody; and (iii) a molecular weight and an acidic isoelectric point similar to that of the E1A T antigens synthesized in Ad12-infected and transformed mammalian cells. The T antigens were purified to near homogeneity in yields of 100-200 micrograms per g wet weight of transformed E. coli cells. Images PMID:6387701

  12. Expression and In Silico Analysis of the Recombinant Bovine Papillomavirus E6 Protein as a Model for Viral Oncoproteins Studies

    PubMed Central

    Mazzuchelli-de-Souza, J.; Carvalho, R. F.; Ruiz, R. M.; Melo, T. C.; Araldi, R. P.; Carvalho, E.; Thompson, C. E.; Sircili, M. P.; Beçak, W.; Stocco, R. C.

    2013-01-01

    Bovine papillomaviruses (BPVs) are recognized as the causal agents of economical relevant diseases in cattle, associated with the development of tumors in skin and mucosa. The oncogenesis process is mainly associated with different viral oncoprotein expressions, which are involved in cell transformation. The expression and characterization of recombinant viral oncoproteins represent an attractive strategy to obtain biotechnological products as antibodies and potential vaccines, Thus, the aim of this work was to clone and express the BPV-1 and BPV-2 E6 recombinant proteins and perform in silico analysis in order to develop a strategy for the systematic study of other papillomaviruses oncoproteins. The results demonstrated that BPV-1 and BPV-2 E6 recombinant proteins were expressed and purified from bacterial system as well as its in silico analysis was performed in order to explore and predict biological characteristics of these proteins. PMID:23878806

  13. Expression and in Silico analysis of the recombinant bovine papillomavirus E6 protein as a model for viral oncoproteins studies.

    PubMed

    Mazzuchelli-de-Souza, J; Carvalho, R F; Ruiz, R M; Melo, T C; Araldi, R P; Carvalho, E; Thompson, C E; Sircili, M P; Beçak, W; Stocco, R C

    2013-01-01

    Bovine papillomaviruses (BPVs) are recognized as the causal agents of economical relevant diseases in cattle, associated with the development of tumors in skin and mucosa. The oncogenesis process is mainly associated with different viral oncoprotein expressions, which are involved in cell transformation. The expression and characterization of recombinant viral oncoproteins represent an attractive strategy to obtain biotechnological products as antibodies and potential vaccines, Thus, the aim of this work was to clone and express the BPV-1 and BPV-2 E6 recombinant proteins and perform in silico analysis in order to develop a strategy for the systematic study of other papillomaviruses oncoproteins. The results demonstrated that BPV-1 and BPV-2 E6 recombinant proteins were expressed and purified from bacterial system as well as its in silico analysis was performed in order to explore and predict biological characteristics of these proteins.

  14. Structure of adenovirus bound to cellular receptor car

    DOEpatents

    Freimuth, Paul I.

    2004-05-18

    Disclosed is a mutant adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain wherein the mutation causes the encoded viral particle to have significantly weakened binding affinity for CARD1 relative to wild-type adenovirus. Such mutations may be in sequences which encode either the AB loop, or the HI loop of the fiber protein knob domain. Specific residues and mutations are described. Also disclosed is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type. In the method, residues of the adenovirus fiber protein knob domain which are predicted to alter D1 binding when mutated, are identified from the crystal structure coordinates of the AD12knob:CAR-D1 complex. A mutation which alters one or more of the identified residues is introduced into the genome of the adenovirus to generate a mutant adenovirus. Whether or not the mutant produced exhibits altered adenovirus-CAR binding properties is then determined.

  15. Mechanisms of pathogenesis of emerging adenoviruses

    PubMed Central

    Cook, James; Radke, Jay

    2017-01-01

    Periodic outbreaks of human adenovirus infections can cause severe illness in people with no known predisposing conditions. The reasons for this increased viral pathogenicity are uncertain. Adenoviruses are constantly undergoing mutation during circulation in the human population, but related phenotypic changes of the viruses are rarely detected because of the infrequency of such outbreaks and the limited biological studies of the emergent strains. Mutations and genetic recombinations have been identified in these new strains. However, the linkage between these genetic changes and increased pathogenicity is poorly understood. It has been observed recently that differences in virus-induced immunopathogenesis can be associated with altered expression of non-mutant viral genes associated with changes in viral modulation of the host innate immune response. Initial small animal studies indicate that these changes in viral gene expression can be associated with enhanced immunopathogenesis in vivo. Available evidence suggests the hypothesis that there is a critical threshold of expression of certain viral genes that determines both the sustainability of viral transmission in the human population and the enhancement of immunopathogenesis. Studies of this possibility will require extension of the analysis of outbreak viral strains from a sequencing-based focus to biological studies of relationships between viral gene expression and pathogenic responses. Advances in this area will require increased coordination among public health organizations, diagnostic microbiology laboratories, and research laboratories to identify, catalog, and systematically study differences between prototype and emergent viral strains that explain the increased pathogenicity that can occur during clinical outbreaks. PMID:28184296

  16. Isolation and Epidemiology of Falcon Adenovirus

    PubMed Central

    Oaks, J. Lindsay; Schrenzel, Mark; Rideout, Bruce; Sandfort, Cal

    2005-01-01

    An adenovirus was detected by electron microscopy in tissues from falcons that died during an outbreak of inclusion body hepatitis and enteritis that affected neonatal Northern aplomado (Falco femoralis septentrionalis) and peregrine (Falco peregrinus anatum) falcons. Molecular characterization has identified the falcon virus as a new member of the aviadenovirus group (M. Schrenzel, J. L. Oaks, D. Rotstein, G. Maalouf, E. Snook, C. Sandfort, and B. Rideout, J. Clin. Microbiol. 43:3402-3413, 2005). In this study, the virus was successfully isolated and propagated in peregrine falcon embryo fibroblasts, in which it caused visible and reproducible cytopathology. Testing for serum neutralizing antibodies found that infection with this virus was limited almost exclusively to falcons. Serology also found that wild and captive peregrine falcons had high seropositivity rates of 80% and 100%, respectively, although clinical disease was rarely reported in this species. These data implicate peregrine falcons as the natural host and primary reservoir for the virus. Other species of North American falcons, including aplomado falcons, had lower seropositivity rates of 43 to 57%. Falcon species of tropical and/or island origin were uniformly seronegative, although deaths among adults of these species have been described, suggesting they are highly susceptible. Chickens and quail were uniformly seronegative and not susceptible to infection, indicating that fowl were not the source of infection. Based on the information from this study, the primary control of falcon adenovirus infections should be based on segregation of carrier and susceptible falcon species. PMID:16000467

  17. Proinflammatory Effects of Interferon Gamma in Mouse Adenovirus 1 Myocarditis

    PubMed Central

    McCarthy, Mary K.; Procario, Megan C.; Twisselmann, Nele; Wilkinson, J. Erby; Archambeau, Ashley J.; Michele, Daniel E.; Day, Sharlene M.

    2014-01-01

    ABSTRACT Adenoviruses are frequent causes of pediatric myocarditis. Little is known about the pathogenesis of adenovirus myocarditis, and the species specificity of human adenoviruses has limited the development of animal models, which is a significant barrier to strategies for prevention or treatment. We have developed a mouse model of myocarditis following mouse adenovirus 1 (MAV-1) infection to study the pathogenic mechanisms of this important cause of pediatric myocarditis. Following intranasal infection of neonatal C57BL/6 mice, we detected viral replication and induction of interferon gamma (IFN-γ) in the hearts of infected mice. MAV-1 caused myocyte necrosis and induced substantial cellular inflammation that was composed predominantly of CD3+ T lymphocytes. Depletion of IFN-γ during acute infection reduced cardiac inflammation in MAV-1-infected mice without affecting viral replication. We observed decreased contractility during acute infection of neonatal mice, and persistent viral infection in the heart was associated with cardiac remodeling and hypertrophy in adulthood. IFN-γ is a proinflammatory mediator during adenovirus-induced myocarditis, and persistent adenovirus infection may contribute to ongoing cardiac dysfunction. IMPORTANCE Studying the pathogenesis of myocarditis caused by different viruses is essential in order to characterize both virus-specific and generalized factors that contribute to disease. Very little is known about the pathogenesis of adenovirus myocarditis, which is a significant impediment to the development of treatment or prevention strategies. We used MAV-1 to establish a mouse model of human adenovirus myocarditis, providing the means to study host and pathogen factors contributing to adenovirus-induced cardiac disease during acute and persistent infection. The MAV-1 model will enable fundamental studies of viral myocarditis, including IFN-γ modulation as a therapeutic strategy. PMID:25320326

  18. Human T-cell leukemia virus type-1 Tax oncoprotein regulates G-protein signaling.

    PubMed

    Twizere, Jean-Claude; Springael, Jean-Yves; Boxus, Mathieu; Burny, Arsène; Dequiedt, Franck; Dewulf, Jean-François; Duchateau, Julie; Portetelle, Daniel; Urbain, Patrice; Van Lint, Carine; Green, Patrick L; Mahieux, Renaud; Parmentier, Marc; Willems, Luc; Kettmann, Richard

    2007-02-01

    Human T-cell leukemia virus type-1 (HTLV-1) is associated with adult T-cell leukemia (ATL) and neurological syndromes. HTLV-1 encodes the oncoprotein Tax-1, which modulates viral and cellular gene expression leading to T-cell transformation. Guanine nucleotide-binding proteins (G proteins) and G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins known and are involved in the regulation of most biological functions. Here, we report an interaction between HTLV-1 Tax oncoprotein and the G-protein beta subunit. Interestingly, though the G-protein beta subunit inhibits Tax-mediated viral transcription, Tax-1 perturbs G-protein beta subcellular localization. Functional evidence for these observations was obtained using conditional Tax-1-expressing transformed T-lymphocytes, where Tax expression correlated with activation of the SDF-1/CXCR4 axis. Our data indicated that HTLV-1 developed a strategy based on the activation of the SDF-1/CXCR4 axis in the infected cell; this could have tremendous implications for new therapeutic strategies.

  19. Degenerate cysteine patterns mediate two redox sensing mechanisms in the papillomavirus E7 oncoprotein.

    PubMed

    Camporeale, Gabriela; Lorenzo, Juan R; Thomas, Maria G; Salvatierra, Edgardo; Borkosky, Silvia S; Risso, Marikena G; Sánchez, Ignacio E; de Prat Gay, Gonzalo; Alonso, Leonardo G

    2017-04-01

    Infection with oncogenic human papillomavirus induces deregulation of cellular redox homeostasis. Virus replication and papillomavirus-induced cell transformation require persistent expression of viral oncoproteins E7 and E6 that must retain their functionality in a persistent oxidative environment. Here, we dissected the molecular mechanisms by which E7 oncoprotein can sense and manage the potentially harmful oxidative environment of the papillomavirus-infected cell. The carboxy terminal domain of E7 protein from most of the 79 papillomavirus viral types of alpha genus, which encloses all the tumorigenic viral types, is a cysteine rich domain that contains two classes of cysteines: strictly conserved low reactive Zn(+2) binding and degenerate reactive cysteine residues that can sense reactive oxygen species (ROS). Based on experimental data obtained from E7 proteins from the prototypical viral types 16, 18 and 11, we identified a couple of low pKa nucleophilic cysteines that can form a disulfide bridge upon the exposure to ROS and regulate the cytoplasm to nucleus transport. From sequence analysis and phylogenetic reconstruction of redox sensing states we propose that reactive cysteine acquisition through evolution leads to three separate E7s protein families that differ in the ROS sensing mechanism: non ROS-sensitive E7s; ROS-sensitive E7s using only a single or multiple reactive cysteine sensing mechanisms and ROS-sensitive E7s using a reactive-resolutive cysteine couple sensing mechanism.

  20. Epstein-Barr Virus oncoprotein super-enhancers control B cell growth

    PubMed Central

    Zhou, Hufeng; Schmidt, Stefanie CS; Jiang, Sizun; Willox, Bradford; Bernhardt, Katharina; Liang, Jun; Johannsen, Eric C; Kharchenko, Peter; Gewurz, Benjamin E; Kieff, Elliott; Zhao, Bo

    2015-01-01

    Summary Super-enhancers are clusters of gene-regulatory sites bound by multiple transcription factors that govern cell transcription, development, phenotype, and oncogenesis. By examining Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs), we identified four EBV oncoproteins and five EBV-activated NF-κB subunits co-occupying ~1800 enhancer sites. Of these, 187 had markedly higher and broader histone H3K27ac signals characteristic of super-enhancers, and were designated “EBV super-enhancers”. EBV super-enhancer-associated genes included the MYC and BCL2 oncogenes, enabling LCL proliferation and survival. EBV super-enhancers were enriched for B cell transcription factor motifs and had a high co-occupancy of the transcription factors STAT5 and NFAT. EBV super-enhancer-associated genes were more highly expressed than other LCL genes. Disrupting EBV super-enhancers by the bromodomain inhibitor, JQ1 or conditionally inactivating an EBV oncoprotein or NF-κB decreased MYC or BCL2 expression and arrested LCL growth. These findings provide insight into mechanisms of EBV-induced lymphoproliferation and identify potential therapeutic interventions. PMID:25639793

  1. A p53-independent apoptotic mechanism of adenoviral mutant E1A was involved in its selective antitumor activity for human cancer

    PubMed Central

    Fang, Lin; Cheng, Qian; Zhao, Jingjing; Ge, Yan; Zhu, Qi; Zhao, Min; Zhang, Jie; Zhang, Qi; Li, Liantao; Liu, Junjie; Zheng, Junnian

    2016-01-01

    The conserved regions (CR) of adenoviral E1A had been shown to be necessary for disruption of pRb-E2F transcription factor complexes and induction of the S phase. Here we constructed a mutant adenoviral E1A with Rb-binding ability absent (E1A 30-60aa and 120-127aa deletion, mE1A) and investigated its antitumor capacities in vitro and in vivo. The mE1A suppressed the viability of tumor cells as efficiently as the wild type E1A, and there was no cytotoxic effect on normal cells. Although the mE1A arrested tumor cell cycle with the same manner as E1A, the former played a different role on cell cycle regulation compared with E1A in normal cells, which might contribute to its selective antitumor activity. E1A and mE1A had accumulated inactive p53, decreased the expression of mdm2, Cdkn1a (also named p21), increased p21's nuclear distribution and induced tumor cell apoptosis in a p53-indenpent manner. Further, E1A or mE1A significantly suppressed tumor growth in subcutaneous hepatocellular carcinoma xenograft models. Especially, tumor-bearing mice treated with mE1A had higher survival rate than those treated with E1A. Our data demonstrated that mutant adenoviral E1A significantly induced tumor cell apoptosis in a p53-indenpednt manner and had selective tumor suppressing ability. The observations of adenoviral E1A mutant had provided a novel mechanism for E1A's complex activities during infection. PMID:27340782

  2. A NOTCH-sensitive uPAR-regulated oncolytic adenovirus effectively suppresses pancreatic tumor growth and triggers synergistic anticancer effects with gemcitabine and nab-paclitaxel.

    PubMed

    Mato-Berciano, Ana; Raimondi, Giulia; Maliandi, Maria Victoria; Alemany, Ramon; Montoliu, Lluis; Fillat, Cristina

    2017-02-07

    Notch signaling pathway is an embryonic program that becomes reactivated in pancreatic cancer and contributes to cancer stem cell (CSC) maintenance. We explored the concept of oncolytic adenoviral activity in response to Notch activation signaling, in the context of a chimeric promoter with uPAR regulatory sequences, as a strategy to drive its activity in neoplastic and CSC. We explored the advantages of a chemo-virotherapy approach based on synergistic combinations. Regulatory sequences recognized by the transcriptional factor CSL upstream a minimal uPAR promoter were engineered in adenoviral vectors and in the oncolytic adenovirus AdNuPARmE1A. Viral response to Notch signaling, and viral potency in cell lines and pancreatic cancer stem cells (PCSC) was tested. Preclinical toxicity and antitumor efficacy in xenografts and Patient-derived xenografts (PDX) mouse models was evaluated, as unimodal or in combination with gemcitabine+nab-paclitaxel. Mechanistic studies were conducted to explore the synergism of combined therapies.We demonstrate that CSL-binding site optimized-engineered sequences respond to Notch activation in AdNuPARmLuc and AdNuPARmE1A. AdNuPARmE1A showed strong lytic effects in pancreatic cancer cell lines and PCSC. AdNuPARmE1A displayed attenuated activity in normal tissues, but robust antitumor effects in xenograft and PDX models, leading to a reduced capacity of treated tumors to form tumorspheres. Chemo-virotherapy treatment enlarged therapeutic response in both tumor models. Synergistic effects of the combination resulted from viral sensitization of apoptotic cell death triggered by chemotherapy.In summary we present a novel effective oncolytic adenovirus, AdNuPARmE1A that reduces PCSC and presents synergistic effects with gemcitabine and nab-paclitaxel, supporting further clinical development.

  3. Structure of adenovirus bound to cellular receptor car

    DOEpatents

    Freimuth, Paul I.

    2007-01-02

    Disclosed is a mutant CAR-DI-binding adenovirus which has a genome comprising one or more mutations in sequences which encode the fiber protein knob domain wherein the mutation causes the encoded viral particle to have a significantly weakened binding affinity for CAR-DI relative to wild-type adenovirus. Such mutations may be in sequences which encode either the AB loop, or the HI loop of the fiber protein knob domain. Specific residues and mutations are described. Also disclosed is a method for generating a mutant adenovirus which is characterized by a receptor binding affinity or specificity which differs substantially from wild type.

  4. IMMUNOFLUORESCENT STUDIES OF THE POTENTIATION OF AN ADENOVIRUS-ASSOCIATED VIRUS BY ADENOVIRUS 7

    PubMed Central

    Blacklow, Neil R.; Hoggan, M. David; Rowe, Wallace P.

    1967-01-01

    A quantitative immunofluorescent procedure for detection of viral antigen was used to study the potentiation of AAV-1 by Ad.7. AAV viral antigen formed only when the cells were also infected with adenovirus, and only in cell culture systems in which the adenovirus infection proceeded to completion. Ad. 7 infection of AGMK. cell cultures did not potentiate AAV unless the Ad. 7 infection was itself potentiated by SV40. Dose-response studies indicated that a single AAV particle and a single infectious Ad. 7 particle sufficed to initiate AAV antigen synthesis. Sequential inoculation studies showed that AAV antigen formed simultaneously with Ad. 7 viral antigen when the AAV was inoculated any time between 15 hr before to 10 hr after the Ad. 7, both antigens appearing about 15 hr after inoculation of Ad. 7. The AAV-1 antigen formation had a minimum latent period of 5 hr, as seen with Ad. 7 preinfection of 10 hr or more. When UV-irradiated Ad. 7 was used as helper, the AAV antigen still appeared simultaneously with the Ad. 7 viral antigen, even though the latter was delayed by 23 hr compared to nonirradiated virus. When the early replicative events of both viruses were allowed to proceed in FUDR-inhibited cells, and then the FUDR inhibition was reversed, AAV antigen formed within 2 hr, which was 3 hr before the Ad. 7 viral antigen appeared. It was inferred that the event in the adenovirus cycle that renders a cell competent to synthesize AAV occurs after the 10th hr and may be temporally associated with replication of the adenovirus DNA. PMID:4225814

  5. Regulation of the Wnt/β-Catenin Signaling Pathway by Human Papillomavirus E6 and E7 Oncoproteins

    PubMed Central

    Muñoz Bello, Jesus Omar; Olmedo Nieva, Leslie; Contreras Paredes, Adriana; Fuentes Gonzalez, Alma Mariana; Rocha Zavaleta, Leticia; Lizano, Marcela

    2015-01-01

    Cell signaling pathways are the mechanisms by which cells transduce external stimuli, which control the transcription of genes, to regulate diverse biological effects. In cancer, distinct signaling pathways, such as the Wnt/β-catenin pathway, have been implicated in the deregulation of critical molecular processes that affect cell proliferation and differentiation. For example, changes in β-catenin localization have been identified in Human Papillomavirus (HPV)-related cancers as the lesion progresses. Specifically, β-catenin relocates from the membrane/cytoplasm to the nucleus, suggesting that this transcription regulator participates in cervical carcinogenesis. The E6 and E7 oncoproteins are responsible for the transforming activity of HPV, and some studies have implicated these viral oncoproteins in the regulation of the Wnt/β-catenin pathway. Nevertheless, new interactions of HPV oncoproteins with cellular proteins are emerging, and the study of the biological effects of such interactions will help to understand HPV-related carcinogenesis. This review addresses the accumulated evidence of the involvement of the HPV E6 and E7 oncoproteins in the activation of the Wnt/β-catenin pathway. PMID:26295406

  6. A model of latent adenovirus 5 infection in the guinea pig (Cavia porcellus).

    PubMed

    Vitalis, T Z; Keicho, N; Itabashi, S; Hayashi, S; Hogg, J C

    1996-03-01

    A model of adenovirus 5 (Ad5) infection was developed in guinea pigs to begin to study its role in the pathogenesis of peripheral lung inflammation. Forty animals were inoculated intranasally with 10(7.0) pfu of Ad5/animal, and 15 animals inoculated with sterile culture media served as controls. Viral titres were 10(4.4), 10(6.1), 10(5.2), and 10(2.9) pfu/animal, on days 1, 3, 4, and 7 after infection, respectively. In situ hybridization to viral DNA and immunocytochemistry for Ad5 E1A protein localized the virus to airway and alveolar epithelial cells. Histologic examination showed an extensive inflammatory cell infiltration around the airways, with epithelial necrosis and an alveolar exudate that caused localized alveolar collapse in the infected areas. Immunocytochemistry identified the cells in the infiltrate as cytotoxic T cells. Although all animals 20 and 47 days after infection had seroconverted to Ad5, virus was not detected in these groups either by viral plaque assay or in situ hybridization. Ad5 E1A DNA was detected by polymerase chain reaction in five of six animals 20 days after infection and in five of five animals 47 days after infection. In these same animals, E1A protein was detected 20 days after infection in two and 47 days after infection in one while persistent bronchiolitis was observed in four and three animals 20 and 47 days after infection, respectively. These results demonstrate that the guinea pig provides a useful model to study the role of Ad5 infection in chronic airway inflammation.

  7. Temporal regulation of adenovirus major late alternative RNA splicing.

    PubMed

    Akusjarvi, Goran

    2008-05-01

    Adenovirus makes extensive use of alternative RNA splicing to produce a complex set of spliced mRNAs during replication. The accumulation of viral mRNAs is subjected to a temporal regulation, a mechanism that ensures that proteins that are needed at certain stages of the virus life cycle are produced in a timely fashion. The complex interactions between the virus and the host cell RNA splicing machinery has been studied in detail during the last decade. These studies have resulted in the characterization of two viral proteins, E4-ORF4 and L4-33K, that adenovirus uses to remodel the host cell RNA splicing machinery. Here I will review the current knowledge of how mRNA expression from the adenovirus major late transcription unit is controlled with a particular emphasis on how cis-acting sequence element, trans-acting factors and mechanisms regulating adenovirus major late L1 alternative RNA splicing is controlled.

  8. An Oncotropic Adenovirus Vector System for Breast Cancer Treatment

    DTIC Science & Technology

    2005-09-01

    AD Award Number: DAMD17-03-1-0629 TITLE: An Oncotropic Adenovirus Vector System for Breast Cancer Treatment PRINCIPAL INVESTIGATOR: Igor P. Dmitriev...Aug 2005 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER An Oncotropic Adenovirus Vector System for Breast Cancer Treatment 5b. GRANT NUMBER DAMD17-03-1...epithelial cells, the origin of most human cancers. However, realization of the full potential of Ad vectors for targeted cancer treatment is currently

  9. Adenovirus-receptor interaction with human lymphocytes.

    PubMed

    Mentel, R; Döpping, G; Wegner, U; Seidel, W; Liebermann, H; Döhner, L

    1997-03-01

    Lymphocytes play a key role in cell-mediated immunity and are host cells for several viral and bacterial pathogens. Their importance in adenovirus (Ad) infections is not yet fully understood. The initial event, the attachment of Ad to lymphocytes and their subsets, was examined using flow cytometry. The study included analysis of stimulated T cells in binding assays with FITC-labeled Ad fiber. The results confirm that native peripheral lymphocytes express very small amounts of Ad receptors. Stimulation with PHA and interleukin 2 induced the expression. The presence of Ad DNA as a sign of internalization in stimulated cells was demonstrated using the polymerase chain reaction. The findings suggest that lymphocytes after stimulation can turn into target cells for Ad. This is particularly important if there are indications for persistence of Ad, and in the case of immunocompromised patients severe, life-threatening diseases can develop.

  10. [Gene engineering of the adenovirus vector].

    PubMed

    Kondo, Saki; Terashima, Miho; Fukuda, Hiromitsu; Saito, Izumu; Kanegae, Yumi

    2007-06-01

    The adenovirus vector is very attractive tool not only for the gene therapy but also for the basic sciences. However, because a construction method of this vector had been complex, only limited scientists had constructed and enjoyed the benefits. Recently, various methods were developed and the researchers came to be able to choose an efficient method, which is the COS-TPC method, or a concise procedure, which is the intact-genome transfection method (in vitro ligation method). Here we described not only these methods but also new method to construct the various Ads simultaneously using the recombinase-mediated cassette exchange (RMCE) by the site-specific recombinase. And also we want to refer the possibility to the worth of the vector, especially the vector of the expression-switch.

  11. Adenovirus Infections in Immunocompetent and Immunocompromised Patients

    PubMed Central

    2014-01-01

    SUMMARY Human adenoviruses (HAdVs) are an important cause of infections in both immunocompetent and immunocompromised individuals, and they continue to provide clinical challenges pertaining to diagnostics and treatment. The growing number of HAdV types identified by genomic analysis, as well as the improved understanding of the sites of viral persistence and reactivation, requires continuous adaptions of diagnostic approaches to facilitate timely detection and monitoring of HAdV infections. In view of the clinical relevance of life-threatening HAdV diseases in the immunocompromised setting, there is an urgent need for highly effective treatment modalities lacking major side effects. The present review summarizes the recent progress in the understanding and management of HAdV infections. PMID:24982316

  12. Vaccine Design: Replication-Defective Adenovirus Vectors.

    PubMed

    Zhou, Xiangyang; Xiang, Zhiquan; Ertl, Hildegund C J

    2016-01-01

    Replication-defective adenovirus (Ad) vectors were initially developed for gene transfer for correction of genetic diseases. Although Ad vectors achieved high levels of transgene product expression in a variety of target cells, expression of therapeutic proteins was found to be transient as vigorous T cell responses directed to components of the vector as well as the transgene product rapidly eliminate Ad vector-transduced cells. This opened the use of Ad vectors as vaccine carriers and by now a multitude of preclinical as well as clinical studies has shown that Ad vectors induce very potent and sustained transgene product-specific T and B cell responses. This chapter provides guidance on developing E1-deleted Ad vectors based on available viral molecular clones. Specifically, it describes methods for cloning, viral rescue and purification as well as quality control studies.

  13. Oncoprotein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2001-02-27

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  14. The E6 Oncoprotein from HPV16 Enhances the Canonical Wnt/β-catenin Pathway in Skin Epidermis in vivo

    PubMed Central

    Bonilla-Delgado, José; Bulut, Gülay; Liu, Xuefeng; Cortés-Malagón, Enoc M.; Schlegel, Richard; Flores-Maldonado, Catalina; Contreras, Rubén G.; Chung, Sang-Hyuk; Lambert, Paul F.; Üren, Aykut; Gariglio, Patricio

    2012-01-01

    The contribution of the Wnt signaling pathway to HPV-induced carcinogenesis is poorly understood. In high-grade dysplastic lesions that are caused by high-risk human papilloma viruses (HR-HPVs), β-catenin is often located in the cell nucleus, which suggests that Wnt pathway may be involved in the development of HPV-related carcinomas. Most of the oncogenic potential of HR-HPVs resides on the E6 protein’s PDZ-binding domain. We hypothesized that the PDZ-binding domain of the HPV16-E6 oncoprotein induces the nuclear accumulation of β-catenin due to its capacity to degrade PDZ-containing cellular targets. To test this hypothesis, we evaluated the staining pattern of β-catenin in the skin epidermis of transgenic mice expressing the full-length E6 oncoprotein (K14E6 mice) and measured LacZ gene expression in K14E6 mice that were crossed with a strain expressing LacZ that was knocked into the Axin2 locus (Axin2+/LacZ mice). Here, we show that the E6 oncoprotein enhances the nuclear accumulation of β-catenin, the accumulation of cellularβ-catenin-responsive genes and the expression of LacZ. None of these effects were observed when a truncated E6 oncoprotein that lacks the PDZ-binding domain was expressed alone (K14E6ΔPDZ mice) or in combination with Axin2+/LacZ. Conversely, co-transfection with either E6 or E6ΔPDZ similarly enhanced canonical Wnt signaling in short-term in vitro assays that utilized a luciferase Wnt/β-catenin/TCF-dependent promoter. We propose that the activation of canonical Wnt signaling could be induced by the HPV16-E6 oncoprotein; however, the participation of the E6 PDZ-binding domain seems to be important in in vivo models only. PMID:22160870

  15. An oncolytic adenovirus enhances antiangiogenic and antitumoral effects of a replication-deficient adenovirus encoding endostatin by rescuing its selective replication in nasopharyngeal carcinoma cells

    SciTech Connect

    Liu, Ran-yi; Zhou, Ling; Zhang, Yan-ling; Huang, Bi-jun; Ke, Miao-la; Chen, Jie-min; Li, Li-xia; Fu, Xiang; Wu, Jiang-xue; Huang, Wenlin

    2013-12-13

    Highlights: •H101 promotes endostatin expression by Ad-Endo via rescuing Ad-Endo replication. •H101 rescued Ad-Endo replication by supplying E1A and E1B19k proteins. •Ad-Endo enhanced the cytotoxicity of H101 in NPC cells. •Ad-Endo and oncolytic Ad H101 have synergistic antitumor effects on NPC. -- Abstract: A replication-deficient adenovirus (Ad) encoding secreted human endostatin (Ad-Endo) has been demonstrated to have promising antiangiogenic and antitumoral effects. The E1B55k-deleted Ad H101 can selectively lyse cancer cells. In this study, we explored the antitumor effects and cross-interactions of Ad-Endo and H101 on nasopharyngeal carcinoma (NPC). The results showed that H101 dramatically promoted endostatin expression by Ad-Endo via rescuing Ad-Endo replication in NPC cells, and the expressed endostatin proteins significantly inhibited the proliferation of human umbilical vein endothelial cells. E1A and E1B19k products are required for the rescuing of H101 to Ad-Endo replication in CNE-1 and CNE-2 cells, but not in C666-1 cells. On the other hand, Ad-Endo enhanced the cytotoxicity of H101 by enhancing Ad replication in NPC cells. The combination of H101 and Ad-Endo significantly inhibited CNE-2 xenografts growth through the increased endostatin expression and Ad replication. These findings indicate that the combination of Ad-Endo gene therapy and oncolytic Ad therapeutics could be promising in comprehensive treatment of NPC.

  16. Karyopherin {beta}3: A new cellular target for the HPV-16 E5 oncoprotein

    SciTech Connect

    Krawczyk, Ewa; Hanover, John A.; Schlegel, Richard; Suprynowicz, Frank A.

    2008-07-11

    Epidemiological and experimental studies have shown that high-risk human papillomaviruses (HPVs) are the causative agents of cervical cancer worldwide, and that HPV-16 is associated with more than half of these cases. In addition to the well-characterized E6 and E7 oncoproteins of HPV-16, recent evidence increasingly has implicated the HPV-16 E5 protein (16E5) as an important mediator of oncogenic transformation. Since 16E5 has no known intrinsic enzymatic activity, its effects on infected cells are most likely mediated by interactions with various cellular proteins and/or its documented association with lipid rafts. In the present study, we describe a new cellular target that binds to 16E5 in COS cells and in stable human ectocervical cell lines. This target is karyopherin {beta}3, a member of the nuclear import receptor family with critical roles in the nuclear import of ribosomal proteins and in the secretory pathway.

  17. An oncolytic adenovirus enhances antiangiogenic and antitumoral effects of a replication-deficient adenovirus encoding endostatin by rescuing its selective replication in nasopharyngeal carcinoma cells.

    PubMed

    Liu, Ran-Yi; Zhou, Ling; Zhang, Yan-Ling; Huang, Bi-Jun; Ke, Miao-la; Chen, Jie-Min; Li, Li-Xia; Fu, Xiang; Wu, Jiang-Xue; Huang, Wenlin

    2013-12-13

    A replication-deficient adenovirus (Ad) encoding secreted human endostatin (Ad-Endo) has been demonstrated to have promising antiangiogenic and antitumoral effects. The E1B55k-deleted Ad H101 can selectively lyse cancer cells. In this study, we explored the antitumor effects and cross-interactions of Ad-Endo and H101 on nasopharyngeal carcinoma (NPC). The results showed that H101 dramatically promoted endostatin expression by Ad-Endo via rescuing Ad-Endo replication in NPC cells, and the expressed endostatin proteins significantly inhibited the proliferation of human umbilical vein endothelial cells. E1A and E1B19k products are required for the rescuing of H101 to Ad-Endo replication in CNE-1 and CNE-2 cells, but not in C666-1 cells. On the other hand, Ad-Endo enhanced the cytotoxicity of H101 by enhancing Ad replication in NPC cells. The combination of H101 and Ad-Endo significantly inhibited CNE-2 xenografts growth through the increased endostatin expression and Ad replication. These findings indicate that the combination of Ad-Endo gene therapy and oncolytic Ad therapeutics could be promising in comprehensive treatment of NPC.

  18. Identification of the nuclear localization and export signals of high risk HPV16 E7 oncoprotein

    SciTech Connect

    Knapp, Alixandra A.; McManus, Patrick M.; Bockstall, Katy; Moroianu, Junona

    2009-01-05

    The E7 oncoprotein of high risk human papillomavirus type 16 (HPV16) binds and inactivates the retinoblastoma (RB) family of proteins. Our previous studies suggested that HPV16 E7 enters the nucleus via a novel Ran-dependent pathway independent of the nuclear import receptors (Angeline, M., Merle, E., and Moroianu, J. (2003). The E7 oncoprotein of high-risk human papillomavirus type 16 enters the nucleus via a nonclassical Ran-dependent pathway. Virology 317(1), 13-23.). Here, analysis of the localization of specific E7 mutants revealed that the nuclear localization of E7 is independent of its interaction with pRB or of its phosphorylation by CKII. Fluorescence microscopy analysis of enhanced green fluorescent protein (EGFP) and 2xEGFP fusions with E7 and E7 domains in HeLa cells revealed that E7 contains a novel nuclear localization signal (NLS) in the N-terminal domain (aa 1-37). Interestingly, treatment of transfected HeLa cells with two specific nuclear export inhibitors, Leptomycin B and ratjadone, changed the localization of 2xEGFP-E7{sub 38-98} from cytoplasmic to mostly nuclear. These data suggest the presence of a leucine-rich nuclear export signal (NES) and a second NLS in the C-terminal domain of E7 (aa 38-98). Mutagenesis of critical amino acids in the putative NES sequence ({sub 76}IRTLEDLLM{sub 84}) changed the localization of 2xEGFP-E7{sub 38-98} from cytoplasmic to mostly nuclear suggesting that this is a functional NES. The presence of both NLSs and an NES suggests that HPV16 E7 shuttles between the cytoplasm and nucleus which is consistent with E7 having functions in both of these cell compartments.

  19. The oncoprotein HBXIP enhances angiogenesis and growth of breast cancer through modulating FGF8 and VEGF.

    PubMed

    Liu, Fabao; You, Xiaona; Wang, Yue; Liu, Qian; Liu, Yunxia; Zhang, Shuqin; Chen, Lingyi; Zhang, Xiaodong; Ye, Lihong

    2014-05-01

    Tumor angiogenesis plays an important role in the development of cancer. Previously, we reported that hepatitis B X-interacting protein (HBXIP) functioned as an oncoprotein in breast cancer. However, the role of HBXIP in angiogenesis in breast cancer remains poorly understood. In the present study, we show that the oncoprotein HBXIP plays crucial roles in the event. We observed that the expression levels of HBXIP were positively correlated with those of fibroblast growth factor 8 (FGF8) or vascular endothelial growth factor (VEGF) in clinical breast cancer tissues. Then, we demonstrated that HBXIP was able to upregulate FGF8 through activation of its promoter involving direct binding to cAMP response element-binding protein (CREB) in breast cancer cells and thereby increased its secretion. Strikingly, we identified another pathway that HBXIP upregulated FGF8 and VEGF through inhibiting miRNA-503, which directly targeted 3' untranslated region of FGF8 or VEGF mRNA in the cells. Moreover, we revealed that HBXIP-induced FGF8 could upregulate VEGF expression through activating phosphoinositide 3-kinase (PI3K)/Akt/hypoxia-inducible factor 1-alpha (HIF1α) signaling and increase its secretion. In function, matrigel angiogenesis assay and hemoglobin content analysis uncovered that HBXIP-enhanced FGF8/VEGF boosted tumor angiogenesis and growth in breast cancer in vitro and in vivo in a paracrine/autocrine manner. Thus, we conclude that HBXIP enhances angiogenesis and growth of breast cancer through modulating FGF8 and VEGF. Our finding provides new insights into the mechanism of tumor angiogenesis in breast cancer. Therapeutically, HBXIP may serve as a novel target of tumor angiogenesis.

  20. Isolation and Characterization of Adenoviruses Persistently Shed from the Gastrointestinal Tract of Non-Human Primates

    PubMed Central

    Kryazhimskiy, Sergey; Grant, Rebecca; Calcedo, Roberto; Yuan, Xin; Keough, Martin; Sandhu, Arbans; Wang, Qiang; Medina-Jaszek, C. Angelica; Plotkin, Joshua B.; Wilson, James M.

    2009-01-01

    Adenoviruses are important human pathogens that have been developed as vectors for gene therapies and genetic vaccines. Previous studies indicated that human infections with adenoviruses are self-limiting in immunocompetent hosts with evidence of some persistence in adenoid tissue. We sought to better understand the natural history of adenovirus infections in various non-human primates and discovered that healthy populations of great apes (chimpanzees, bonobos, gorillas, and orangutans) and macaques shed substantial quantities of infectious adenoviruses in stool. Shedding in stools from asymptomatic humans was found to be much less frequent, comparable to frequencies reported before. We purified and fully sequenced 30 novel adenoviruses from apes and 3 novel adenoviruses from macaques. Analyses of the new ape adenovirus sequences (as well as the 4 chimpanzee adenovirus sequences we have previously reported) together with 22 complete adenovirus genomes available from GenBank revealed that (a) the ape adenoviruses could clearly be classified into species corresponding to human adenovirus species B, C, and E, (b) there was evidence for intraspecies recombination between adenoviruses, and (c) the high degree of phylogenetic relatedness of adenoviruses across their various primate hosts provided evidence for cross species transmission events to have occurred in the natural history of B and E viruses. The high degree of asymptomatic shedding of live adenovirus in non-human primates and evidence for zoonotic transmissions warrants caution for primate handling and housing. Furthermore, the presence of persistent and/or latent adenovirus infections in the gut should be considered in the design and interpretation of human and non-human primate studies with adenovirus vectors. PMID:19578438

  1. Insights into Adenovirus Uncoating from Interactions with Integrins and Mediators of Host Immunity

    PubMed Central

    Nemerow, Glen R.; Stewart, Phoebe L.

    2016-01-01

    Human adenoviruses are large (150 MDa) nonenveloped double-stranded DNA (dsDNA) viruses that cause acute respiratory, gastrointestinal and ocular infections. Despite these disease associations, adenovirus has aided basic and clinical research efforts through studies of its association with cells and as a target of host antiviral responses. This review highlights the knowledge of adenovirus disassembly and nuclear transport gleaned from structural, biophysical and functional analyses of adenovirus interactions with soluble and membrane-associated host molecules. PMID:28009821

  2. ERK Signaling Pathway Is Involved in HPV-16 E6 but not E7 Oncoprotein-Induced HIF-1α Protein Accumulation in NSCLC Cells.

    PubMed

    Liu, Fei; Lin, Bihua; Liu, Xin; Zhang, Wenzhang; Zhang, Erying; Hu, Liang; Ma, Yuefan; Li, Xiangyong; Tang, Xudong

    2016-01-01

    Extracellular signal-regulated kinase (ERK)1/2 signaling pathway plays a critical role in regulating tumor angiogenesis. Our previous studies have demonstrated that HPV-16 oncoproteins enhanced hypoxia-inducible factor-1α (HIF-1α) protein accumulation and vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) expression in non-small cell lung cancer (NSCLC) cells, thus contributing to angiogenesis. In this study, we further investigated the role of ERK1/2 signaling pathway in HPV-16 oncoprotein-induced HIF-1α, VEGF, and IL-8 expression and in vitro angiogenesis in NSCLC cells. Our results showed that HPV-16 E6 and HPV-16 E7 oncoproteins promoted the activation of ERK1/2 signaling pathway in A549 and NCI-H460 cells. Moreover, PD98059, a specific inhibitor of ERK1/2, blocked in vitro angiogenesis stimulated by HPV-16 E6 but not E7 oncoprotein. Additionally, HIF-1α protein accumulation and VEGF and IL-8 expression in NSCLC cells induced by HPV-16 E6 but not E7 oncoprotein were significantly inhibited by PD98059. Taken together, our results suggest that ERK1/2 signaling pathway is involved in HPV-16 E6 but not E7 oncoprotein-induced HIF-1α, VEGF, and IL-8 expression in NSCLC cells, leading to the enhanced angiogenesis in vitro.

  3. Pertussis-like syndrome associated with adenovirus presenting with hyperleukocytosis: Case report

    PubMed Central

    Sarbay, Hakan; Polat, Aziz; Mete, Emin; Balci, Yasemin Isik; Akin, Mehmet

    2016-01-01

    Adenovirus is an infectious viral agent that causes variety of clinical presentations such as respiratory disease, conjunctivitis, and gastroenteritis. Hepatitis, pancreatitis, myocarditis, encephalitis, and disseminated infection are primarily seen in immunocompromised patients. Rarely, adenovirus infection can present with pertussis-like syndrome. Described here is case of pertussis-like syndrome associated with adenovirus presenting with hyperleukocytosis. PMID:28058402

  4. Exploiting features of adenovirus replication to support mammalian kinase production

    PubMed Central

    Cotten, Matt; Stegmueller, Kerstin; Eickhoff, Jan; Hanke, Miriam; Herzberger, Katrin; Herget, Thomas; Choidas, Axel; Daub, Henrik; Godl, Klaus

    2003-01-01

    Faced with the current wealth of genomic data, it is essential to have robust and reliable methods of converting DNA sequences into their functional gene products. We demonstrate here that when conditions are established that take advantage of the replication-associated virus amplification, the virus-induced shutdown of host protein synthesis as well as the activation of signalling pathways that normally occur during virus replication, adenovirus biology can be exploited to generate a potent kinase expression system. Residual virus in the protein production has always been a limitation for adenovirus systems and we describe a DNA intercalator/ultraviolet light treatment that eliminates residual adenovirus in protein preparations that has no deleterious effect on enzyme activity. The use of mammalian cells in combination with adenovirus generated a variety of active enzymes which could not be produced in Escherichia coli or baculovirus-infected insect cells. Thus, the utility of adenovirus-mediated enzyme expression as a versatile alternative to established protein production technologies is demonstrated. PMID:14576328

  5. Exploiting features of adenovirus replication to support mammalian kinase production.

    PubMed

    Cotten, Matt; Stegmueller, Kerstin; Eickhoff, Jan; Hanke, Miriam; Herzberger, Katrin; Herget, Thomas; Choidas, Axel; Daub, Henrik; Godl, Klaus

    2003-11-01

    Faced with the current wealth of genomic data, it is essential to have robust and reliable methods of converting DNA sequences into their functional gene products. We demonstrate here that when conditions are established that take advantage of the replication-associated virus amplification, the virus-induced shutdown of host protein synthesis as well as the activation of signalling pathways that normally occur during virus replication, adenovirus biology can be exploited to generate a potent kinase expression system. Residual virus in the protein production has always been a limitation for adenovirus systems and we describe a DNA intercalator/ultraviolet light treatment that eliminates residual adenovirus in protein preparations that has no deleterious effect on enzyme activity. The use of mammalian cells in combination with adenovirus generated a variety of active enzymes which could not be produced in Escherichia coli or baculovirus-infected insect cells. Thus, the utility of adenovirus-mediated enzyme expression as a versatile alternative to established protein production technologies is demonstrated.

  6. Oncolytic virotherapy for osteosarcoma using midkine promoter-regulated adenoviruses.

    PubMed

    Takagi-Kimura, M; Yamano, T; Tagawa, M; Kubo, S

    2014-03-01

    Oncolytic virotherapy using adenoviruses has potential therapeutic benefits for a variety of cancers. We recently developed MOA5, a tumor-specific midkine promoter-regulated oncolytic vector based on human adenovirus serotype 5 (Ad5). We modified the binding tropism of MOA5 by replacing the cell-binding domain of the Ad5 fiber knob with that from another adenovirus serotype 35 (Ad35); the resulting vector was designated MOA35. Here we evaluated the therapeutic efficacies of MOA5 and MOA35 for human osteosarcoma. Midkine mRNA expression and its promoter activity was significantly high in five human osteosarcoma cell lines, but was restricted in normal cells. Very low levels of adenovirus cellular receptor coxsackievirus/adenovirus receptor (CAR) (Ad5 receptor) expression were observed in MNNG-HOS and MG-63 cells, whereas high levels of CAR expression were seen in the other osteosarcoma cell lines. By contrast, CD46 (Ad35 receptor) was highly expressed in all osteosarcoma cell lines. Infectivity and in vitro cytocidal effect of MOA35 was significantly enhanced in MNNG-HOS and MG-63 cells compared with MOA5, although the cytocidal effects of MOA5 were sometimes higher in high CAR-expressing cell lines. In MG-63 xenograft models, MOA35 significantly enhanced antitumor effects compared with MOA5. Our findings indicate that MOA5 and MOA35 allow tailored virotherapy and facilitate more effective treatments for osteosarcoma.

  7. Retargeted adenoviruses for radiation-guided gene delivery

    PubMed Central

    Kaliberov, S A; Kaliberova, L N; Yan, H; Kapoor, V; Hallahan, D E

    2016-01-01

    The combination of radiation with radiosensitizing gene delivery or oncolytic viruses promises to provide an advantage that could improve the therapeutic results for glioblastoma. X-rays can induce significant molecular changes in cancer cells. We isolated the GIRLRG peptide that binds to radiation-inducible 78 kDa glucose-regulated protein (GRP78), which is overexpressed on the plasma membranes of irradiated cancer cells and tumor-associated microvascular endothelial cells. The goal of our study was to improve tumor-specific adenovirus-mediated gene delivery by selectively targeting the adenovirus binding to this radiation-inducible protein. We employed an adenoviral fiber replacement approach to conduct a study of the targeting utility of GRP78-binding peptide. We have developed fiber-modified adenoviruses encoding the GRP78-binding peptide inserted into the fiber-fibritin. We have evaluated the reporter gene expression of fiber-modified adenoviruses in vitro using a panel of glioma cells and a human D54MG tumor xenograft model. The obtained results demonstrated that employment of the GRP78-binding peptide resulted in increased gene expression in irradiated tumors following infection with fiber-modified adenoviruses, compared with untreated tumor cells. These studies demonstrate the feasibility of adenoviral retargeting using the GRP78-binding peptide that selectively recognizes tumor cells responding to radiation treatment. PMID:27492853

  8. Bovine adenovirus-3 as a vaccine delivery vehicle.

    PubMed

    Ayalew, Lisanework E; Kumar, Pankaj; Gaba, Amit; Makadiya, Niraj; Tikoo, Suresh K

    2015-01-15

    The use of vaccines is an effective and relatively inexpensive means of controlling infectious diseases, which cause heavy economic losses to the livestock industry through animal loss, decreased productivity, treatment expenses and decreased carcass quality. However, some vaccines produced by conventional means are imperfect in many respects including virulence, safety and efficacy. Moreover, there are no vaccines for some animal diseases. Although genetic engineering has provided new ways of producing effective vaccines, the cost of production for veterinary use is a critical criterion for selecting the method of production and delivery of vaccines. The cost effective production and intrinsic ability to enter cells has made adenovirus vectors a highly efficient tool for delivery of vaccine antigens. Moreover, adenoviruses induce both humoral and cellular immune responses to expressed vaccine antigens. Since nonhuman adenoviruses are species specific, the development of animal specific adenoviruses as vaccine delivery vectors is being evaluated. This review summarizes the work related to the development of bovine adenovirus-3 as a vaccine delivery vehicle in animals, particularly cattle.

  9. Gene targeting with a replication-defective adenovirus vector.

    PubMed Central

    Fujita, A; Sakagami, K; Kanegae, Y; Saito, I; Kobayashi, I

    1995-01-01

    Wide application of the gene-targeting technique has been hampered by its low level of efficiency. A replication-defective adenovirus vector was used for efficient delivery of donor DNA in order to bypass this problem. Homologous recombination was selected between a donor neo gene inserted in the adenovirus vector and a target mutant neo gene on a nuclear papillomavirus plasmid. These recombinant adenoviruses allowed gene transfer to 100% of the treated cells without impairing their viability. Homologous recombinants were obtained at a level of frequency much higher than that obtained by electroporation or a calcium phosphate procedure. The structure of the recombinants was analyzed in detail after recovery in an Escherichia coli strain. All of the recombinants examined had experienced a precise correction of the mutant neo gene. Some of them had a nonhomologous rearrangement of their sequences as well. One type of nonhomologous recombination took place at the end of the donor-target homology. The vector adenovirus DNA was inserted into some of the products obtained at a high multiplicity of infection. The insertion was at the end of the donor-target homology with a concomitant insertion of a 10-bp-long filler sequence in one of the recombinants. The possible relationship between these rearrangements and the homologous recombination is discussed. These results demonstrate the applicability of adenovirus-mediated gene delivery in gene targeting and gene therapy. PMID:7666520

  10. Latest insights on adenovirus structure and assembly.

    PubMed

    San Martín, Carmen

    2012-05-01

    Adenovirus (AdV) capsid organization is considerably complex, not only because of its large size (~950 Å) and triangulation number (pseudo T = 25), but also because it contains four types of minor proteins in specialized locations modulating the quasi-equivalent icosahedral interactions. Up until 2009, only its major components (hexon, penton, and fiber) had separately been described in atomic detail. Their relationships within the virion, and the location of minor coat proteins, were inferred from combining the known crystal structures with increasingly more detailed cryo-electron microscopy (cryoEM) maps. There was no structural information on assembly intermediates. Later on that year, two reports described the structural differences between the mature and immature adenoviral particle, starting to shed light on the different stages of viral assembly, and giving further insights into the roles of core and minor coat proteins during morphogenesis [1,2]. Finally, in 2010, two papers describing the atomic resolution structure of the complete virion appeared [3,4]. These reports represent a veritable tour de force for two structural biology techniques: X-ray crystallography and cryoEM, as this is the largest macromolecular complex solved at high resolution by either of them. In particular, the cryoEM analysis provided an unprecedented clear picture of the complex protein networks shaping the icosahedral shell. Here I review these latest developments in the field of AdV structural studies.

  11. Latest Insights on Adenovirus Structure and Assembly

    PubMed Central

    San Martín, Carmen

    2012-01-01

    Adenovirus (AdV) capsid organization is considerably complex, not only because of its large size (~950 Å) and triangulation number (pseudo T = 25), but also because it contains four types of minor proteins in specialized locations modulating the quasi-equivalent icosahedral interactions. Up until 2009, only its major components (hexon, penton, and fiber) had separately been described in atomic detail. Their relationships within the virion, and the location of minor coat proteins, were inferred from combining the known crystal structures with increasingly more detailed cryo-electron microscopy (cryoEM) maps. There was no structural information on assembly intermediates. Later on that year, two reports described the structural differences between the mature and immature adenoviral particle, starting to shed light on the different stages of viral assembly, and giving further insights into the roles of core and minor coat proteins during morphogenesis [1,2]. Finally, in 2010, two papers describing the atomic resolution structure of the complete virion appeared [3,4]. These reports represent a veritable tour de force for two structural biology techniques: X-ray crystallography and cryoEM, as this is the largest macromolecular complex solved at high resolution by either of them. In particular, the cryoEM analysis provided an unprecedented clear picture of the complex protein networks shaping the icosahedral shell. Here I review these latest developments in the field of AdV structural studies. PMID:22754652

  12. Adenovirus 36 and Obesity: An Overview

    PubMed Central

    Ponterio, Eleonora; Gnessi, Lucio

    2015-01-01

    There is an epidemic of obesity starting about 1980 in both developed and undeveloped countries definitely associated with multiple etiologies. About 670 million people worldwide are obese. The incidence of obesity has increased in all age groups, including children. Obesity causes numerous diseases and the interaction between genetic, metabolic, social, cultural and environmental factors are possible cofactors for the development of obesity. Evidence emerging over the last 20 years supports the hypothesis that viral infections may be associated with obesity in animals and humans. The most widely studied infectious agent possibly linked to obesity is adenovirus 36 (Adv36). Adv36 causes obesity in animals. In humans, Adv36 associates with obesity both in adults and children and the prevalence of Adv36 increases in relation to the body mass index. In vivo and in vitro studies have shown that the viral E4orf1 protein (early region 4 open reading frame 1, Adv) mediates the Adv36 effect including its adipogenic potential. The Adv36 infection should therefore be considered as a possible risk factor for obesity and could be a potential new therapeutic target in addition to an original way to understand the worldwide rise of the epidemic of obesity. Here, the data indicating a possible link between viral infection and obesity with a particular emphasis to the Adv36 will be reviewed. PMID:26184280

  13. An adenoviral vector-based expression and delivery system for the inhibition of wild-type adenovirus replication by artificial microRNAs

    PubMed Central

    Ibrišimović, Mirza; Kneidinger, Doris; Lion, Thomas; Klein, Reinhard

    2013-01-01

    Human adenoviruses are rarely associated with life-threatening infections in healthy individuals. However, immunocompromised patients, and particularly allogeneic hematopoietic stem cell transplant recipients, are at high risk of developing disseminated and potentially fatal disease. The efficacy of commonly used drugs to treat adenovirus infections (i.e., cidofovir in most cases) is limited, and alternative treatment options are needed. Artificial microRNAs (amiRNAs) are a class of synthetic RNAs resembling cellular miRNAs, and, similar to their natural relatives, can mediate the knockdown of endogenous gene expression. This process, termed RNA interference, can be harnessed to target and potentially silence both cellular and viral genes. In this study, we designed amiRNAs directed against adenoviral E1A, DNA polymerase, and preterminal protein (pTP) mRNAs in order to inhibit adenoviral replication in vitro. For the expression of amiRNA-encoding sequences, we utilized replication-deficient adenoviral vectors. In cells transduced with the recombinant vectors and infected with the wild-type (wt) adenovirus, one particular amiRNA that was directed against the pTP mRNA was capable of decreasing the output of infectious wt virus progeny by 2.6 orders of magnitude. This inhibition rate could be achieved by concatemerizing amiRNA-encoding sequences to allow for high intracellular amiRNA concentrations. Because superinfecting wt virus induces the replication and amplification of the recombinant adenoviral vector, amiRNA concentrations were increased in cells infected with wt adenovirus. Furthermore, a combination of amiRNA expression and treatment of infected cells with cidofovir resulted in additive effects that manifested as a total reduction of infectious virus progeny by greater than 3 orders of magnitude. PMID:23127366

  14. An adenoviral vector-based expression and delivery system for the inhibition of wild-type adenovirus replication by artificial microRNAs.

    PubMed

    Ibrišimović, Mirza; Kneidinger, Doris; Lion, Thomas; Klein, Reinhard

    2013-01-01

    Human adenoviruses are rarely associated with life-threatening infections in healthy individuals. However, immunocompromised patients, and particularly allogeneic hematopoietic stem cell transplant recipients, are at high risk of developing disseminated and potentially fatal disease. The efficacy of commonly used drugs to treat adenovirus infections (i.e., cidofovir in most cases) is limited, and alternative treatment options are needed. Artificial microRNAs (amiRNAs) are a class of synthetic RNAs resembling cellular miRNAs, and, similar to their natural relatives, can mediate the knockdown of endogenous gene expression. This process, termed RNA interference, can be harnessed to target and potentially silence both cellular and viral genes. In this study, we designed amiRNAs directed against adenoviral E1A, DNA polymerase, and preterminal protein (pTP) mRNAs in order to inhibit adenoviral replication in vitro. For the expression of amiRNA-encoding sequences, we utilized replication-deficient adenoviral vectors. In cells transduced with the recombinant vectors and infected with the wild-type (wt) adenovirus, one particular amiRNA that was directed against the pTP mRNA was capable of decreasing the output of infectious wt virus progeny by 2.6 orders of magnitude. This inhibition rate could be achieved by concatemerizing amiRNA-encoding sequences to allow for high intracellular amiRNA concentrations. Because superinfecting wt virus induces the replication and amplification of the recombinant adenoviral vector, amiRNA concentrations were increased in cells infected with wt adenovirus. Furthermore, a combination of amiRNA expression and treatment of infected cells with cidofovir resulted in additive effects that manifested as a total reduction of infectious virus progeny by greater than 3 orders of magnitude.

  15. MYB3Rs, plant homologs of Myb oncoproteins, control cell cycle-regulated transcription and form DREAM-like complexes.

    PubMed

    Kobayashi, Kosuke; Suzuki, Toshiya; Iwata, Eriko; Magyar, Zoltán; Bögre, László; Ito, Masaki

    2015-01-01

    Plant MYB3R transcription factors, homologous to Myb oncoproteins, regulate the genes expressed at G2 and M phases in the cell cycle. Recent studies showed that MYB3Rs constitute multiprotein complexes that may correspond to animal complexes known as DREAM or dREAM. Discovery of the putative homologous complex in plants uncovered their significant varieties in structure, function, dynamics, and heterogeneity, providing insight into conserved and diversified aspects of cell cycle-regulated gene transcription.

  16. Flat cells come full sphere: Are mutant cytoskeletal-related proteins oncoprotein-monsters or useful immunogens?

    PubMed

    Parry, Michele L; Blanck, George

    2016-01-01

    Osteogenesis imperfecta is inherited as a dominant disease because if one allele is mutated, it contributes a mutant, destructive subunit polypeptide to collagen, which requires many subunits to form normal, polymeric, collagenous structures. Recent cancer genome atlas (TCGA) data indicate that cytoskeletal-related proteins are among the most commonly mutated proteins in human cancers, in distinct mutation frequency groups, i.e., including low mutation frequency groups. Part of the explanation for this observation is likely to be the fact that many of the coding regions for these proteins are very large, and indeed, it is likely these coding regions are mutated in many cells that never become cancerous. However, it would not be surprising if mutations in cytoskeletal proteins, when combined with oncoprotein or tumor suppressor protein mutations, had significant impacts on cancer development, for a number of reasons, including results obtained almost 5 decades ago indicating that well-spread cells in tissue culture, with well-formed cytoskeletons, were less tumorigenic than spherical cells with disrupted cytoskeletons. This raises the question, are mutant cytoskeletal proteins, which would likely interfere with polymer formation, a new class of oncoproteins, in particular, dominant negative oncoproteins? If these proteins are so commonly mutant, could they be the bases for common cancer vaccines?

  17. Complete genome sequences of pigeon adenovirus 1 and duck adenovirus 2 extend the number of species within the genus Aviadenovirus.

    PubMed

    Marek, Ana; Kaján, Győző L; Kosiol, Carolin; Harrach, Balázs; Schlötterer, Christian; Hess, Michael

    2014-08-01

    Complete genomes of the first isolates of pigeon adenovirus 1 (PiAdV-1) and Muscovy duck adenovirus (duck adenovirus 2, DAdV-2) were sequenced. The PiAdV-1 genome is 45,480bp long, and has a gene organization most similar to turkey adenovirus 1. Near the left end of the genome, it lacks ORF0, ORF1A, ORF1B and ORF1C, and possesses ORF52, whereas six novel genes were found near the right end. The DAdV-2 genome is 43,734bp long, and has a gene organization similar to that of goose adenovirus 4 (GoAdV-4). It lacks ORF51, ORF1C and ORF54, and possesses ORF55A and five other novel genes. PiAdV-1 and DAdV-2 genomes contain two and one fiber genes, respectively. Genome organization, G+C content, molecular phylogeny and host type confirm the need to establish two novel species (Pigeon aviadenovirus A and Duck aviadenovirus B) within the genus Aviadenovirus. Phylogenetic data show that DAdV-2 is most closely related to GoAdV-4.

  18. Inhibition of adenovirus replication in vitro by trifluridine.

    PubMed

    Lennette, D A; Eiferman, R A

    1978-09-01

    At present, there is no effective chemotherapeutic agent available for the treatment of adenoviral keratoconjunctivitis. Recent evidence suggests that trifluridine (3FT) may effectively inhibit the replication of some adenovirus serotypes known to cause keratoconjunctivitis. The ability of 3FT to inhibit two reference strains of adenoviruses, type 8 and type 19, was examined using cell cultures. Two second-passage isolates of adenoviruses, identified as serotype 13, were also tested. Compared with untreated, virusinfected cell cultures, drug-treated cell cultures developed a lesser degree of cytopathic effect following infection with all three serotypes. Virus production was reduced in the drug-treated cell cultures: approximately tenfold for type 8, more than 1,000-fold for type 19, and 5,000-fold for the type 13 isolates.

  19. Capsid-like Arrays in Crystals of Chimpanzee Adenovirus Hexon

    SciTech Connect

    Xue,F.; Burnett, R.

    2006-01-01

    The major coat protein, hexon, from a chimpanzee adenovirus (AdC68) is of interest as a target for vaccine vector modification. AdC68 hexon has been crystallized in the orthorhombic space group C222 with unit cell dimensions of a = 90.8 Angstroms, b = 433.0 Angstroms, c = 159.3 Angstroms, and one trimer (3 x 104,942 Da) in the asymmetric unit. The crystals diffract to 2.1 Angstroms resolution. Initial studies reveal that the molecular arrangement is quite unlike that in hexon crystals for human adenovirus. In the AdC68 crystals, hexon trimers are parallel and pack closely in two-dimensional continuous arrays similar to those formed on electron microscope grids. The AdC68 crystals are the first in which adenovirus hexon has molecular interactions that mimic those used in constructing the viral capsid.

  20. Genetic Relatedness Studies with Adenovirus-associated Viruses

    PubMed Central

    Rose, James A.; Hoggan, M. David; Koczot, Frank; Shatkin, Aaron J.

    1968-01-01

    Adenovirus-associated viruses (AAV) contain double-stranded deoxyribonucleic acid (DNA). DNA from each of the four AAV serotypes was used as template for the in vitro synthesis of complementary 3H-ribonucleic acids(RNA). An estimation of genetic interrelatedness was made on the basis of hybridization reactions between synthetic AAV RNA and AAV DNA. Heterologous reactions were 27 to 37% of homologous reactions, suggesting that the AAV serotypes are related to about the same extent. AAV-1 synthetic RNA was also reacted with DNA from helper adenovirus types 2, 7, and SV15. Very low levels of RNA binding were observed, but it is not likely that these reactions represent AAV-adenovirus genetic relatedness. PMID:5739847

  1. Characterization of a novel adenovirus isolated from a skunk.

    PubMed

    Kozak, Robert A; Ackford, James G; Slaine, Patrick; Li, Aimin; Carman, Susy; Campbell, Doug; Welch, M Katherine; Kropinski, Andrew M; Nagy, Éva

    2015-11-01

    Adenoviruses are a ubiquitous group of viruses that have been found in a wide range of hosts. A novel adenovirus from a skunk suffering from acute hepatitis was isolated and its DNA genome sequenced. The analysis revealed this virus to be a new member of the genus Mastadenovirus, with a genome of 31,848 bp in length containing 30 genes predicted to encode proteins, and with a G+C content of 49.0%. Global genomic organization indicated SkAdV-1 was similar in organization to bat and canine adenoviruses, and phylogenetic comparison suggested these viruses shared a common ancestor. SkAdV-1 demonstrated an ability to replicate in several mammalian liver cell lines suggesting a potential tropism for this virus.

  2. Effects of cold atmospheric plasmas on adenoviruses in solution

    NASA Astrophysics Data System (ADS)

    Zimmermann, J. L.; Dumler, K.; Shimizu, T.; Morfill, G. E.; Wolf, A.; Boxhammer, V.; Schlegel, J.; Gansbacher, B.; Anton, M.

    2011-12-01

    Experiments were performed with cold atmospheric plasma (CAP) to inactivate adenovirus, a non-enveloped double stranded DNA virus, in solution. The plasma source used was a surface micro-discharge technology operating in air. Various plasma diagnostic measurements and tests were performed in order to determine the efficacy of CAPs and to understand the inactivation mechanism(s). Different stages of the adenovirus ‘life cycle’ were investigated—infectivity and gene expression as well as viral replication and spread. Within 240 s of CAP treatment, inactivation of up to 6 decimal log levels can be achieved.

  3. Transport of human adenoviruses in porous media

    NASA Astrophysics Data System (ADS)

    Kokkinos, Petros; Syngouna, Vasiliki I.; Tselepi, Maria A.; Bellou, Maria; Chrysikopoulos, Constantinos V.; Vantarakis, Apostolos

    2015-04-01

    Groundwater may be contaminated with infective human enteric viruses from various wastewater discharges, sanitary landfills, septic tanks, agricultural practices, and artificial groundwater recharge. Coliphages have been widely used as surrogates of enteric viruses, because they share many fundamental properties and features. Although a large number of studies focusing on various factors (i.e. pore water solution chemistry, fluid velocity, moisture content, temperature, and grain size) that affect biocolloid (bacteria, viruses) transport have been published over the past two decades, little attention has been given toward human adenoviruses (hAdVs). The main objective of this study was to evaluate the effect of pore water velocity on hAdV transport in water saturated laboratory-scale columns packed with glass beads. The effects of pore water velocity on virus transport and retention in porous media was examined at three pore water velocities (0.39, 0.75, and 1.22 cm/min). The results indicated that all estimated average mass recovery values for hAdV were lower than those of coliphages, which were previously reported in the literature by others for experiments conducted under similar experimental conditions. However, no obvious relationship between hAdV mass recovery and water velocity could be established from the experimental results. The collision efficiencies were quantified using the classical colloid filtration theory. Average collision efficiency, α, values decreased with decreasing flow rate, Q, and pore water velocity, U, but no significant effect of U on α was observed. Furthermore, the surface properties of viruses and glass beads were used to construct classical DLVO potential energy profiles. The results revealed that the experimental conditions of this study were unfavorable to deposition and that no aggregation between virus particles is expected to occur. A thorough understanding of the key processes governing virus transport is pivotal for public

  4. Adenovirus Respiratory Tract Infections in Peru

    PubMed Central

    Ampuero, Julia S.; Ocaña, Víctor; Gómez, Jorge; Gamero, María E.; Garcia, Josefina; Halsey, Eric S.; Laguna-Torres, V. Alberto

    2012-01-01

    Background Currently, there is a paucity of data regarding human adenovirus (HAdv) circulation in Andean regions of South America. To address this shortcoming, we report the clinical, phylogenetic, and epidemiologic characteristics of HAdv respiratory tract infection from a large sentinel surveillance study conducted among adults and children in Peru. Methods/Principal Findings Oropharyngeal swabs were collected from participants visiting any of 38 participating health centers, and viral pathogens were identified by immunofluorescence assay in cell culture. In addition, molecular characterization was performed on 226 randomly selected HAdv samples. Between 2000 and 2010, a total of 26,375 participants with influenza-like illness (ILI) or severe acute respiratory infection (SARI) were enrolled in the study. HAdv infection was identified in 2.5% of cases and represented 6.2% of all viral pathogens. Co-infection with a heterologous virus was found in 15.5% of HAdv cases. HAdv infection was largely confined to children under the age of 15, representing 88.6% of HAdv cases identified. No clinical characteristics were found to significantly distinguish HAdv infection from other respiratory viruses. Geographically, HAdv infections were more common in sites from the arid coastal regions than in the jungle or highland regions. Co-circulation of subgroups B and C was observed each year between 2006 and 2010, but no clear seasonal patterns of transmission were detected. Conclusions/Significance HAdv accounted for a significant fraction of those presenting with ILI and SARI in Peru and tended to affect the younger population disproportionately. Longitudinal studies will help better characterize the clinical course of patients with HAdv in Peru, as well as determine the role of co-infections in the evolution of illness. PMID:23056519

  5. Adenovirus infection stimulates the Raf/MAPK signaling pathway and induces interleukin-8 expression.

    PubMed Central

    Bruder, J T; Kovesdi, I

    1997-01-01

    Previous studies have shown that airway administration of adenovirus or adenovirus vectors results in a dose-dependent inflammatory response which limits the duration of transgene expression. We explored the possibility that adenovirus infection triggers signal transduction pathways that induce the synthesis of cytokines and thus contribute to the early inflammatory response. Since stimulation of the Raf/mitogen-activated protein kinase (MAPK) pathway activates transcription factors that control the expression of inflammatory cytokines, we examined the activation of this pathway following adenovirus infection. Adenovirus infection induced the rapid activation of Raf-1 and a transient increase in the tyrosine phosphorylation and activation of p42mapk at early times postinfection. Activation of the Raf/MAPK pathway by adenovirus is likely triggered by the infection process, since it occurred rapidly and with various mutant adenoviruses and adenovirus vectors. Moreover, interleukin-8 (IL-8) mRNA accumulation was evident at 20 min postinfection and was induced even in the presence of cycloheximide. Both MAPK activation and IL-8 production were inhibited by forskolin, a potent inhibitor of Raf-1. These results suggest that adenovirus-induced Raf/MAPK activation contributes to IL-8 production. Adenovirus-induced activation of the Raf/MAPK signaling pathway and IL-8 production may play critical roles in the inflammation observed following in vivo administration of adenovirus vectors for gene therapy. PMID:8985363

  6. Identification and characterization of a novel adenovirus in the cloacal bursa of gulls

    SciTech Connect

    Bodewes, R.; Bildt, M.W.G. van de; Schapendonk, C.M.E.; Leeuwen, M. van; Boheemen, S. van; Jong, A.A.W. de; Osterhaus, A.D.M.E.; Smits, S.L.; Kuiken, T.

    2013-05-25

    Several viruses of the family of Adenoviridae are associated with disease in birds. Here we report the detection of a novel adenovirus in the cloacal bursa of herring gulls (Larus argentatus) and lesser black-backed gulls (Larus fuscus) that were found dead in the Netherlands in 2001. Histopathological analysis of the cloacal bursa revealed cytomegaly and karyomegaly with basophilic intranuclear inclusions typical for adenovirus infection. The presence of an adenovirus was confirmed by electron microscopy. By random PCR in combination with deep sequencing, sequences were detected that had the best hit with known adenoviruses. Phylogenetic analysis of complete coding sequences of the hexon, penton and polymerase genes indicates that this novel virus, tentatively named Gull adenovirus, belongs to the genus Aviadenovirus. The present study demonstrates that birds of the Laridae family are infected by family-specific adenoviruses that differ from known adenoviruses in other bird species. - Highlights: ► Lesions typical for adenovirus infection detected in cloacal bursa of dead gulls. ► Confirmation of adenovirus infection by electron microscopy and deep sequencing. ► Sequence analysis indicates that it is a novel adenovirus in the genus Aviadenovirus. ► The novel (Gull) adenovirus was detected in multiple organs of two species of gulls.

  7. A novel tetracycline-controlled transactivator-transrepressor system enables external control of oncolytic adenovirus replication.

    PubMed

    Fechner, H; Wang, X; Srour, M; Siemetzki, U; Seltmann, H; Sutter, A P; Scherübl, H; Zouboulis, C C; Schwaab, R; Hillen, W; Schultheiss, H-P; Poller, W

    2003-09-01

    The use of restricted replication-competent adenoviruses (RRCAs) inducing tumor cell-specific lysis is a promising approach in cancer gene therapy. However, the use of RRCAs in humans carries considerable risk, since after injection into the patient, further regulation or inhibition of virus replication from the outside is impossible. Therefore, we have developed a novel system allowing external pharmacological control of RRCA replication. We show here that a tumor-selective E1B-deleted RRCA can be tightly regulated by use of doxycycline (dox)-controlled adenoviral E1A gene expression, which in turn determines vector replication. RRCA replication is switched on by addition and switched off by withdrawal of dox. The system results in efficient tumor cell killing after induction by dox, whereas cells are unaffected by the uninduced system. It was also employed for efficient external control of transgene expression from cotransfected replication-deficient adenovectors. Furthermore, the use of a liver cell-specific human alpha1-antitrypsin (hAAT)-promoter driving a tetracycline-controlled transcriptional silencer allowed specific protection of cells with hAAT-promoter activity in the absence of dox in vitro and in vivo, delineating a new principle of 'tissue protective' gene therapy. The concept of external control of RRCAs may help to improve the safety of cancer gene therapy.

  8. Phylogenetic and pathogenic characterization of novel adenoviruses isolated from long-tailed ducks (Clangula hyemalis).

    PubMed

    Counihan, Katrina L; Skerratt, Lee F; Franson, J Christian; Hollmén, Tuula E

    2015-11-01

    Novel adenoviruses were isolated from a long-tailed duck (Clangula hyemalis) mortality event near Prudhoe Bay, Alaska in 2000. The long-tailed duck adenovirus genome was approximately 27 kb. A 907 bp hexon gene segment was used to design primers specific for the long-tailed duck adenovirus. Nineteen isolates were phylogenetically characterized based on portions of their hexon gene and 12 were most closely related to Goose adenovirus A. The remaining 7 shared no hexon sequences with any known adenoviruses. Experimental infections of mallards with a long-tailed duck reference adenovirus caused mild lymphoid infiltration of the intestine and paint brush hemorrhages of the mucosa and dilation of the intestine. This study shows novel adenoviruses from long-tailed ducks are diverse and provides further evidence that they should be considered in cases of morbidity and mortality in sea ducks. Conserved and specific primers have been developed that will help screen sea ducks for adenoviral infections.

  9. C-erbB-2 onco-protein expression in breast cancer: relationship to tumour characteristics and short-term survival in Universiti Kebansaan Malaysia Medical Centre.

    PubMed

    Sharifah, N A; Lee, B R; Clarence-Ko, C H; Tan, G C; Shiran, M S; Naqiyah, I; Rohaizak, M; Fuad, I; Tamil, A M

    2008-01-01

    Breast cancer is the commonest cancer affecting females in Malaysia, contributing 31% of all newly diagnosed cases amongst Malaysian women. The present retrospective cohort study evaluated the relationship between cerbB- 2 onco-protein overexpression with various tumour characteristics and survival rate of breast cancer patients treated at the Universiti Kebangsaan Malaysia Medical Centre (UKMMC) between 1996-2000. CerbB- 2 oncoprotein overexpression was determined by immunohistochemistry (IHC) and tumors showing 2+ positivity were verified by Fluorescence In Situ Hybridization (FISH). One hundred and seventy two patients were eligible for the study with a short-term follow-up (median) of 5.1 years. C-erbB-2 oncoprotein overexpression correlated with lymph node positivity, oestrogen receptor (ER) and progesterone receptor (PR) negativity. Univariate analyses showed shorter disease free survival (DFS) and overall survival (OS) in patients with cerbB- 2 oncoprotein overexpression, Malay ethnicity, higher tumour grade, lymph node positivity, ER and PR negativity. In a subgroup of patients with c-erbB-2 oncoprotein overexpression, a shorter OS was observed in those with lymph node positivity, ER and PR negativity. In multivariate prognostic analysis, lymph node status, ER status and tumour grading were the strongest independent prognostic factors for both OS and DFS. However, c-erbB-2 status was not a significantly independent prognostic factor, even in subsets with lymph node positive or negative group. C-erbB-2 oncoprotein overexpression correlated well with lymph node status, ER and PR. Shorter OS and DFS were significantly observed in patients with c-erbB-2 oncoprotein overexpression. Lymph node status, ER status and tumour grading were the only three independent prognostic factors for OS and DFS in this study. Although c-erbB-2 expression is obviously important from a biological standpoint, multivariate analysis showed that it is not an independent prognostic

  10. The oncoprotein HBXIP promotes migration of breast cancer cells via GCN5-mediated microtubule acetylation.

    PubMed

    Li, Leilei; Liu, Bowen; Zhang, Xiaodong; Ye, Lihong

    2015-03-13

    We have documented that the oncoprotein hepatitis B X-interacting protein (HBXIP) is able to promote migration of breast cancer cells. A subset of acetylated microtubules that accumulates in the cell leading edge is necessary for cell polarization and directional migration. In this study, we explored the hypothesis that HBXIP contributes to migration of breast cancer cells by supporting microtubule acetylation in breast cancer cells. We found that HBXIP could induce acetylated microtubules accumulating into the leading protrusion in wound-induced directional migration in breast cancer cells by immunofluorescence staining analysis. Interestingly, HBXIP was able to increase the acetylation of α-tubulin in the cells by immunofluorescence staining and Western blot analysis. Furthermore, we observed that acetyltransferase GCN5 was involved in the event that HBXIP induced increase of acetylated microtubules and their expansion in protrusions in breast cancer cells by Western blot analysis and immunofluorescence staining. Moreover, GCN5 was required for the HBXIP-enhanced migration of breast cancer cells by wound healing assay. Thus, we conclude that HBXIP promotes the migration of breast cancer cells through modulating microtubule acetylation mediated by GCN5. Therapeutically, HBXIP may serve as a novel target in breast cancer.

  11. Structure of the replicative helicase of the oncoprotein SV40 large tumour antigen.

    SciTech Connect

    Li, D.; Zhao, R.; Lilyestrom, W.; Gai, D.; Zhang, R.; DeCaprio, J. A.; Fanning, E.; Joachimiak, A.; Szakonyi, G.; Chen, X. S.; Univ. of Colorado Health Science Center; Dana-Farber Cancer Ins.; Vanderbilt Univ.

    2003-05-29

    The oncoprotein large tumour antigen (LTag) is encoded by the DNA tumour virus simian virus 40. LTag transforms cells and induces tumours in animals by altering the functions of tumour suppressors (including pRB and p53) and other key cellular proteins. LTag is also a molecular machine that distorts/melts the replication origin of the viral genome and unwinds duplex DNA. LTag therefore seems to be a functional homologue of the eukaryotic minichromosome maintenance (MCM) complex. Here we present the X-ray structure of a hexameric LTag with DNA helicase activity. The structure identifies the p53-binding surface and reveals the structural basis of hexamerization. The hexamer contains a long, positively charged channel with an unusually large central chamber that binds both single-stranded and double-stranded DNA. The hexamer organizes into two tiers that can potentially rotate relative to each other through connecting alpha-helices to expand/constrict the channel, producing an 'iris' effect that could be used for distorting or melting the origin and unwinding DNA at the replication fork.

  12. Structural characterization of a C-terminally truncated E5 oncoprotein from papillomavirus in lipid bilayers.

    PubMed

    Windisch, Dirk; Ziegler, Colin; Bürck, Jochen; Ulrich, Anne S

    2014-12-01

    E5 is the major transforming oncoprotein of bovine papillomavirus, which activates the platelet-derived growth factor receptor β in a highly specific manner. The short transmembrane protein E5 with only 44 residues interacts directly with the transmembrane segments of the receptor, but structural details are not available. Biophysical investigations are challenging, because the hydrophobic E5 protein tends to aggregate and get cross-linked non-specifically via two Cys residues near its C-terminus. Here, we demonstrate that a truncation by 10 amino acids creates a more manageable protein that can be conveniently used for structure analysis. Synchrotron radiation circular dichroism and solid-state (15)N- and (31)P-nuclear magnetic resonance spectroscopy show that this E5 variant serves as a representative model for the wild-type protein. The helical conformation of the transmembrane segment, its orientation in the lipid bilayer, and the ability to form homodimers in the membrane are not affected by the C-terminal truncation.

  13. Bridging Links between Long Noncoding RNA HOTAIR and HPV Oncoprotein E7 in Cervical Cancer Pathogenesis

    PubMed Central

    Sharma, Sweta; Mandal, Paramita; Sadhukhan, Tamal; Roy Chowdhury, Rahul; Ranjan Mondal, Nidhu; Chakravarty, Biman; Chatterjee, Tanmay; Roy, Sudipta; Sengupta, Sharmila

    2015-01-01

    Human Papillomavirus (HPV) type 16 oncoprotein E7 plays a major role in cervical carcinogenesis by interacting with and functionally inactivating various host regulatory molecules. Long noncoding RNA (lncRNA) HOTAIR is one such regulator that recruits chromatin remodelling complex PRC2, creating gene silencing H3K27 me3 marks. Hence, we hypothesized that HOTAIR could be a potential target of E7, in HPV16 related cervical cancers (CaCx). We identified significant linear trend of progressive HOTAIR down-regulation through HPV negative controls, HPV16 positive non-malignants and CaCx samples. Majority of CaCx cases portrayed HOTAIR down-regulation in comparison to HPV negative controls, with corresponding up-regulation of HOTAIR target, HOXD10, and enrichment of cancer related pathways. However, a small subset had significantly higher HOTAIR expression, concomitant with high E7 expression and enrichment of metastatic pathways. Expression of HOTAIR and PRC2-complex members (EZH2 and SUZ12), showed significant positive correlation with E7 expression in CaCx cases and E7 transfected C33A cell line, suggestive of interplay between E7 and HOTAIR. Functional inactivation of HOTAIR by direct interaction with E7 could also be predicted by in silico analysis and confirmed by RNA-Immunoprecipitation. Our study depicts one of the causal mechanisms of cervical carcinogenesis by HPV16 E7, through modulation of HOTAIR expression and function. PMID:26152361

  14. Akt Pathway Activation by Human T-cell Leukemia Virus Type 1 Tax Oncoprotein.

    PubMed

    Cherian, Mathew A; Baydoun, Hicham H; Al-Saleem, Jacob; Shkriabai, Nikoloz; Kvaratskhelia, Mamuka; Green, Patrick; Ratner, Lee

    2015-10-23

    Human T-cell leukemia virus (HTLV) type 1, the etiological agent of adult T-cell leukemia, expresses the viral oncoprotein Tax1. In contrast, HTLV-2, which expresses Tax2, is non-leukemogenic. One difference between these homologous proteins is the presence of a C-terminal PDZ domain-binding motif (PBM) in Tax1, previously reported to be important for non-canonical NFκB activation. In contrast, this study finds no defect in non-canonical NFκB activity by deletion of the Tax1 PBM. Instead, Tax1 PBM was found to be important for Akt activation. Tax1 attenuates the effects of negative regulators of the PI3K-Akt-mammalian target of rapamycin pathway, phosphatase and tensin homologue (PTEN), and PHLPP. Tax1 competes with PTEN for binding to DLG-1, unlike a PBM deletion mutant of Tax1. Forced membrane expression of PTEN or PHLPP overcame the effects of Tax1, as measured by levels of Akt phosphorylation, and rates of Akt dephosphorylation. The current findings suggest that Akt activation may explain the differences in transforming activity of HTLV-1 and -2.

  15. LMO2 Oncoprotein Stability in T-Cell Leukemia Requires Direct LDB1 Binding

    PubMed Central

    Layer, Justin H.; Alford, Catherine E.; McDonald, W. Hayes

    2015-01-01

    LMO2 is a component of multisubunit DNA-binding transcription factor complexes that regulate gene expression in hematopoietic stem and progenitor cell development. Enforced expression of LMO2 causes leukemia by inducing hematopoietic stem cell-like features in T-cell progenitor cells, but the biochemical mechanisms of LMO2 function have not been fully elucidated. In this study, we systematically dissected the LMO2/LDB1-binding interface to investigate the role of this interaction in T-cell leukemia. Alanine scanning mutagenesis of the LIM interaction domain of LDB1 revealed a discrete motif, R320LITR, required for LMO2 binding. Most strikingly, coexpression of full-length, wild-type LDB1 increased LMO2 steady-state abundance, whereas coexpression of mutant proteins deficient in LMO2 binding compromised LMO2 stability. These mutant LDB1 proteins also exerted dominant negative effects on growth and transcription in diverse leukemic cell lines. Mass spectrometric analysis of LDB1 binding partners in leukemic lines supports the notion that LMO2/LDB1 function in leukemia occurs in the context of multisubunit complexes, which also protect the LMO2 oncoprotein from degradation. Collectively, these data suggest that the assembly of LMO2 into complexes, via direct LDB1 interaction, is a potential molecular target that could be exploited in LMO2-driven leukemias resistant to existing chemotherapy regimens. PMID:26598604

  16. Structural Role of the Conserved Cysteines in the Dimerization of the Viral Transmembrane Oncoprotein E5

    PubMed Central

    Windisch, Dirk; Hoffmann, Silke; Afonin, Sergii; Vollmer, Stefanie; Benamira, Soraya; Langer, Birgid; Bürck, Jochen; Muhle-Goll, Claudia; Ulrich, Anne S.

    2010-01-01

    The E5 oncoprotein is the major transforming protein of bovine papillomavirus type 1. This 44-residue transmembrane protein can interact with the platelet-derived growth factor receptor β, leading to ligand-independent activation and cell transformation. For productive interaction, E5 needs to dimerize via a C-terminal pair of cysteines, though a recent study suggested that its truncated transmembrane segment can dimerize on its own. To analyze the structure of the full protein in a membrane environment and elucidate the role of the Cys-Ser-Cys motif, we produced recombinantly the wild-type protein and four cysteine mutants. Comparison by circular dichroism in detergent micelles and lipid vesicular dispersion and by NMR in trifluoroethanol demonstrates that the absence of one or both cysteines does not influence the highly α-helical secondary structure, nor does it impair the ability of E5 to dimerize, observations that are further supported by sodium dodecylsulfate polyacrylamide gel electrophoresis. We also observed assemblies of higher order. Oriented circular dichroism in lipid bilayers shows that E5 is aligned as a transmembrane helix with a slight tilt angle, and that this membrane alignment is also independent of any cysteines. We conclude that the Cys-containing motif represents a disordered region of the protein that serves as an extra covalent connection for stabilization. PMID:20858420

  17. TRIP-Br1 oncoprotein inhibits autophagy, apoptosis, and necroptosis under nutrient/serum-deprived condition

    PubMed Central

    Duan, Jingjing; Lee, Soonduck; Kim, Kyeri; Park, Yeonji; Yang, Young; Kim, Keun-Il; Lim, Jong-Seok; Cheon, Chung-Il; Kang, Young-Sook; Lee, Myeong-Sok

    2015-01-01

    TRIP-Br1 oncogenic protein has been shown to have multiple biological functions in cells. In this study, we demonstrate that TRIP-Br1 functions as an oncoprotein by inhibiting autophagy, apoptosis, and necroptosis of cancer cells and eventually helping them to survive under the nutrient/serum starved condition. TRIP-Br1 expression level was significantly increased in conditions with low levels of nutrients. Nutrient depleted conditions were induced by culturing cancer cells until they were overcrowded with high cell density or in media deprived of glucose, amino acids, or serum. Among them, serum starvation significantly enhanced the expression of TRIP-Br1 only in all tested breast cancer cell lines (MCF7, MDA-MB-231, T47D, MDA-MB-435, Hs578D, BT549, and MDA-MB-435) but not in the three normal cell lines (MCF10A, HfCH8, and NIH3T3). As compared with the control cells, the introduction of TRIP-Br1 silencing siRNA into MCF7 and MDA-MB-231 cells accelerated cell death by inducing apoptosis and necroptosis. In this process, TRIP-Br1 confers resistance to serum starvation-induced cell deaths by stabilizing the XIAP protein and inhibiting cellular ROS production. Moreover, our data also show that the intracellular increase of TRIP-Br1 protein resulting from serum starvation seems to occur in part through the blockage of PI3K/AKT signaling pathway. PMID:26334958

  18. The HPV E6 oncoprotein targets histone methyltransferases for modulating specific gene transcription

    PubMed Central

    Hsu, C-H; Peng, K-L; Jhang, H-C; Lin, C-H; Wu, S-Y; Chiang, C-M; Lee, S-C; Yu, W C Y; Juan, L-J

    2012-01-01

    Expression of viral proteins causes important epigenetic changes leading to abnormal cell growth. Whether viral proteins directly target histone methyltransferases (HMTs), a key family enzyme for epigenetic regulation, and modulate their enzymatic activities remains elusive. Here we show that the E6 proteins of both low-risk and high-risk human papillomavirus (HPV) interact with three coactivator HMTs, CARM1, PRMT1 and SET7, and downregulate their enzymatic activities in vitro and in HPV-transformed HeLa cells. Furthermore, these three HMTs are required for E6 to attenuate p53 transactivation function. Mechanistically, E6 hampers CARM1- and PRMT1-catalyzed histone methylation at p53-responsive promoters, and suppresses the binding of p53 to chromatinized DNA independently of E6-mediated p53 degradation. p53 pre-methylated at lysine-372 (p53K372 mono-methylation) by SET7 protects p53 from E6-induced degradation. Consistently, E6 downregulates p53K372 mono-methylation and thus reduces p53 protein stability. As a result of the E6-mediated inhibition of HMT activity, expression of p53 downstream genes is suppressed. Together, our results not only reveal a clever approach for the virus to interfere with p53 function, but also demonstrate the modulation of HMT activity as a novel mechanism of epigenetic regulation by a viral oncoprotein. PMID:21963854

  19. The HPV E6 oncoprotein targets histone methyltransferases for modulating specific gene transcription.

    PubMed

    Hsu, C-H; Peng, K-L; Jhang, H-C; Lin, C-H; Wu, S-Y; Chiang, C-M; Lee, S-C; Yu, W C Y; Juan, L-J

    2012-05-03

    Expression of viral proteins causes important epigenetic changes leading to abnormal cell growth. Whether viral proteins directly target histone methyltransferases (HMTs), a key family enzyme for epigenetic regulation, and modulate their enzymatic activities remains elusive. Here we show that the E6 proteins of both low-risk and high-risk human papillomavirus (HPV) interact with three coactivator HMTs, CARM1, PRMT1 and SET7, and downregulate their enzymatic activities in vitro and in HPV-transformed HeLa cells. Furthermore, these three HMTs are required for E6 to attenuate p53 transactivation function. Mechanistically, E6 hampers CARM1- and PRMT1-catalyzed histone methylation at p53-responsive promoters, and suppresses the binding of p53 to chromatinized DNA independently of E6-mediated p53 degradation. p53 pre-methylated at lysine-372 (p53K372 mono-methylation) by SET7 protects p53 from E6-induced degradation. Consistently, E6 downregulates p53K372 mono-methylation and thus reduces p53 protein stability. As a result of the E6-mediated inhibition of HMT activity, expression of p53 downstream genes is suppressed. Together, our results not only reveal a clever approach for the virus to interfere with p53 function, but also demonstrate the modulation of HMT activity as a novel mechanism of epigenetic regulation by a viral oncoprotein.

  20. The oncoprotein gankyrin interacts with RelA and suppresses NF-{kappa}B activity

    SciTech Connect

    Higashitsuji, Hiroaki Higashitsuji, Hisako; Liu, Yu; Masuda, Tomoko; Fujita, Takanori; Abdel-Aziz, H. Ismail; Kongkham, Supranee; Dawson, Simon; John Mayer, R.; Itoh, Yoshito; Sakurai, Toshiharu; Itoh, Katsuhiko; Fujita, Jun

    2007-11-23

    Gankyrin is an oncoprotein commonly overexpressed in hepatocellular carcinomas. It interacts with multiple proteins and accelerates degradation of tumor suppressors Rb and p53. Since gankyrin consists of 7 ankyrin repeats and is structurally similar to I{kappa}Bs, we investigated its interaction with NF-{kappa}B. We found that gankyrin directly binds to RelA. In HeLa and 293 cells, overexpression of gankyrin suppressed the basal as well as TNF{alpha}-induced transcriptional activity of NF-{kappa}B, whereas down-regulation of gankyrin increased it. Gankyrin did not affect the NF-{kappa}B DNA-binding activity or nuclear translocation of RelA induced by TNF{alpha} in these cells. Leptomycin B that inhibits nuclear export of RelA suppressed the NF-{kappa}B activity, which was further suppressed by gankyrin. The inhibitory effect of gankyrin was abrogated by nicotinamide as well as down-regulation of SIRT1, a class III histone deacetylase. Thus, gankyrin binds to NF-{kappa}B and suppresses its activity at the transcription level by modulating acetylation via SIRT1.

  1. Midkine promoter-driven suicide gene expression and -mediated adenovirus replication produced cytotoxic effects to immortalised and tumour cells.

    PubMed

    Yu, L; Hamada, K; Namba, M; Kadomatsu, K; Muramatsu, T; Matsubara, S; Tagawa, M

    2004-07-01

    We examined possible application of a regulatory region of midkine (MK) gene, which is frequently upregulated in a number of human tumours but not in normal cells, to cancer gene therapy. We examined transcriptional activity of the MK genomic fragments in paired cell lines, immortalized cells and their parental normal fibroblasts, and found that the MK fragments activated a fused reporter or a suicide gene preferentially in the immortalized cells. Recombinant adenoviruses (Ad), in which the MK fragment was inserted upstream to the E1A gene (AdMK), replicated preferentially in the immortalized cells and were cytotoxie to them. Human hepatocellular carcinoma cells were significantly susceptible to AdMK compared with human normal fibroblasts in vitro and the replication of AdMK was less than that of wild-type Ad in the infected fibroblasts. Hepatocellular carcinoma cells infected with AdMK did not form tumours in immunocompromised mice and intratumoural injection of AdMK into the hepatocellular carcinoma developed in mice retarded the subsequent tumour growth. Expression of E1A and necrosis of tumours were detected in AdMK-injected but not control Ad-injected cases. The MK promoter-driven suicide gene therapy and -mediated replicative Ad can thereby produce cytotoxic effects to immortalized and tumour cells with minimal damage to normal cells.

  2. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Adenovirus serological reagents. 866.3020 Section 866.3020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3020...

  3. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Adenovirus serological reagents. 866.3020 Section 866.3020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3020...

  4. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Adenovirus serological reagents. 866.3020 Section 866.3020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3020...

  5. 21 CFR 866.3020 - Adenovirus serological reagents.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Adenovirus serological reagents. 866.3020 Section 866.3020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3020...

  6. Mechanics of Viral Chromatin Reveals the Pressurization of Human Adenovirus.

    PubMed

    Ortega-Esteban, Alvaro; Condezo, Gabriela N; Pérez-Berná, Ana J; Chillón, Miguel; Flint, S Jane; Reguera, David; San Martín, Carmen; de Pablo, Pedro J

    2015-11-24

    Tight confinement of naked genomes within some viruses results in high internal pressure that facilitates their translocation into the host. Adenovirus, however, encodes histone-like proteins that associate with its genome resulting in a confined DNA-protein condensate (core). Cleavage of these proteins during maturation decreases core condensation and primes the virion for proper uncoating via unidentified mechanisms. Here we open individual, mature and immature adenovirus cages to directly probe the mechanics of their chromatin-like cores. We find that immature cores are more rigid than the mature ones, unveiling a mechanical signature of their condensation level. Conversely, intact mature particles demonstrate more rigidity than immature or empty ones. DNA-condensing polyamines revert the mechanics of mature capsid and cores to near-immature values. The combination of these experiments reveals the pressurization of adenovirus particles induced by maturation. We estimate a pressure of ∼30 atm by continuous elasticity, which is corroborated by modeling the adenovirus mini-chromosome as a confined compact polymer. We propose this pressurization as a mechanism that facilitates initiating the stepwise disassembly of the mature particle, enabling its escape from the endosome and final genome release at the nuclear pore.

  7. Bioaccumulation of animal adenoviruses in the pink shrimp.

    PubMed

    Luz, Roger B; Staggemeier, Rodrigo; Fabres, Rafael B; Soliman, Mayra C; Souza, Fernanda G; Gonçalves, Raoni; Fausto, Ivone V; Rigotto, Caroline; Heinzelmann, Larissa S; Henzel, Andréia; Fleck, Juliane D; Spilki, Fernando R

    2015-01-01

    Adenoviruses are among the most promising viral markers of fecal contamination. They are frequently found in the water, sediment and soil of regions impacted by human activity. Studies of the bioaccumulation of enteric viruses in shrimp are scarce. The cities located in the northern coast of the lake systems in Southern Brazil have high urbanization and intensive farming rates, and poor sewage collection and treatment. One hundred (n = 100) Farfantepenaeus paulensis pink-shrimp specimens and 48 water samples were collected from coastal lagoons between June 2012 and May 2013. Water samples were concentrated and the shrimp, mashed. After DNA extraction, samples were analyzed by real time polymerase chain reaction (qPCR) in order to detect and quantify viral genomes. Thirty-five percent of shrimp samples were positive for contamination, predominantly by avian adenoviruses. A total of 91.7% of water samples contained adenoviruses DNA, with the human form being the most frequent. Our results provided evidence of significant bioaccumulation of adenoviruses in shrimp, showing the extent of the impact of fecal pollution on aquatic ecosystems.

  8. Serologic and hexon phylogenetic analysis of ruminant adenoviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were to determine the antigenic relationship among ruminant adenoviruses and determine their phylogenetic relationship based on the deduced hexon gene amino acid sequence. Results of reciprocal cross-neutralization tests demonstrated antigenic relationships in either on...

  9. Acute respiratory infection with mouse adenovirus type 1

    PubMed Central

    Weinberg, Jason B.; Stempfle, Gregory S.; Wilkinson, John E.; Younger, John G.; Spindler, Katherine R.

    2005-01-01

    Studies of the pathogenesis of adenovirus respiratory disease are limited by the strict species-specificity of the adenoviruses. Following intranasal inoculation of adult C57BL/6 mice with mouse adenovirus type 1 (MAV-1), we detected MAV-1 early region 3 (E3) and hexon gene expression in the lungs at 7 days post-infection (dpi). We detected MAV-1 E3 protein in the respiratory epithelium 7 dpi. We did not detect viral mRNA or protein at 14 dpi, but MAV-1 DNA was detected by PCR at 21 dpi. Chemokine transcript levels increased between 7 and 14 dpi in the lungs of infected mice. MAV-1 infection induced a patchy cellular infiltrate in lungs at 7 and 14 dpi. This is the first report demonstrating the presence of MAV-1 in the respiratory epithelium of infected mice and describing chemokine responses in the lung induced by MAV-1 respiratory infection. MAV-1 infection of mice has the potential to serve as a model for inflammatory changes seen in human adenovirus respiratory disease. PMID:16054189

  10. Adenovirus infection reverses the antiviral state induced by human interferon.

    PubMed

    Feduchi, E; Carrasco, L

    1987-04-06

    HeLa cells treated with human lymphoblastoid interferon do not synthesize poliovirus proteins. The antiviral state against poliovirus is reversed if cells are previously infected with adenovirus type 5. A late gene product seems to be involved in this reversion, since no effect is observed at early stages of infection or in the presence of aphidicolin.

  11. Bioaccumulation of animal adenoviruses in the pink shrimp

    PubMed Central

    Luz, Roger B.; Staggemeier, Rodrigo; Fabres, Rafael B.; Soliman, Mayra C.; Souza, Fernanda G.; Gonçalves, Raoni; Fausto, Ivone V.; Rigotto, Caroline; Heinzelmann, Larissa S.; Henzel, Andréia; Fleck, Juliane D.; Spilki, Fernando R.

    2015-01-01

    Adenoviruses are among the most promising viral markers of fecal contamination. They are frequently found in the water, sediment and soil of regions impacted by human activity. Studies of the bioaccumulation of enteric viruses in shrimp are scarce. The cities located in the northern coast of the lake systems in Southern Brazil have high urbanization and intensive farming rates, and poor sewage collection and treatment. One hundred (n = 100) Farfantepenaeus paulensis pink-shrimp specimens and 48 water samples were collected from coastal lagoons between June 2012 and May 2013. Water samples were concentrated and the shrimp, mashed. After DNA extraction, samples were analyzed by real time polymerase chain reaction (qPCR) in order to detect and quantify viral genomes. Thirty-five percent of shrimp samples were positive for contamination, predominantly by avian adenoviruses. A total of 91.7% of water samples contained adenoviruses DNA, with the human form being the most frequent. Our results provided evidence of significant bioaccumulation of adenoviruses in shrimp, showing the extent of the impact of fecal pollution on aquatic ecosystems. PMID:26413052

  12. Site-specific nicking within the adenovirus inverted terminal repetition.

    PubMed Central

    Chow, K C; Pearson, G D

    1984-01-01

    Site-specific nicking occurs on the l-strand, but not on the r-strand, of the adenovirus left inverted terminal repeat. Nicks are presumably introduced into double- or single-stranded DNA by a cellular endonuclease in an ATP-independent reaction. The consensus nick site has the sequence: (sequence in text). Images PMID:6322107

  13. Immunoexpression of HPV 16/18 E6 and E7 oncoproteins in high-grade cervical squamous intraepithelial lesions in HIV-positive women.

    PubMed

    Rodrigues, L C; Speck, N M de Gois; Focchi, G R de Azevedo; Schimidt, M A; Marques, R M; Ribalta, J C Lascasas

    2016-02-19

    The aim of this study was to assess the immunoexpression of human papillomavirus genotypes 16 and 18 (E6 and E7) oncoproteins in cervical high-grade squamous intraepithelial lesions (HSIL) of human immunodeficiency virus (HIV)-positive women. These results were also compared to the persistence and/or recurrence of lesions after loop electrosurgical excision procedure. Cervical samples from 158 patients were divided into three groups according to the presence or absence of HSIL in women who were or were not HIV-positive. By using the tissue microarray technique, immunohistochemistry was performed to analyze the expression of HPV 16/18 E6 and E7 oncoproteins. Cervical samples from 95 HIV-positive women and 63 HIV-negative women were studied. A statistically significant difference was found in the immunoexpression of E6 and E7 oncoproteins in samples from HIV-positive women with HSIL and that of women with non-neoplastic tissue (P < 0.001). There was also a statistically significant correlation between the immunoexpression of E6 (P = 0.012) and E7 (P < 0.001) oncoproteins in lesion persistence among HIV-positive women. Within the limitations of this study, the immunoexpression of HPV 16/18 E6 and E7 oncoproteins may have prognostic value regarding lesion persistence in HIV-positive women.

  14. Subcellular localization of the human papillomavirus 16 E7 oncoprotein in CaSki cells and its detection in cervical adenocarcinoma and adenocarcinoma in situ.

    PubMed

    Dreier, Kerstin; Scheiden, René; Lener, Barbara; Ehehalt, Daniela; Pircher, Haymo; Müller-Holzner, Elisabeth; Rostek, Ursula; Kaiser, Andreas; Fiedler, Marc; Ressler, Sigrun; Lechner, Stefan; Widschwendter, Andreas; Even, Jos; Capesius, Catherine; Jansen-Dürr, Pidder; Zwerschke, Werner

    2011-01-05

    E7 is the major oncoprotein of high-risk human papillomaviruses (HPV) which causes cervical cancer. To date E7 oncoproteins have not been investigated in cervical adenocarcinoma. In this study we generated a rabbit monoclonal anti-HPV-16 E7 antibody, RabMab42-3, which recognizes a conformational epitope in the E7 carboxy-terminal zinc-finger resulting in a strong increase in the sensitivity for the detection of cell-associated HPV-16 E7 protein relative to conventional polyclonal anti-HPV-16 E7 antibodies. Using RabMab42-3, we show that the subcellular localization of endogenous HPV-16 E7 oncoprotein varies during the cell cycle in cervical cancer cells. Moreover, we demonstrate for the first time that the HPV-16 E7 oncoprotein is abundantly expressed in cervical adenocarcinoma in situ and adenocarcinoma, suggesting an important role of HPV-16 E7 for the development of these tumors. Our findings suggest that the HPV-16 E7 oncoprotein could be a useful marker for the detection of cervical adenocarcinoma and their precursors.

  15. Transformation Potentials of the Noninfectious (Defective) Component in Pools of Adenoviruses Type 12 and Simian Adenovirus 7

    PubMed Central

    Schaller, J. P.; Yohn, D. S.

    1974-01-01

    Pools of adenovirus 12 and simian adenovirus 7 were separated into four or five fractions by density gradient centrifugation in cesium chloride. Each fraction was analyzed for total in vitro infectivity units, total transformation activity, and for total virus particle (VP) content. Two major subpopulations were separated with mean densities of 1.30 ± 0.02 and 1.34 ± 0.02 g/ml, respectively. Virions in the 1.34 g/ml range were highly infectious (102 to 103 VP per infectivity unit) in contrast to virions at 1.30 g/ml density (104 to 105 VP per infectivity units). Transformation capacity was evenly distributed throughout fractions of both viruses, indicating that genetically incomplete or defective virus particles were not deficient in their ability to induce transformation. The average VP per transformation unit for adenovirus 12 (2.85 × 106) and for simian adenovirus 7 (4.00 × 106) did not vary significantly from fraction to fraction. These values were obtained with optimal input multiplicities of 16 to 64 VP per cell. At higher multiplicities the apparent increase in VP per transformation unit was attributable to the viral cytocidal effect on hamster cells. These studies revealed that quantitation of in vitro transformation based on VP multiplicities was more reliable than on the basis of infectious units. These estimates were independent of method of virus production, extraction, and purification. Images PMID:4211167

  16. Electrostatic environment surrounding the activation loop phosphotyrosine in the oncoprotein v-Fps.

    PubMed

    Leon, B C; Tsigelny, I; Adams, J A

    2001-08-28

    Autophosphorylation of Tyr-1073 in the activation loop of the oncoprotein v-Fps enhances the phosphoryl transfer reaction without influencing substrate, ATP, or metal ion binding affinities [Saylor, P., et al. (1998) Biochemistry 37, 17875-17881]. A structural model of v-Fps, generated from the insulin receptor, indicates that pTyr-1073 chelates two arginines. Mutation of these residues to alanine (R1042A and R1066A) results in weakly phosphorylated enzymes, indicating that one electropositive center is insufficient for attaining maximum loop phosphorylation and concomitant high catalytic activity. While the turnover rate for R1066A is similar to that for a mutant lacking a phosphorylatable residue in the activation loop, the rate for R1042A is 50-fold slower. While solvent perturbation studies suggest that the former is due to a slow phosphoryl transfer step, the latter effect results from a slow conformational change in the mutant, potentially linked to motions in the catalytic loop. Binding of a stoichiometric quantity of Mg(2+) is essential for ATP binding and catalysis, while binding of an additional Mg(2+) ion activates further the wild-type enzyme. The affinity of the R1066A enzyme for the second Mg(2+) ion is 23-fold higher than that of the phosphorylated or unphosphorylated form of wild-type v-Fps, with substrate binding unaffected. Conversely, the affinity of R1066A for a substrate mimic lacking a phosphorylation site is 12-fold higher than that for the phosphorylated or unphosphorylated form of wild-type v-Fps, with binding of the second Mg(2+) ion unaffected. A comparison of these enzyme-independent parameters indicates that Arg-1042 and Arg-1066 induce strain in the active site in the repressed form of the enzyme. While this strain is not relieved in the phosphorylated form, the improvements in catalysis in activated v-Fps compensate for reduced metal and substrate binding affinities.

  17. Modularity of the oncoprotein-like properties of platelet glycoprotein Ibalpha.

    PubMed

    Li, Youjun; Lu, Jie; Prochownik, Edward V

    2009-01-16

    Glycoprotein Ib alpha (GpIbalpha), a trans-membrane glycoprotein, is expressed on the surface of megakaryocytes and platelets, where, in association with glycoprotein Ib beta, glycoprotein V, and glycoprotein IX, it normally forms the von Willebrand factor receptor (vWFR). A fully functional vWFR is necessary for platelet attachment, aggregation, and activation and has also been shown to regulate megakaryocyte ploidy. We have recently shown that the gene encoding GpIbalpha is a transcriptional target for the c-Myc oncoprotein and is more widely expressed than previously thought, with particularly high levels occurring in transformed cells. Indeed, GpIbalpha can substitute for c-Myc in promoting growth, transformation, and genomic instability. In the current work, we have demonstrated that, despite the promiscuous expression of GpIbalpha, other vWFR subunits remain largely restricted to megakaryocytes. We have characterized a panel of GpIbalpha mutants and shown that some regions of the protein essential for vWFR activity are not necessary for c-Myc-like functions. Specifically, the six C-terminal amino acids of the cytoplasmic domain, which mediate vWFR signaling, are entirely dispensible for the c-Myc-like functions of GpIbalpha. Instead, these require a more membrane-proximal filamin-binding domain. Also important is the GpIbalpha signal peptide, which, in the absence of other vWFR subunits, directs GpIbalpha to the endoplasmic reticulum rather than the membrane. Together, these results provide strong evidence that the domains of GpIbalpha mediating c-Myc-like functions are modular, genetically distinct, and independent of those involved in vWFR signaling.

  18. Binding and Internalization of Iron Oxide Nanoparticles Targeted to Nuclear Oncoprotein

    PubMed Central

    Knight, Linda C.; Romano, Jan E.; Krynska, Barbara; Faro, Scott; Mohamed, Feroze B.; Gordon, Jennifer

    2013-01-01

    A targeted nanoconjugate is being developed for non-invasive detection of gene expression in cells expressing the JC virus oncoprotein, T-antigen, which has been associated with medulloblastoma and other cancers. JC virus T-antigen localizes predominantly to the nucleus via a classical monopartite nuclear localization signal (NLS). An antibody fragment which recognizes JC virus T-antigen was attached to cross-linked dextran coated iron oxide nanoparticles. Radiolabeled conjugates were added to mouse medulloblastoma cells expressing the target T-antigen to test their ability to bind to tumor cells and be internalized by the cells. All conjugates containing targeting antibody bound to cells and were internalized, with increasing levels over time. There was no difference in cell binding or internalization among conjugates containing 2, 4, 6 or 8 antibody fragments per nanoparticle. Conjugates with only nonspecific antibody on nanoparticles, or unconjugated nonspecific antibody, had significantly lower total binding and internalization than conjugates with targeting antibody. Unconjugated targeting antibody had equivalent or lower cell uptake compared with targeted nanoparticle conjugates. Specificity of uptake was demonstrated by >80% reduction of nanoconjugate uptake in the presence of 100 fold excess of unconjugated antibody. The presence of a membrane translocation peptide (Tat) on the nanoparticles in addition to targeting antibody did not improve nanoconjugate internalization over the internalization caused by the antibody alone. This antibody nanoconjugate demonstrates feasibility of targeting a nuclear protein and suggests that a minimum number of antibody fragments per nanoparticle are sufficient for achieving binding specificity and efficient uptake into living cells. PMID:23487404

  19. Lurbinectedin Inactivates the Ewing Sarcoma Oncoprotein EWS-FLI1 by Redistributing It within the Nucleus.

    PubMed

    Harlow, Matt L; Maloney, Nichole; Roland, Joseph; Guillen Navarro, Maria Jose; Easton, Matthew K; Kitchen-Goosen, Susan M; Boguslawski, Elissa A; Madaj, Zachary B; Johnson, Ben K; Bowman, Megan J; D'Incalci, Maurizio; Winn, Mary E; Turner, Lisa; Hostetter, Galen; Galmarini, Carlos María; Aviles, Pablo M; Grohar, Patrick J

    2016-11-15

    There is a great need to develop novel approaches to target oncogenic transcription factors with small molecules. Ewing sarcoma is emblematic of this need, as it depends on the continued activity of the EWS-FLI1 transcription factor to maintain the malignant phenotype. We have previously shown that the small molecule trabectedin interferes with EWS-FLI1. Here, we report important mechanistic advances and a second-generation inhibitor to provide insight into the therapeutic targeting of EWS-FLI1. We discovered that trabectedin functionally inactivated EWS-FLI1 by redistributing the protein within the nucleus to the nucleolus. This effect was rooted in the wild-type functions of the EWSR1, compromising the N-terminal half of the chimeric oncoprotein, which is known to be similarly redistributed within the nucleus in the presence of UV light damage. A second-generation trabectedin analogue lurbinectedin (PM01183) caused the same nuclear redistribution of EWS-FLI1, leading to a loss of activity at the promoter, mRNA, and protein levels of expression. Tumor xenograft studies confirmed this effect, and it was increased in combination with irinotecan, leading to tumor regression and replacement of Ewing sarcoma cells with benign fat cells. The net result of combined lurbinectedin and irinotecan treatment was a complete reversal of EWS-FLI1 activity and elimination of established tumors in 30% to 70% of mice after only 11 days of therapy. Our results illustrate the preclinical safety and efficacy of a disease-specific therapy targeting the central oncogenic driver in Ewing sarcoma. Cancer Res; 76(22); 6657-68. ©2016 AACR.

  20. Multistage modeling of protein dynamics with monomeric Myc oncoprotein as an example

    NASA Astrophysics Data System (ADS)

    Liu, Jiaojiao; Dai, Jin; He, Jianfeng; Niemi, Antti J.; Ilieva, Nevena

    2017-03-01

    We propose to combine a mean-field approach with all-atom molecular dynamics (MD) into a multistage algorithm that can model protein folding and dynamics over very long time periods yet with atomic-level precision. As an example, we investigate an isolated monomeric Myc oncoprotein that has been implicated in carcinomas including those in colon, breast, and lungs. Under physiological conditions a monomeric Myc is presumed to be an example of intrinsically disordered proteins that pose a serious challenge to existing modeling techniques. We argue that a room-temperature monomeric Myc is in a dynamical state, it oscillates between different conformations that we identify. For this we adopt the C α backbone of Myc in a crystallographic heteromer as an initial ansatz for the monomeric structure. We construct a multisoliton of the pertinent Landau free energy to describe the C α profile with ultrahigh precision. We use Glauber dynamics to resolve how the multisoliton responds to repeated increases and decreases in ambient temperature. We confirm that the initial structure is unstable in isolation. We reveal a highly degenerate ground-state landscape, an attractive set towards which Glauber dynamics converges in the limit of vanishing ambient temperature. We analyze the thermal stability of this Glauber attractor using room-temperature molecular dynamics. We identify and scrutinize a particularly stable subset in which the two helical segments of the original multisoliton align in parallel next to each other. During the MD time evolution of a representative structure from this subset, we observe intermittent quasiparticle oscillations along the C-terminal α helix, some of which resemble a translating Davydov's Amide-I soliton. We propose that the presence of oscillatory motion is in line with the expected intrinsically disordered character of Myc.

  1. Protease activity of the API2-MALT1 fusion oncoprotein in MALT lymphoma development and treatment.

    PubMed

    Rosebeck, Shaun; Lucas, Peter C; McAllister-Lucas, Linda M

    2011-05-01

    Gastric mucosa-associated lymphoid tissue (MALT) lymphoma is a prototypical cancer that occurs in the setting of chronic inflammation and an important model for understanding how deregulated NF-κB transcriptional activity contributes to malignancy. Most gastric MALT lymphomas require ongoing antigenic stimulation for continued tumor growth, and Stage I disease is usually cured by eradicating the causative microorganism, Helicobacter pylori, with antibiotics. However, in a subset of MALT lymphomas, chromosomal translocations are acquired that render the lymphoma antigen-independent. The recurrent translocation t(11;18)(q21;q21) is associated with failure to respond to antibiotic therapy and increased rate of dissemination. This translocation creates the API2-MALT1 fusion oncoprotein, which comprises the amino terminus of inhibitor of apoptosis 2 (API2 or cIAP2) fused to the carboxy terminus of MALT1. A common characteristic of chromosomal translocations in MALT lymphoma, including t(11;18), is that genes involved in the regulation of the NF-κB transcription factor are targeted by the translocations, and these genetic perturbations thereby result in deregulated, constitutive NF-κB stimulation. In the last decade, great insights into the roles of API2 and MALT1 in NF-κB signaling have been made. For example, recent pivotal studies have uncovered the long sought-after proteolytic activity of MALT1 and have demonstrated its critical involvement in the survival of certain lymphomas. Here, we review the current understanding of the role of MALT1 in normal lymphocyte function and lymphomagenesis. We then highlight our recent work that has revealed an intriguing link between the proteolytic activity of the API2-MALT1 fusion and its ability to influence lymphomagenesis by cleaving a key NF-κB regulatory protein, NF-κB-inducing kinase.

  2. Nucleocytoplasmic shuttling of oncoprotein Hdm2 is required for Hdm2-mediated degradation of p53

    PubMed Central

    Tao, Weikang; Levine, Arnold J.

    1999-01-01

    The Hdm2 oncoprotein inhibits p53 functions by two means: (i) it blocks p53’s transactivation activity and (ii) it targets p53 for degradation in a proteasome-dependent manner. Recent data indicate that Hdm2 shuttles between the nucleus and the cytoplasm and that the regulation of p53 levels by Hdm2 requires its nuclear export activity. Two different models are consistent with these observations. In the first, Hdm2 binds to p53 in the nucleus and shuttles p53 from the nucleus to the cytoplasm, and then it targets p53 to the cytoplasmic proteasome. Alternatively, Hdm2 and p53 could be exported separately from the nucleus and then associate in the cytoplasm, where Hdm2 promotes the degradation of p53. To distinguish between these two models, several Hdm2 mutants were employed. Hdm2NLS lacks the ability to enter the nucleus, whereas Hdm2NES is deficient in nuclear export. Hdm2NLS, Hdm2NES, or the combination of both mutants were unable to promote p53 degradation in the cotransfected 2KO cells (which were null for both the p53 and mdm2 genes), although wild-type Hdm2 efficiently reduced p53 levels under the same conditions. This observation is not a result of the differences in expression levels or stability between Hdm2 and these mutants. Moreover, coexpression of these mutants had no effect on wild-type Hdm-2-induced p53 destabilization. Thus, Hdm2 must shuttle p53 from the nucleus to the cytoplasm to target it for degradation in the cytoplasm. PMID:10077639

  3. miR-24 and miR-205 expression is dependent on HPV onco-protein expression in keratinocytes

    SciTech Connect

    McKenna, Declan J.; Patel, Daksha; McCance, Dennis J.

    2014-01-05

    A screen of microRNA (miRNA) expression following differentiation in human foreskin keratinocytes (HFKs) identified changes in several miRNAs, including miR-24 and miR-205. We investigated how expression of Human Papilloma Virus Type-16 (HPV16) onco-proteins E6 and E7 affected expression of miR-24 and miR-205 during proliferation and differentiation of HFKs. We show that the induction of both miR-24 and miR-205 observed during differentiation of HFKs is lost in HFKs expressing E6 and E7. We demonstrate that the effect on miR-205 is due to E7 activity, as miR-205 expression is dependent on pRb expression. Finally, we provide evidence that miR-24 effects in the cell may be due to targeting of cyclin dependent kinase inhibitor p27. In summary, these results indicate that expression of both miR-24 and miR-205 are impacted by E6 and/or E7 expression, which may be one mechanism by which HPV onco-proteins can disrupt the balance between proliferation and differentiation in keratinocytes. - Highlights: • miR-24 and miR-205 are induced during keratinocyte differentiation. • This induction is lost in keratinocytes expressing HPV onco-proteins E6 and E7. • miR-205 is dependent upon pRb expression. • miR-24 targets p27 in cycling keratinocytes.

  4. HPV16 Oncoproteins Induce MMPs/RECK-TIMP-2 Imbalance in Primary Keratinocytes: Possible Implications in Cervical Carcinogenesis

    PubMed Central

    Cardeal, Laura Beatriz da Silva; Boccardo, Enrique; Termini, Lara; Rabachini, Tatiana; Andreoli, Maria Antonieta; di Loreto, Celso; Filho, Adhemar Longatto; Villa, Luisa Lina; Maria-Engler, Silvya Stuchi

    2012-01-01

    Cervical cancer is the third most common cancer in women worldwide. Persistent infection with high-risk HPV types, principally HPV16 and 18 is the main risk factor for the development of this malignancy. However, the onset of invasive tumor occurs many years after initial exposure in a minority of infected women. This suggests that other factors beyond viral infection are necessary for tumor establishment and progression. Tumor progression is characterized by an increase in secretion and activation of matrix metalloproteinases (MMPs) produced by either the tumor cells themselves or tumor-associated fibroblasts or macrophages. Increased MMPs expression, including MMP-2, MMP-9 and MT1-MMP, has been observed during cervical carcinoma progression. These proteins have been associated with degradation of ECM components, tumor invasion, metastasis and recurrence. However, few studies have evaluated the interplay between HPV infection and the expression and activity of MMPs and their regulators in cervical cancer. We analyzed the effect of HPV16 oncoproteins on the expression and activity of MMP-2, MMP-9, MT1-MMP, and their inhibitors TIMP-2 and RECK in cultures of human keratinocytes. We observed that E7 expression is associated with increased pro-MMP-9 activity in the epithelial component of organotypic cultures, while E6 and E7 oncoproteins co-expression down-regulates RECK and TIMP-2 levels in organotypic and monolayers cultures. Finally, a study conducted in human cervical tissues showed a decrease in RECK expression levels in precancer and cancer lesions. Our results indicate that HPV oncoproteins promote MMPs/RECK-TIMP-2 imbalance which may be involved in HPV-associated lesions outcome. PMID:22438955

  5. Nuclear export of cutaneous HPV8 E7 oncoprotein is mediated by a leucine-rich nuclear export signal via a CRM1 pathway.

    PubMed

    Onder, Zeynep; Chang, Vivian; Moroianu, Junona

    2015-01-01

    We recently determined that the nuclear import of cutaneous beta genus HPV8 E7 oncoprotein it is mediated by its zinc-binding domain via direct hydrophobic interactions with the FG nucleoporins Nup62 and Nup153 (Onder and Moroianu, 2014). Here we investigated the nuclear export of HPV8 E7 oncoprotein using confocal microscopy after transfections of HeLa cells with EGFP-8cE7 and mutant plasmids and treatment with Ratjadone A nuclear export inhibitor. We determined that HPV8 E7 contains a leucine-rich nuclear export signal (NES), 76IRTFQELLF84, within its zinc-binding domain that mediates its nuclear export via a CRM1 pathway. We found that HPV8 E7 interacts with CRM1 and that the hydrophobic amino acid residues I76, F79 and L82 of the NES are essential for this interaction and for nuclear export of HPV8 E7 oncoprotein.

  6. Naive and radiolabeled antibodies to E6 and E7 HPV-16 oncoproteins show pronounced antitumor activity in experimental cervical cancer

    PubMed Central

    Phaëton, R; Gutierrez, J; Jiang, Z; Karabakhtsian, RG; Albanese, J; Sunkara, J; Fisher, DR; Goldberg, GL; Dadachova, E

    2015-01-01

    Background In spite of profound reduction in incidence, cervical cancer claims >275,000 lives annually. Previously we demonstrated efficacy and safety of radioimmunotherapy directed at HPV16 E6 oncoprotein in experimental cervical cancer. Materials & methods We undertook a direct comparison of targeting E7 and E6 oncoproteins with specific 188Rhenium-labeled monoclonal antibodies in CasKi subcutaneous xenografts of cervical cancer cells in mice. Results The most significant tumor inhibition was seen in radioimmunotherapy-treated mice, followed by the unlabeled monoclonal antibodies to E6 and E7. No hematological toxicity was observed. Immunohistochemistry suggests that the effect of unlabeled antibodies is C3 complement mediated. Conclusion We have demonstrated for the first time that radioimmunotherapy directed toward E7 oncoprotein inhibits experimental tumors growth, decreases E7 expression and may offer a novel approach to cervical cancer therapy. PMID:26098137

  7. Routine monitoring of adenovirus and norovirus within the health care environment.

    PubMed

    Pankhurst, Louise; Cloutman-Green, Elaine; Canales, Melisa; D'Arcy, Nikki; Hartley, John C

    2014-11-01

    This study investigated the presence of adenovirus and norovirus on ward surfaces using real-time polymerase chain reaction (PCR) to assist in the development of evidence-based infection control policy. Screening was carried out weekly for 6 months in the common areas of 2 pediatric wards. Additionally, a one-off screening was undertaken for adenovirus and norovirus on a day unit and for adenovirus only in patient cubicles while occupied. Over the 6-month screening of common areas, 2.4% of samples were positive for adenovirus or norovirus. In rooms occupied with adenovirus-infected children, all cubicle screening sites and almost all swabs were contaminated with adenovirus. In the day unit, 13% of samples were positive. Cleaning and environmental interaction strategies must therefore be designed to control nosocomial transmission of viruses outside of outbreak scenarios.

  8. Effect of adenovirus infection on expression of human histone genes.

    PubMed Central

    Flint, S J; Plumb, M A; Yang, U C; Stein, G S; Stein, J L

    1984-01-01

    The influence of adenovirus type 2 infection of HeLa cells upon expression of human histone genes was examined as a function of the period of infection. Histone RNA synthesis was assayed after run-off transcription in nuclei isolated from mock-infected cells and after various periods of adenovirus infection. Histone protein synthesis was measured by [3H]leucine labeling of intact cells and fluorography of electrophoretically fractionated nuclear and cytoplasmic proteins. The cellular representation of RNA species complementary to more than 13 different human histone genes was determined by RNA blot analysis of total cellular, nuclear or cytoplasmic RNA by using a series of 32P-labeled cloned human histone genes as hybridization probes and also by analysis of 3H-labeled histone mRNA species synthesized in intact cells. By 18 h after infection, HeLa cell DNA synthesis and all parameters of histone gene expression, including transcription and the nuclear and cytoplasmic concentrations of core and H1 mRNA species, were reduced to less than 5 to 10% of the control values. By contrast, transcription and processing of other cellular mRNA sequences have been shown to continue throughout this period of infection. The early period of adenovirus infection was marked by an inhibition of transcription of histone genes that accompanied the reduction in rate of HeLa cell DNA synthesis. These results suggest that the adenovirus-induced inhibition of histone gene expression is mediated in part at the transcriptional level. However, the persistence of histone mRNA species at concentrations comparable to those of mock-infected control cells during the early phase of the infection, despite a reduction in histone gene transcription and histone protein synthesis, implies that histone gene expression is also regulated post-transcriptionally in adenovirus-infected cells. These results suggest that the tight coupling between histone mRNA concentrations and the rate of cellular DNA

  9. Province-wide adenovirus type 3 outbreak with severe cases in New Brunswick

    PubMed Central

    Girouard, Gabriel; Garceau, Richard; Thibault, Louise; Bourque, Christine; Bastien, Nathalie; Li, Yan

    2011-01-01

    Adenovirus is a commonly isolated virus in clinical samples. Life-threatening infections, although rare, are described worldwide. An epidemic spread of an adenovirus type 3 strain occurred in the province of New Brunswick during the fall of 2008 to the winter of 2009; it resulted in three severely ill patients, with one fatality. Adenovirus should be considered as a cause of severe community-acquired viral pneumonia, especially when the influenza test is negative. PMID:22379488

  10. Large Epidemic of Respiratory Illness Due to Adenovirus Types 7 and 3 in Healthy Young Adults

    DTIC Science & Technology

    2008-02-15

    Epidemic of Respiratory fliness Due to Adenovirus Types 7 and 3 in Healthy Young Adults Margaret A. K. Ryan, Gregory C. Gray," Besa Smith, Jamie A...immunization, respiratory infections due to adenoviruses have reemerged to threaten the health of young adults in the military. Shortly after the loss...challenges for young adults in the military in the postvaccine era. The US military has long had concern about the impact adenovirus serotypes 4 and 7

  11. Dramatic Decline of Respiratory Illness Among US Military Recruits After the Renewed Use of Adenovirus Vaccines

    DTIC Science & Technology

    2014-10-01

    Naval Health Research Center Dramatic Decline of Respiratory Illness Among US Military Recruits After the Renewed Use of Adenovirus Vaccines ...Renewed Use of Adenovirus Vaccines Jennifer M. Radin,1,2 Anthony W. Hawksworth,1 Patrick J. Blair,1 Dennis J. Faix,3 Rema Raman,4 Kevin L. Russell,5...hiatus, oral vaccines against adenovirus types 4 (Ad4) and 7 (Ad7) were again produced and administered to US military recruits. This study examined the

  12. Adenovirus urethritis and concurrent conjunctivitis: a case series and review of the literature.

    PubMed

    Liddle, Olivia Louise; Samuel, Mannampallil Itty; Sudhanva, Malur; Ellis, Joanna; Taylor, Chris

    2015-03-01

    We present eight cases and review the literature of concurrent urethritis and conjunctivitis where adenovirus was identified as the causative pathogen. The focus of this review concerns the identification of specific sexual practices, symptoms, signs and any serotypes that seem more commonly associated with such adenovirus infections. We discuss the seasonality of adenovirus infection and provide practical advice for clinicians to give to the patient.

  13. Overcoming pre-existing adenovirus immunity by genetic engineering of adenovirus-based vectors.

    PubMed

    Seregin, Sergey S; Amalfitano, Andrea

    2009-12-01

    Adenovirus (Ad)-based vectors offer several benefits showing their potential for use in a variety of vaccine applications. Recombinant Ad-based vaccines possess potent immunogenic potential, capable of generating humoral and cellular immune responses to a variety of pathogen-specific antigens expressed by the vectors. Ad5 vectors can be readily produced, allowing for usage in thousands of clinical trial subjects. This is now coupled with a history of safe clinical use in the vaccine setting. However, traditional Ad5-based vaccines may not be generating optimal antigen-specific immune responses, and generate diminished antigen-specific immune responses when pre-existing Ad5 immunity is present. These limitations have driven initiation of several approaches to improve the efficacy of Ad-based vaccines, and/or allow modified vaccines to overcome pre-existing Ad immunity. These include: generation of chemically modified Ad5 capsids; generation of chimeric Ads; complete replacement of Ad5-based vaccine platforms with alternative (human and non-human origin) Ad serotypes, and Ad5 genome modification approaches that attempt to retain the native Ad5 capsid, while simultaneously improving the efficacy of the platform as well as minimizing the effect of pre-existing Ad immunity. Here we discuss recent advances in- and limitations of each of these approaches, relative to their abilities to overcome pre-existing Ad immunity.

  14. Combinatorial treatment with oncolytic adenovirus and helper-dependent adenovirus augments adenoviral cancer gene therapy

    PubMed Central

    Farzad, Lisa; Cerullo, Vincenzo; Yagyu, Shigeki; Bertin, Terry; Hemminki, Akseli; Rooney, Cliona; Lee, Brendan; Suzuki, Masataka

    2014-01-01

    Oncolytic adenoviruses (Onc.Ads) produce significant antitumor effects but as single agents they rarely eliminate tumors. Investigators have therefore incorporated sequences into these vectors that encode immunomodulatory molecules to enhance antitumor immunity. Successful implementation of this strategy requires multiple tumor immune inhibitory mechanisms to be overcome, and insertion of the corresponding multiple functional genes reduces the titer and replication of Onc.Ads, compromising their direct ant-tumor effects. By contrast, helper-dependent (HD) Ads are devoid of viral coding sequences, allowing inclusion of multiple transgenes. HDAds, however, lack replicative capacity. Since HDAds encode the adenoviral packaging signal, we hypothesized that the coadministration of Onc.Ad with HDAd would allow to be amplified and packaged during replication of Onc.Ad in transduced cancer cells. This combination could provide immunostimulation without losing oncolytic activity. We now show that coinfection of Onc.Ad with HDAd subsequently replicates HDAd vector DNA in trans in human cancer cell lines in vitro and in vivo, amplifying the transgenes the HDAd encode. This combinatorial treatment significantly suppresses the tumor growth compared to treatment with a single agent in an immunocompetent mouse model. Hence, combinatorial treatment of Onc.Ad with HDAd should overcome the inherent limitations of each agent and provide a highly immunogenic oncolytic therapy. PMID:27119096

  15. The E1B 19,000-molecular-weight protein of group C adenoviruses prevents tumor necrosis factor cytolysis of human cells but not of mouse cells.

    PubMed Central

    Gooding, L R; Aquino, L; Duerksen-Hughes, P J; Day, D; Horton, T M; Yei, S P; Wold, W S

    1991-01-01

    Tumor necrosis factor (TNF) is a multifunctional immunoregulatory protein that is secreted by activated macrophages and is believed to have antiviral activities. We reported earlier that when mouse C3HA fibroblasts are infected with human adenoviruses, the 289R and 243R proteins encoded by region E1A render the cells susceptible to lysis by TNF, and a 14,700-molecular-weight protein (14.7K protein) encoded by region E3 protects the cells against lysis by TNF. We now report that the 19,000-molecular-weight (19K) (176R) protein encoded by the E1B transcription unit can protect human HEL-299 fibroblasts and human ME-180 cervical carcinoma cells against lysis by TNF. This was determined by infecting cells with adenovirus double mutants that lack region E3 and do or do not express the E1B-19K protein and by measuring cytolysis by using a short-term (18-h) 51Cr-release assay. Under these assay conditions, the 51Cr release was specific to TNF and was not a consequence of the cyt phenotype associated with E1B-19K protein-negative mutants. Also, by using virus double mutants that lack E3 in combination with other early regions, we found that E1A, the E1B-55K protein-encoding gene, E3, and E4 are not required to protect HEL-299 cells against TNF cytolysis. Three additional human cancer cell lines (HeLa, HCT8, and RC29) and a simian virus 40-transformed WI38 cell line (VA-13) also required E1B for protection against TNF cytolysis, indicating that the E1B-19K protein is required to protect many if not all human cell types against lysis by TNF when infected by adenovirus. The E1B-19K protein was not able to protect six different adenovirus-infected mouse cell lines against TNF lysis, even though the protein was shown to be efficiently expressed in one of the cell lines. HEL-299 or ME-180 cells infected by a mutant that lacks the E1B-19K protein but retains region E3 were not lysed by TNF, indicating that one or more of the E3 proteins can protect these cells against TNF lysis

  16. Phylogenetic analyses of novel squamate adenovirus sequences in wild-caught Anolis lizards.

    PubMed

    Ascher, Jill M; Geneva, Anthony J; Ng, Julienne; Wyatt, Jeffrey D; Glor, Richard E

    2013-01-01

    Adenovirus infection has emerged as a serious threat to the health of captive snakes and lizards (i.e., squamates), but we know relatively little about this virus' range of possible hosts, pathogenicity, modes of transmission, and sources from nature. We report the first case of adenovirus infection in the Iguanidae, a diverse family of lizards that is widely-studied and popular in captivity. We report adenovirus infections from two closely-related species of Anolis lizards (anoles) that were recently imported from wild populations in the Dominican Republic to a laboratory colony in the United States. We investigate the evolution of adenoviruses in anoles and other squamates using phylogenetic analyses of adenovirus polymerase gene sequences sampled from Anolis and a range of other vertebrate taxa. These phylogenetic analyses reveal that (1) the sequences detected from each species of Anolis are novel, and (2) adenoviruses are not necessarily host-specific and do not always follow a co-speciation model under which host and virus phylogenies are perfectly concordant. Together with the fact that the Anolis adenovirus sequences reported in our study were detected in animals that became ill and subsequently died shortly after importation while exhibiting clinical signs consistent with acute adenovirus infection, our discoveries suggest the need for renewed attention to biosecurity measures intended to prevent the spread of adenovirus both within and among species of snakes and lizards housed in captivity.

  17. Addition of Polyadenylate Sequences to Virus-Specific RNA during Adenovirus Replication

    PubMed Central

    Philipson, L.; Wall, R.; Glickman, G.; Darnell, J. E.

    1971-01-01

    Adenovirus-specific nuclear and polysomal RNA, both early and late in the infectious cycle, contain a covalently linked region of polyadenylic acid 150-250 nucleotides long. A large proportion of the adenovirus-specific messenger RNA contains poly(A). As revealed by hybridization experiments, the poly(A) is not transcribed from adenovirus DNA. Furthermore, an adenosine analogue, cordycepin, blocks the synthesis of poly(A) and also inhibits the accumulation of adenovirus messenger RNA on polysomes. Addition of poly(A) to viral RNA may involve a host-controlled mechanism that regulates the processing and transport of messenger RNA. PMID:5315962

  18. Addition of polyadenylate sequences to virus-specific RNA during adenovirus replication.

    PubMed

    Philipson, L; Wall, R; Glickman, G; Darnell, J E

    1971-11-01

    Adenovirus-specific nuclear and polysomal RNA, both early and late in the infectious cycle, contain a covalently linked region of polyadenylic acid 150-250 nucleotides long. A large proportion of the adenovirus-specific messenger RNA contains poly(A). As revealed by hybridization experiments, the poly(A) is not transcribed from adenovirus DNA. Furthermore, an adenosine analogue, cordycepin, blocks the synthesis of poly(A) and also inhibits the accumulation of adenovirus messenger RNA on polysomes. Addition of poly(A) to viral RNA may involve a host-controlled mechanism that regulates the processing and transport of messenger RNA.

  19. Antiviral antibodies target adenovirus to phagolysosomes and amplify the innate immune response.

    PubMed

    Zaiss, Anne K; Vilaysane, Akosua; Cotter, Matthew J; Clark, Sharon A; Meijndert, H Christopher; Colarusso, Pina; Yates, Robin M; Petrilli, Virginie; Tschopp, Jurg; Muruve, Daniel A

    2009-06-01

    Adenovirus is a nonenveloped dsDNA virus that activates intracellular innate immune pathways. In vivo, adenovirus-immunized mice displayed an enhanced innate immune response and diminished virus-mediated gene delivery following challenge with the adenovirus vector AdLacZ suggesting that antiviral Abs modulate viral interactions with innate immune cells. Under naive serum conditions in vitro, adenovirus binding and internalization in macrophages and the subsequent activation of innate immune mechanisms were inefficient. In contrast to the neutralizing effect observed in nonhematopoietic cells, adenovirus infection in the presence of antiviral Abs significantly increased FcR-dependent viral internalization in macrophages. In direct correlation with the increased viral internalization, antiviral Abs amplified the innate immune response to adenovirus as determined by the expression of NF-kappaB-dependent genes, type I IFNs, and caspase-dependent IL-1beta maturation. Immune serum amplified TLR9-independent type I IFN expression and enhanced NLRP3-dependent IL-1beta maturation in response to adenovirus, confirming that antiviral Abs specifically amplify intracellular innate pathways. In the presence of Abs, confocal microscopy demonstrated increased targeting of adenovirus to LAMP1-positive phagolysosomes in macrophages but not epithelial cells. These data show that antiviral Abs subvert natural viral tropism and target the adenovirus to phagolysosomes and the intracellular innate immune system in macrophages. Furthermore, these results illustrate a cross-talk where the adaptive immune system positively regulates the innate immune system and the antiviral state.

  20. Phylogenetic Analyses of Novel Squamate Adenovirus Sequences in Wild-Caught Anolis Lizards

    PubMed Central

    Ascher, Jill M.; Geneva, Anthony J.; Ng, Julienne; Wyatt, Jeffrey D.; Glor, Richard E.

    2013-01-01

    Adenovirus infection has emerged as a serious threat to the health of captive snakes and lizards (i.e., squamates), but we know relatively little about this virus' range of possible hosts, pathogenicity, modes of transmission, and sources from nature. We report the first case of adenovirus infection in the Iguanidae, a diverse family of lizards that is widely-studied and popular in captivity. We report adenovirus infections from two closely-related species of Anolis lizards (anoles) that were recently imported from wild populations in the Dominican Republic to a laboratory colony in the United States. We investigate the evolution of adenoviruses in anoles and other squamates using phylogenetic analyses of adenovirus polymerase gene sequences sampled from Anolis and a range of other vertebrate taxa. These phylogenetic analyses reveal that (1) the sequences detected from each species of Anolis are novel, and (2) adenoviruses are not necessarily host-specific and do not always follow a co-speciation model under which host and virus phylogenies are perfectly concordant. Together with the fact that the Anolis adenovirus sequences reported in our study were detected in animals that became ill and subsequently died shortly after importation while exhibiting clinical signs consistent with acute adenovirus infection, our discoveries suggest the need for renewed attention to biosecurity measures intended to prevent the spread of adenovirus both within and among species of snakes and lizards housed in captivity. PMID:23593364

  1. First detection of adenovirus in the vampire bat (Desmodus rotundus) in Brazil.

    PubMed

    Lima, Francisco Esmaile de Sales; Cibulski, Samuel Paulo; Elesbao, Felipe; Carnieli Junior, Pedro; Batista, Helena Beatriz de Carvalho Ruthner; Roehe, Paulo Michel; Franco, Ana Cláudia

    2013-10-01

    This paper describes the first detection of adenovirus in a Brazilian Desmodus rotundus bat, the common vampire bat. As part of a continuous rabies surveillance program, three bat specimens were captured in Southern Brazil. Total DNA was extracted from pooled organs and submitted to a nested PCR designed to amplify a 280 bp long portion of the DNA polymerase gene of adenoviruses. One positive sample was subjected to nucleotide sequencing, confirming that this DNA fragment belongs to a member of the genus Mastadenovirus. This sequence is approximately 25 % divergent at the nucleotide level from equine adenovirus 1 and two other recently characterized bat adenoviruses.

  2. Adenovirus vector delivery stimulates natural killer cell recognition

    PubMed Central

    Tomasec, Peter; Wang, Eddie C. Y.; Groh, Veronika; Spies, Thomas; McSharry, Brian P.; Aicheler, Rebecca J.; Stanton, Richard J.; Wilkinson, Gavin W. G.

    2007-01-01

    We report that delivery of first-generation replication-deficient adenovirus (RDAd) vectors into primary human fibroblasts is associated with the induction of natural killer (NK) cell-mediated cytolysis in vitro. RDAd vector delivery induced cytolysis by a range of NK cell populations including the NK cell clone NKL, primary polyclonal NK lines and a proportion of NK clones (36 %) in autologous HLA-matched assays. Adenovirus-induced cytolysis was inhibited by antibody blocking of the NK-activating receptor NKG2D, implicating this receptor in this function. NKG2D is ubiquitously expressed on NK cells and CD8+ T cells. Significantly, γ-irradiation of the vector eliminated the effect, suggesting that breakthrough expression from the vector induces at least some of the pro-inflammatory responses of unknown aetiology following the application of RDAd vectors during in vivo gene delivery. PMID:17374753

  3. Adenovirus-derived vectors for prostate cancer gene therapy.

    PubMed

    de Vrij, Jeroen; Willemsen, Ralph A; Lindholm, Leif; Hoeben, Rob C; Bangma, Chris H; Barber, Chris; Behr, Jean-Paul; Briggs, Simon; Carlisle, Robert; Cheng, Wing-Shing; Dautzenberg, Iris J C; de Ridder, Corrina; Dzojic, Helena; Erbacher, Patrick; Essand, Magnus; Fisher, Kerry; Frazier, April; Georgopoulos, Lindsay J; Jennings, Ian; Kochanek, Stefan; Koppers-Lalic, Daniela; Kraaij, Robert; Kreppel, Florian; Magnusson, Maria; Maitland, Norman; Neuberg, Patrick; Nugent, Regina; Ogris, Manfred; Remy, Jean-Serge; Scaife, Michelle; Schenk-Braat, Ellen; Schooten, Erik; Seymour, Len; Slade, Michael; Szyjanowicz, Pio; Totterman, Thomas; Uil, Taco G; Ulbrich, Karel; van der Weel, Laura; van Weerden, Wytske; Wagner, Ernst; Zuber, Guy

    2010-07-01

    Prostate cancer is a leading cause of death among men in Western countries. Whereas the survival rate approaches 100% for patients with localized cancer, the results of treatment in patients with metastasized prostate cancer at diagnosis are much less successful. The patients are usually presented with a variety of treatment options, but therapeutic interventions in prostate cancer are associated with frequent adverse side effects. Gene therapy and oncolytic virus therapy may constitute new strategies. Already a wide variety of preclinical studies has demonstrated the therapeutic potential of such approaches, with oncolytic prostate-specific adenoviruses as the most prominent vector. The state of the art and future prospects of gene therapy in prostate cancer are reviewed, with a focus on adenoviral vectors. We summarize advances in adenovirus technology for prostate cancer treatment and highlight areas where further developments are necessary.

  4. Dielectrophoresis and dielectrophoretic impedance detection of adenovirus and rotavirus

    NASA Astrophysics Data System (ADS)

    Nakano, Michihiko; Ding, Zhenhao; Suehiro, Junya

    2016-01-01

    The aim of this study is the electrical detection of pathogenic viruses, namely, adenovirus and rotavirus, using dielectrophoretic impedance measurement (DEPIM). DEPIM consists of two simultaneous processes: dielectrophoretic trapping of the target and measurement of the impedance change and increase in conductance with the number of trapped targets. This is the first study of applying DEPIM, which was originally developed to detect bacteria suspended in aqueous solutions, to virus detection. The dielectric properties of the viruses were also investigated in terms of their dielectrophoretic behavior. Although their estimated dielectric properties were different from those of bacteria, the trapped viruses increased the conductance of the microelectrode in a manner similar to that in bacteria detection. We demonstrated the electrical detection of viruses within 60 s at concentrations as low as 70 ng/ml for adenovirus and 50 ng/ml for rotavirus.

  5. Critical Role for Arginine Methylation in Adenovirus-Infected Cells▿

    PubMed Central

    Iacovides, Demetris C.; O'Shea, Clodagh C.; Oses-Prieto, Juan; Burlingame, Alma; McCormick, Frank

    2007-01-01

    During the late stages of adenovirus infection, the 100K protein (100K) inhibits the translation of cellular messages in the cytoplasm and regulates hexon trimerization and assembly in the nucleus. However, it is not known how it switches between these two functions. Here we show that 100K is methylated on arginine residues at its C terminus during infection and that this region is necessary for binding PRMT1 methylase. Methylated 100K is exclusively nuclear. Mutation of the third RGG motif (amino acids 741 to 743) prevents localization to the nucleus during infection, suggesting that methylation of that sequence is important for 100K shuttling. Treatment of infected cells with methylation inhibitors inhibits expression of late structural proteins. These data suggest that arginine methylation of 100K is necessary for its localization to the nucleus and is a critical cellular function necessary for productive adenovirus infection. PMID:17686851

  6. An Analysis of Adenovirus Genomes Using Whole Genome Software Tools

    PubMed Central

    Mahadevan, Padmanabhan

    2016-01-01

    The evolution of sequencing technology has lead to an enormous increase in the number of genomes that have been sequenced. This is especially true in the field of virus genomics. In order to extract meaningful biological information from these genomes, whole genome data mining software tools must be utilized. Hundreds of tools have been developed to analyze biological sequence data. However, only some of these tools are user-friendly to biologists. Several of these tools that have been successfully used to analyze adenovirus genomes are described here. These include Artemis, EMBOSS, pDRAW, zPicture, CoreGenes, GeneOrder, and PipMaker. These tools provide functionalities such as visualization, restriction enzyme analysis, alignment, and proteome comparisons that are extremely useful in the bioinformatics analysis of adenovirus genomes. PMID:28293072

  7. Therapy of Breast Cancers Using Conditionally Replicating Adenovirus

    DTIC Science & Technology

    2005-08-01

    virotherapy on breast cancer cells in vitro. We have developed a CRAd using the fit-I promoter element for specific EIA gene expression (CRAdflt), RGD...replicating adenoviruses (CRAd) and investigate effects of CRAd virotherapy on endothelial cells and breast cancer cells in vitro. Vascular targeting...determined the capacity of CRAdRGDflt-mda-7 virotherapy to induce breast cancer cell death. To verify the levels of MDA-7/IL-24 protein expression in vitro

  8. Oncolytic adenovirus-mediated therapy for prostate cancer

    PubMed Central

    Sweeney, Katrina; Halldén, Gunnel

    2016-01-01

    Prostate cancer is a leading cause of cancer-related death and morbidity in men in the Western world. Tumor progression is dependent on functioning androgen receptor signaling, and initial administration of antiandrogens and hormone therapy (androgen-deprivation therapy) prevent growth and spread. Tumors frequently develop escape mechanisms to androgen-deprivation therapy and progress to castration-resistant late-stage metastatic disease that, in turn, inevitably leads to resistance to all current therapeutics, including chemotherapy. In spite of the recent development of more effective inhibitors of androgen–androgen receptor signaling such as enzalutamide and abiraterone, patient survival benefits are still limited. Oncolytic adenoviruses have proven efficacy in prostate cancer cells and cause regression of tumors in preclinical models of numerous drug-resistant cancers. Data from clinical trials demonstrate that adenoviral mutants have limited toxicity to normal tissues and are safe when administered to patients with various solid cancers, including prostate cancer. While efficacy in response to adenovirus administration alone is marginal, findings from early-phase trials targeting local-ized and metastatic prostate cancer suggest improved efficacy in combination with cytotoxic drugs and radiation therapy. Here, we review recent progress in the development of multimodal oncolytic adenoviruses as biological therapeutics to improve on tumor elimination in prostate cancer patients. These optimized mutants target cancer cells by several mechanisms including viral lysis and by expression of cytotoxic transgenes and immune-stimulatory factors that activate the host immune system to destroy both infected and noninfected prostate cancer cells. Additional modifications of the viral capsid proteins may support future systemic delivery of oncolytic adenoviruses. PMID:27579296

  9. [Adenovirus vectors and their clinical application in gene therapy].

    PubMed

    Adám, E; Nász, I

    2001-09-23

    The potential therapeutic application of the gene transfer technology with adenovirus vectors seems to be enormous. Adenovirus vectors offer several advantages over other vectors, but several important limitations of adenovirus mediated gene transfer are also known. Great number of studies in inherited diseases and in different cancer therapy clinical trials have provided information of critical importance for design of efficient clinical protocols. Clinical trials have been extended to the treatment of many other diseases, too. There are about thirty currently active gene therapy protocols for the treatment only of HIV-1 infection in the USA. These programs aim to confer protective immunity against HIV-1 transmission to individuals who are in risk of infection, to develop preventive or therapeutic vaccines for patients with AIDS and other infectious diseases. Gene therapy represents one of the most important developments in oncology, however, before this can be realised as standard treatment the technical problems of gene delivery and higher safety must be overcome. The early--first and second generation--adenovirus vectors are now likely to be phased out for most diseases, and further experiments seem to be necessary. It might be change to the third generation or other, more modern vector application in clinical trials, as the helper dependent vectors. Almost all transcriptional unit is removed from the DNA of these vectors ("gutless vectors"), therefore they cannot reproduce, give higher gene expression and far less inflammatory. Despite the latest achievement reported in vector design it is not possible to predict yet to what extent and when gene therapy will be effective.

  10. The S100A4 Oncoprotein Promotes Prostate Tumorigenesis in a Transgenic Mouse Model

    PubMed Central

    Siddique, Hifzur R.; Adhami, Vaqar M.; Parray, Aijaz; Johnson, Jeremy J.; Siddiqui, Imtiaz A.; Shekhani, Mohammad T.; Murtaza, Imtiyaz; Ambartsumian, Noona; Konety, Badrinath R.; Mukhtar, Hasan

    2013-01-01

    S100A4, a calcium-binding protein, is known for its role in the metastatic spread of tumor cells, a late event of cancer disease. This is the first report showing that S100A4 is not merely a metastatic protein but also an oncoprotein that plays a critical role in the development of tumors. We earlier showed that S100A4 expression progressively increases in prostatic tissues with the advancement of prostate cancer (CaP) in TRAMP, an autochthonous mouse model. To study the functional significance of S100A4 in CaP, we generated a heterozygously deleted S100A4 (TRAMP/S100A4+/−) genotype by crossing TRAMP with S100A4−/− mice. TRAMP/S100A4+/− did not show a lethal phenotype, and transgenes were functional. As compared to age-matched TRAMP littermates, TRAMP/S100A4+/− mice exhibited 1) an increased tumor latency period (P < 0.001), 2) a 0% incidence of metastasis, and 3) reduced prostatic weights (P < 0.001). We generated S100A4-positive clones from S100A4-negative CaP cells and tested their potential. S100A4-positive tumors grew at a faster rate than S100A4-negative tumors in vitro and in a xenograft mouse model. The S100A4 protein exhibited growth factor–like properties in multimode (intracellular and extracellular) forms. We observed that 1) the growth-promoting effect of S100A4 is due to its activation of NFκB, 2) S100A4-deficient tumors exhibit reduced NFκB activity, 3) S100A4 regulates NFκB through the RAGE receptor, and 4) S100A4 and RAGE co-localize in prostatic tissues of mice. Keeping in view its growth-promoting role, we suggest that S100A4 qualifies as an excellent candidate to be exploited for therapeutic agents to treat CaP in humans. PMID:24069509

  11. An outbreak of lethal adenovirus infection among different otariid species.

    PubMed

    Inoshima, Yasuo; Murakami, Tomoaki; Ishiguro, Naotaka; Hasegawa, Kazuhiro; Kasamatsu, Masahiko

    2013-08-30

    An outbreak of fatal fulminant hepatitis at a Japanese aquarium involved 3 otariids: a California sea lion (Zalophus californianus), a South African fur seal (Arctocephalus pusillus) and a South American sea lion (Otaria flavescens). In a span of about a week in February 2012, 3 otariids showed diarrhea and were acutely low-spirited; subsequently, all three animals died within a period of 3 days. Markedly increased aspartate amino transferase and alanine amino transferase activities were observed. Necrotic hepatitis and eosinophilic intranuclear inclusion bodies in liver hepatocytes and intestinal epithelial cells were observed in the South American sea lion on histological examination. Otarine adenovirus DNA was detected from the livers of all three animals by polymerase chain reaction and determination of the sequences showed that all were identical. These results suggest that a single otarine adenovirus strain may have been the etiological agent of this outbreak of fatal fulminant hepatitis among the different otariid species, and it may be a lethal threat to wild and captive otariids. This is the first evidence of an outbreak of lethal adenovirus infection among different otariid species.

  12. Effect of CD4 gene expression on adenovirus replication.

    PubMed Central

    Hotta, J; Shi, L; Ginsberg, H S

    1994-01-01

    The gene encoding the CD4 receptor was introduced into KB cells to establish the KBT4 cell line, a cell line susceptible to infection with human immunodeficiency virus type 1. Adenovirus replication was found to be significantly less in these cells than in the parental KB cells. Similar decreased adenovirus type 5 (Ad5) replication occurred in HeLaT4 cells compared with the original HeLa cells. The presence of CD4 did not alter the cell surface population of KB cell adenovirus receptors, since viral adsorption was similar in the two cell lines. Moreover, addition of soluble CD4 did not reduce viral replication in either KB or KBT4 infected cells. Uncoating of viral DNA was also unchanged in KBT4 cells compared with the parental KB cells. In contrast, migration to or entrance of viral DNA into nuclei and synthesis of early viral RNAs was delayed and reduced in KBT4 cells. These effects were more pronounced for Ad7 than for Ad5. The yields of infectious viruses were the same in both cell lines, however, after transfection of naked viral DNAs to initiate infection. These results imply that the expression of the CD4 gene in KBT4 cells interfered with passage of uncoated virus across endosomal vesicles and/or transfer of uncoated core viral DNA into the nucleus. Images PMID:7933112

  13. Effect of CD4 gene expression on adenovirus replication.

    PubMed

    Hotta, J; Shi, L; Ginsberg, H S

    1994-11-01

    The gene encoding the CD4 receptor was introduced into KB cells to establish the KBT4 cell line, a cell line susceptible to infection with human immunodeficiency virus type 1. Adenovirus replication was found to be significantly less in these cells than in the parental KB cells. Similar decreased adenovirus type 5 (Ad5) replication occurred in HeLaT4 cells compared with the original HeLa cells. The presence of CD4 did not alter the cell surface population of KB cell adenovirus receptors, since viral adsorption was similar in the two cell lines. Moreover, addition of soluble CD4 did not reduce viral replication in either KB or KBT4 infected cells. Uncoating of viral DNA was also unchanged in KBT4 cells compared with the parental KB cells. In contrast, migration to or entrance of viral DNA into nuclei and synthesis of early viral RNAs was delayed and reduced in KBT4 cells. These effects were more pronounced for Ad7 than for Ad5. The yields of infectious viruses were the same in both cell lines, however, after transfection of naked viral DNAs to initiate infection. These results imply that the expression of the CD4 gene in KBT4 cells interfered with passage of uncoated virus across endosomal vesicles and/or transfer of uncoated core viral DNA into the nucleus.

  14. Ion etching of human adenovirus 2: structure of the core

    SciTech Connect

    Newcomb, W.W.; Boring, J.W.; Brown, J.C.

    1984-07-01

    The surface of human adenovirus 2 was etched by irradiating intact virions with low-energy (1-keV) Ar/sup +/ ions in a Technics Hummer V sputter coater. Viral structures exposed by the etching process were shadowed and then examined in the electron microscope. Periods of etching that were sufficient to reduce the viral diameter by 20 to 30 nm revealed distinct substructural elements in the virion core. Cores were found to consist of a cluster of 12 large, uniformly sized spheres which abutted one another in the intact virion. The spheres, for which we suggest the name adenosomes, had a diameter of 23.0 +/- 2.3 nm, and they were related to each other by two-, three-, and fivefold axes of rotational symmetry. The results support the view, originally suggested by Brown et al. that the adenovirus 2 core is composed of 12 large spheres packed tightly together in such a way that each is directed toward the vertex of an icosahedron. Such a structure, constructed of 23.0-nm-diameter spheres, would have an outside diameter (vertex-to-vertex distance) of 67.0 nm and a face-to-face distance of 58.2 nm. It could be accommodated inside the icosahedral adenovirus capsid if each large sphere were located beneath a capsid vertex.

  15. Progress on adenovirus-vectored universal influenza vaccines.

    PubMed

    Xiang, Kui; Ying, Guan; Yan, Zhou; Shanshan, Yan; Lei, Zhang; Hongjun, Li; Maosheng, Sun

    2015-01-01

    Influenza virus (IFV) infection causes serious health problems and heavy financial burdens each year worldwide. The classical inactivated influenza virus vaccine (IIVV) and live attenuated influenza vaccine (LAIV) must be updated regularly to match the new strains that evolve due to antigenic drift and antigenic shift. However, with the discovery of broadly neutralizing antibodies that recognize conserved antigens, and the CD8(+) T cell responses targeting viral internal proteins nucleoprotein (NP), matrix protein 1 (M1) and polymerase basic 1 (PB1), it is possible to develop a universal influenza vaccine based on the conserved hemagglutinin (HA) stem, NP, and matrix proteins. Recombinant adenovirus (rAd) is an ideal influenza vaccine vector because it has an ideal stability and safety profile, induces balanced humoral and cell-mediated immune responses due to activation of innate immunity, provides 'self-adjuvanting' activity, can mimic natural IFV infection, and confers seamless protection against mucosal pathogens. Moreover, this vector can be developed as a low-cost, rapid-response vaccine that can be quickly manufactured. Therefore, an adenovirus vector encoding conserved influenza antigens holds promise in the development of a universal influenza vaccine. This review will summarize the progress in adenovirus-vectored universal flu vaccines and discuss future novel approaches.

  16. Progress on adenovirus-vectored universal influenza vaccines

    PubMed Central

    Xiang, Kui; Ying, Guan; Yan, Zhou; Shanshan, Yan; Lei, Zhang; Hongjun, Li; Maosheng, Sun

    2015-01-01

    Influenza virus (IFV) infection causes serious health problems and heavy financial burdens each year worldwide. The classical inactivated influenza virus vaccine (IIVV) and live attenuated influenza vaccine (LAIV) must be updated regularly to match the new strains that evolve due to antigenic drift and antigenic shift. However, with the discovery of broadly neutralizing antibodies that recognize conserved antigens, and the CD8+ T cell responses targeting viral internal proteins nucleoprotein (NP), matrix protein 1 (M1) and polymerase basic 1 (PB1), it is possible to develop a universal influenza vaccine based on the conserved hemagglutinin (HA) stem, NP, and matrix proteins. Recombinant adenovirus (rAd) is an ideal influenza vaccine vector because it has an ideal stability and safety profile, induces balanced humoral and cell-mediated immune responses due to activation of innate immunity, provides ‘self-adjuvanting’ activity, can mimic natural IFV infection, and confers seamless protection against mucosal pathogens. Moreover, this vector can be developed as a low-cost, rapid-response vaccine that can be quickly manufactured. Therefore, an adenovirus vector encoding conserved influenza antigens holds promise in the development of a universal influenza vaccine. This review will summarize the progress in adenovirus-vectored universal flu vaccines and discuss future novel approaches. PMID:25876176

  17. Structural organization and polypeptide composition of the avian adenovirus core.

    PubMed Central

    Li, P; Bellett, A J; Parish, C R

    1984-01-01

    CELO virus (fowl adenovirus 1) contained three core polypeptides of molecular weights 20,000, 12,000, and 9,500. The core was similar to that of human adenoviruses, with some evidence of compact subcore domains. Micrococcal nuclease digestion of CELO virus cores produced a smear of DNA fragments of gradually decreasing size, with no nucleosome subunit or repeat pattern. Moreover, when digested cores were analyzed without protease treatment, there was again no evidence of a nucleosome substructure; neither DNA fragments nor core proteins entered a 4% polyacrylamide gel. The organization of the core is thus quite unlike that of chromatin. Restriction endonuclease analysis of the DNA from digested cores showed that the right end was on the outside of the core. We suggest that adenovirus DNA is condensed into the core by cross-linking and neutralization by the core proteins, beginning with the packaging sequence at the center of the core and ending with the right end of the DNA on the outside. Images PMID:6092686

  18. Template requirements for the initiation of adenovirus DNA replication.

    PubMed Central

    Challberg, M D; Rawlins, D R

    1984-01-01

    The first step in the replication of the adenovirus genome is the covalent attachment of the 5'-terminal nucleotide, dCMP, to the virus-encoded terminal protein precursor (pTP). This reaction can be observed in vitro and has been previously shown to be dependent upon either viral DNA or linearized plasmid DNA containing viral terminal sequences. Plasmids containing deletions or point mutations within the viral terminal sequence were constructed by site-directed mutagenesis. In the case of linear double-stranded templates, pTP-dCMP formation required sequences located within the first 18 base pairs of the viral genome. This sequence contains a segment of 10 base pairs that is conserved in all human adenovirus serotypes. Point mutations within the conserved segment greatly reduced the efficiency of initiation, while a point mutation at a nonconserved position within the first 18 base pairs had little effect. Single-stranded DNAs can also support pTP-dCMP formation in vitro. In contrast to the results obtained with duplex templates, experiments with a variety of single-stranded templates, including phage M13-adenovirus recombinants, denatured plasmids, and synthetic oligodeoxynucleotides, failed to reveal any requirements for specific nucleotide sequences. With single-stranded templates containing no dG residues, the specific deoxynucleoside triphosphate requirements of the initiation reaction were altered. Images PMID:6320160

  19. Prevalence of human adenoviruses in raw and treated water.

    PubMed

    van Heerden, J; Ehlers, M M; van Zyl, W B; Grabow, W O K

    2004-01-01

    Human adenoviruses (HAds), of which there are 51 antigenic types, are associated aetiologically with gastrointestinal, respiratory, urinary tract and eye infections. The clinical importance of HAds and the potential health risks constituted by HAds in water environments are widely recognised. This study was conducted to assess the use of an optimised integrated cell culture molecular-based technique to determine the prevalence of HAds in raw and treated drinking-water supplies in South Africa. Selected supplies were monitored weekly for the presence of adenoviruses over a one-year period (July 2001 to June 2002). Drinking-water supplies were derived from acceptable quality surface water sources using treatment processes that conformed to international standards for the production of safe drinking water. Adenoviruses were detected by amplification in cell cultures, followed by amplifying the extracted nucleic acids using molecular techniques (nested PCR). HAds were detected in 29.8% (59/198) of the treated drinking water, 16% (8/50) of dam water and 44% (22/50) of river-water samples tested. The results of this study confirmed the presence of HAds in some raw and treated drinking water supplies in South Africa.

  20. An Update on Canine Adenovirus Type 2 and Its Vectors

    PubMed Central

    Bru, Thierry; Salinas, Sara; Kremer, Eric J.

    2010-01-01

    Adenovirus vectors have significant potential for long- or short-term gene transfer. Preclinical and clinical studies using human derived adenoviruses (HAd) have demonstrated the feasibility of flexible hybrid vector designs, robust expression and induction of protective immunity. However, clinical use of HAd vectors can, under some conditions, be limited by pre-existing vector immunity. Pre-existing humoral and cellular anti-capsid immunity limits the efficacy and duration of transgene expression and is poorly circumvented by injections of larger doses and immuno-suppressing drugs. This review updates canine adenovirus serotype 2 (CAV-2, also known as CAdV-2) biology and gives an overview of the generation of early region 1 (E1)-deleted to helper-dependent (HD) CAV-2 vectors. We also summarize the essential characteristics concerning their interaction with the anti-HAd memory immune responses in humans, the preferential transduction of neurons, and its high level of retrograde axonal transport in the central and peripheral nervous system. CAV-2 vectors are particularly interesting tools to study the pathophysiology and potential treatment of neurodegenerative diseases, as anti-tumoral and anti-viral vaccines, tracer of synaptic junctions, oncolytic virus and as a platform to generate chimeric vectors. PMID:21994722

  1. History of the restoration of adenovirus type 4 and type 7 vaccine, live oral (Adenovirus Vaccine) in the context of the Department of Defense acquisition system.

    PubMed

    Hoke, Charles H; Snyder, Clifford E

    2013-03-15

    Respiratory pathogens cause morbidity and mortality in US military basic trainees. Following the influenza pandemic of 1918, and stimulated by WWII, the need to protect military personnel against epidemic respiratory disease was evident. Over several decades, the US military elucidated etiologies of acute respiratory diseases and invented and deployed vaccines to prevent disease caused by influenza, meningococcus, and adenoviruses. In 1994, the Adenovirus Vaccine manufacturer stopped its production. By 1999, supplies were exhausted and adenovirus-associated disease, especially serotype 4-associated febrile respiratory illness, returned to basic training installations. Advisory bodies persuaded Department of Defense leaders to initiate restoration of Adenovirus Vaccine. In 2011, after 10 years of effort by government and contractor personnel and at a cost of about $100 million, the Adenovirus Vaccine was restored to use at all military basic training installations. Disease and adenovirus serotype 4 isolation rates have fallen dramatically since vaccinations resumed in October 2011 and remain very low. Mindful of the adage that "The more successful a vaccine is, the more quickly the need for it will be forgotten.", sustainment of the supply of the Adenovirus Vaccine may be a challenge, and careful management will be required for such sustainment.

  2. Tropism modification of adenovirus vectors by peptide ligand insertion into various positions of the adenovirus serotype 41 short-fiber knob domain.

    PubMed

    Hesse, Andrea; Kosmides, Daniela; Kontermann, Roland E; Nettelbeck, Dirk M

    2007-03-01

    Recombinant adenoviruses have emerged as promising agents in therapeutic gene transfer, genetic vaccination, and viral oncolysis. Therapeutic applications of adenoviruses, however, would benefit substantially from targeted virus cell entry, for example, into cancer or immune cells, as opposed to the broad tropism that adenoviruses naturally possess. Such tropism modification of adenoviruses requires the deletion of their natural cell binding properties and the incorporation of cell binding ligands. The short fibers of subgroup F adenoviruses have recently been suggested as a tool for genetic adenovirus detargeting based on the reduced infectivity of corresponding adenovectors with chimeric fibers in vitro and in vivo. The goal of our study was to determine functional insertion sites for peptide ligands in the adenovirus serotype 41 (Ad41) short fiber knob. With a model peptide, CDCRGDCFC, we could demonstrate that ligand incorporation into three of five analyzed loops of the knob, namely, EG, HI, and IJ, is feasible without a loss of fiber trimerization. The resulting adenovectors showed enhanced infectivity for various cell types, which was superior to that of viruses with the same peptide fused to the fiber C terminus. Strategies to further augment gene transfer efficacy by extension of the fiber shaft, insertion of tandem copies of the ligand peptide, or extension of the ligand-flanking linkers failed, indicating that precise ligand positioning is pivotal. Our study establishes that internal ligand incorporation into a short-shafted adenovirus fiber is feasible and suggests the Ad41 short fiber with ligand insertion into the top (IJ loop) or side (EG and HI loops) of the knob domain as a novel platform for genetic targeting of therapeutic adenoviruses.

  3. Genomic and phylogenetic analyses of an adenovirus isolated from a corn snake (Elaphe guttata) imply a common origin with members of the proposed new genus Atadenovirus.

    PubMed

    Farkas, Szilvia L; Benko, Mária; Elo, Péter; Ursu, Krisztina; Dán, Adám; Ahne, Winfried; Harrach, Balázs

    2002-10-01

    Approximately 60% of the genome of an adenovirus isolated from a corn snake (Elaphe guttata) was cloned and sequenced. The results of homology searches showed that the genes of the corn snake adenovirus (SnAdV-1) were closest to their counterparts in members of the recently proposed new genus ATADENOVIRUS: In phylogenetic analyses of the complete hexon and protease genes, SnAdV-1 indeed clustered together with the atadenoviruses. The characteristic features in the genome organization of SnAdV-1 included the presence of a gene homologous to that for protein p32K, the lack of structural proteins V and IX and the absence of homologues of the E1A and E3 regions. These characteristics are in accordance with the genus-defining markers of atadenoviruses. Comparison of the cleavage sites of the viral protease in core protein pVII also confirmed SnAdV-1 as a candidate member of the genus ATADENOVIRUS: Thus, the hypothesis on the possible reptilian origin of atadenoviruses (Harrach, Acta Veterinaria Hungarica 48, 484-490, 2000) seems to be supported. However, the base composition of DNA sequence (>18 kb) determined from the SnAdV-1 genome showed an equilibrated GC content of 51%, which is unusual for an atadenovirus.

  4. The Evaluation of Polyhexamethylene Biguanide (PHMB) as a Disinfectant for Adenovirus

    PubMed Central

    Romanowski, Eric G.; Yates, Kathleen A.; O’Connor, Katherine E.; Mah, Francis S.; Shanks, Robert M. Q.; Kowalski, Regis P.

    2013-01-01

    Purpose Swimming pools can be a vector for transmission of adenovirus ocular infections. Polyhexamethylene biguanide (PHMB) is a disinfectant used in swimming pools and hot tubs. The current study determined whether PHMB is an effective disinfectant against ocular adenovirus serotypes at a concentration used to disinfect swimming pools and hot tubs. Methods The direct disinfecting activity of PHMB was determined in triplicate assays by incubating nine human adenovirus types (1, 2, 3, 4, 5, 7a, 8, 19, and 37) with 50 and 0 PPM (µg/ml) of PHMB for 24 hours at room temperature, to simulate swimming pool temperatures, or 40°C, to simulate hot tub temperatures. Plaque assays determined adenovirus titers after incubation. Titers were Log10 converted and mean ± standard deviation Log10 reductions from controls were calculated. Virucidal (greater than 99.9%) decreases in mean adenovirus titers after PHMB treatment were determined for each adenovirus type and temperature tested. Results At room temperature, 50 PPM of PHMB produced mean reductions in titers less than 1 Log10 for all adenovirus types tested. At 40°C, 50 PPM of PHMB produced mean reductions in titers less than 1 Log10 for two adenovirus types and greater than 1 Log10, but less than 3 Log10, for seven of nine adenovirus types. Conclusions 50 PPM of PHMB was not virucidal against adenovirus at temperatures consistent with swimming pools or hot tubs. Clinical Relevance Recreational water maintained and sanitized with PHMB has the potential to serve as a vector for the transmission of ocular adenovirus infections. PMID:23450376

  5. Effects of adeno-associated virus on adenovirus replication and gene expression during coinfection.

    PubMed

    Timpe, Jennifer M; Verrill, Kristin C; Trempe, James P

    2006-08-01

    Adeno-associated virus (AAV) is a nonpathogenic parvovirus that requires adenovirus (Ad) or another helper virus for a fully permissive infection. AAV-mediated inhibition of Ad is well documented, yet many details of this interaction remain unclear. In this study, we observed a maximum 50-fold decrease in infectious virus production and a 10- to 40-fold reduction in Ad DNA synthesis during coinfections with AAV. With the exception of the E3 gene, AAV decreased all steady-state Ad mRNA levels at 24 h postinfection (hpi) in a dose-dependent manner. However, not all transcription units were affected equally. E4 and late transcription were the most strongly inhibited, and E1A and E2A were the least affected. The temporal effects of AAV on Ad mRNA transcript levels also varied among the Ad genes. Ad protein expression paralleled mRNA levels at 24 hpi, suggesting that coinfecting AAV does not exert substantial effects on translation. In plasmid transfection assays, Rep78 protein most effectively limited Ad amplification, while Rep40 had no effect. Since E2a and E4 proteins are essential for efficient Ad DNA amplification, we examined the relationship between reduced E2A and E4 expression and decreased DNA amplification. Transfected Rep78 did not reduce E2A and E4 transcript levels prior to DNA replication. Also, AAV-induced inhibition of E2A and E4 mRNA production did not occur in the presence of hydroxyurea. It is therefore unlikely that decreased early gene expression is solely responsible for AAV's suppression of Ad DNA replication. Our results suggest that AAV amplification and/or Rep gene expression inhibits Ad DNA synthesis.

  6. Effects of Adeno-Associated Virus on Adenovirus Replication and Gene Expression during Coinfection

    PubMed Central

    Timpe, Jennifer M.; Verrill, Kristin C.; Trempe, James P.

    2006-01-01

    Adeno-associated virus (AAV) is a nonpathogenic parvovirus that requires adenovirus (Ad) or another helper virus for a fully permissive infection. AAV-mediated inhibition of Ad is well documented, yet many details of this interaction remain unclear. In this study, we observed a maximum 50-fold decrease in infectious virus production and a 10- to 40-fold reduction in Ad DNA synthesis during coinfections with AAV. With the exception of the E3 gene, AAV decreased all steady-state Ad mRNA levels at 24 h postinfection (hpi) in a dose-dependent manner. However, not all transcription units were affected equally. E4 and late transcription were the most strongly inhibited, and E1A and E2A were the least affected. The temporal effects of AAV on Ad mRNA transcript levels also varied among the Ad genes. Ad protein expression paralleled mRNA levels at 24 hpi, suggesting that coinfecting AAV does not exert substantial effects on translation. In plasmid transfection assays, Rep78 protein most effectively limited Ad amplification, while Rep40 had no effect. Since E2a and E4 proteins are essential for efficient Ad DNA amplification, we examined the relationship between reduced E2A and E4 expression and decreased DNA amplification. Transfected Rep78 did not reduce E2A and E4 transcript levels prior to DNA replication. Also, AAV-induced inhibition of E2A and E4 mRNA production did not occur in the presence of hydroxyurea. It is therefore unlikely that decreased early gene expression is solely responsible for AAV's suppression of Ad DNA replication. Our results suggest that AAV amplification and/or Rep gene expression inhibits Ad DNA synthesis. PMID:16873238

  7. Eliminating established tumor in nu/nu nude mice by a TRAIL-armed oncolytic adenovirus

    PubMed Central

    Dong, Fengqin; Wang, Li; Davis, John J.; Hu, Wenxian; Zhang, Lidong; Guo, Wei; Teraishi, Fuminori; Ji, Lin; Fang, Bingliang

    2006-01-01

    Purpose The tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) and oncolytic viruses have recently been investigated extensively for cancer therapy. However, preclinical and clinical studies have revealed that their clinical application is hampered by either weak anticancer activity or systemic toxicity. We examined whether the weaknesses of the two strategies can be overcome by integrating the TRAIL gene into an oncolytic vector. Experimental Design We constructed a TRAIL-expressing oncolytic adenovector designated Ad/TRAIL-E1. The expression of both the TRAIL and viral E1A genes is under the control of a synthetic promoter consisting of sequences from the human telomerase reverse transcriptase promoter and a minimal cytomegalovirus early promoter. The transgene expression, apoptosis induction, viral replication, antitumor activity and toxicity of Ad/TRAIL-E1 were determined in vitro and in vivo in comparison with control vectors. Results Ad/TRAIL-E1 elicited enhanced viral replication and/or stronger oncolytic effect in vitro in various human cancer cell lines than a TRAIL-expressing replication-defective adenovector or an oncolytic adenovector expressing green fluorescent protein. Intralesional administration of Ad/TRAIL-E1 eliminated all subcutaneous xenograft tumors established from a human non-small cell lung cancer cell line, H1299, on nu/nu nude mice, resulting in long-term tumor-free survival. Furthermore, we found no treatment-related toxicity. Conclusions Viral replication and antitumor activity of oncolytic adenovirus can be enhanced by the TRAIL gene and Ad/TRAIL-E1 could become a potent therapeutic agent for cancer therapy. PMID:16951242

  8. Alteration of a single amino acid in the basic domain of Marek's disease virus Meq oncoprotein plays an important role in T-cell transformation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Marek’s disease virus encoded oncoprotein, Meq, has been shown to play a major role in transformation of T-lymphocytes. We have earlier shown that replacement of the meq gene in the very virulent strain Md5 with that of vaccine strain CVI988/Rispens resulted in virus attenuation in chickens. To dete...

  9. NUP98 fusion oncoproteins interact with the APC/C(Cdc20) as a pseudosubstrate and prevent mitotic checkpoint complex binding.

    PubMed

    Salsi, Valentina; Fantini, Sebastian; Zappavigna, Vincenzo

    2016-09-01

    NUP98 is a recurrent partner gene in translocations causing acute myeloid leukemias and myelodisplastic syndrome. The expression of NUP98 fusion oncoproteins has been shown to induce mitotic spindle defects and chromosome missegregation, which correlate with the capability of NUP98 fusions to cause mitotic checkpoint attenuation. We show that NUP98 oncoproteins physically interact with the APC/C(Cdc20) in the absence of the NUP98 partner protein RAE1, and prevent the binding of the mitotic checkpoint complex to the APC/C(Cdc20). NUP98 oncoproteins require the GLEBS-like domain present in their NUP98 moiety to bind the APC/C(Cdc20). We found that NUP98 wild-type is a substrate of APC/C(Cdc20) prior to mitotic entry, and that its binding to APC/C(Cdc20) is controlled via phosphorylation of a PEST sequence located within its C-terminal portion. We identify S606, within the PEST sequence, as a key target site, whose phosphorylation modulates the capability of NUP98 to interact with APC/C(Cdc20). We finally provide evidence for an involvement of the peptidyl-prolyl isomerase PIN1 in modulating the possible conformational changes within NUP98 that lead to its dissociation from the APC/C(Cdc20) during mitosis. Our results provide novel insight into the mechanisms underlying the aberrant capability of NUP98 oncoproteins to interact with APC/C(Cdc20) and to interfere with its function.

  10. E4orf6 variants with separate abilities to augment adenovirus replication and direct nuclear localization of the E1B 55-kilodalton protein.

    PubMed

    Orlando, Joseph S; Ornelles, David A

    2002-02-01

    The E4orf6 protein of group C adenovirus is an oncoprotein that, in association with the E1B 55-kDa protein and by E1B-independent means, promotes virus replication. An arginine-faced amphipathic alpha-helix in the E4orf6 protein is required for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein and to enhance replication of an E4 deletion virus. In this study, E4orf6 protein variants containing arginine substitutions in the amphipathic alpha-helix were analyzed. Two of the six arginine residues within the alpha-helix, arginine-241 and arginine-243, were critical for directing nuclear localization of the E1B 55-kDa protein. The four remaining arginine residues appear to provide a net positive charge for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein. The molecular determinants of the arginine-faced amphipathic alpha-helix that were required for the functional interaction between the E4orf6 and E1B 55-kDa proteins seen in the transfected cell differed from those required to support a productive infection. Several E4orf6 protein variants with arginine-to-glutamic acid substitutions that failed to direct nuclear localization of the E1B 55-kDa protein restored replication of an E4 deletion virus. Additionally, a variant containing an arginine-to-alanine substitution at position 243 that directed nuclear localization of the E1B 55-kDa protein failed to enhance virus replication. These results indicate that the ability of the E4orf6 protein to relocalize the E1B 55-kDa protein to the nucleus can be separated from the ability of the E4orf6 protein to support a productive infection.

  11. 9 CFR 113.305 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Type 2 Vaccine. 113.305 Section 113.305 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION... STANDARD REQUIREMENTS Live Virus Vaccines § 113.305 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine. Canine Hepatitis Vaccine and Canine Adenovirus Type 2 Vaccine shall be prepared from virus-bearing...

  12. 9 CFR 113.305 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Type 2 Vaccine. 113.305 Section 113.305 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION... STANDARD REQUIREMENTS Live Virus Vaccines § 113.305 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine. Canine Hepatitis Vaccine and Canine Adenovirus Type 2 Vaccine shall be prepared from virus-bearing...

  13. 9 CFR 113.202 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... Type 2 Vaccine, Killed Virus. 113.202 Section 113.202 Animals and Animal Products ANIMAL AND PLANT...; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.202 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus. Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed...

  14. 9 CFR 113.202 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Type 2 Vaccine, Killed Virus. 113.202 Section 113.202 Animals and Animal Products ANIMAL AND PLANT...; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.202 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus. Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed...

  15. 9 CFR 113.202 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Type 2 Vaccine, Killed Virus. 113.202 Section 113.202 Animals and Animal Products ANIMAL AND PLANT...; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.202 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus. Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed...

  16. 9 CFR 113.305 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... Type 2 Vaccine. 113.305 Section 113.305 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION... STANDARD REQUIREMENTS Live Virus Vaccines § 113.305 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine. Canine Hepatitis Vaccine and Canine Adenovirus Type 2 Vaccine shall be prepared from virus-bearing...

  17. 9 CFR 113.202 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... Type 2 Vaccine, Killed Virus. 113.202 Section 113.202 Animals and Animal Products ANIMAL AND PLANT...; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.202 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus. Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed...

  18. 9 CFR 113.305 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... Type 2 Vaccine. 113.305 Section 113.305 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION... STANDARD REQUIREMENTS Live Virus Vaccines § 113.305 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine. Canine Hepatitis Vaccine and Canine Adenovirus Type 2 Vaccine shall be prepared from virus-bearing...

  19. 9 CFR 113.202 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Type 2 Vaccine, Killed Virus. 113.202 Section 113.202 Animals and Animal Products ANIMAL AND PLANT...; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Killed Virus Vaccines § 113.202 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed Virus. Canine Hepatitis and Canine Adenovirus Type 2 Vaccine, Killed...

  20. 9 CFR 113.305 - Canine Hepatitis and Canine Adenovirus Type 2 Vaccine.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... Type 2 Vaccine. 113.305 Section 113.305 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION... STANDARD REQUIREMENTS Live Virus Vaccines § 113.305 Canine Hepatitis and Canine Adenovirus Type 2 Vaccine. Canine Hepatitis Vaccine and Canine Adenovirus Type 2 Vaccine shall be prepared from virus-bearing...

  1. Recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers.

    PubMed Central

    Weitzman, M D; Fisher, K J; Wilson, J M

    1996-01-01

    Replication of a human parvovirus, adeno-associated virus (AAV), is facilitated by coinfection with adeno-virus to provide essential helper functions. We have used the techniques of in situ hybridization and immunocytochemistry to characterize the localization of AAV replication within infected cells, Previous studies have shown that adenovirus establishes foci called replication centers within the nucleus, where adenoviral replication and transcription occur. Our studies indicate that AAV is colocalized with the adenovirus replication centers, where it may utilize adenovirus and cellular proteins for its own replication. Expression of the AAV Rep protein inhibits the normal maturation of the adenovirus centers. Similar experiments were performed with recombinant AAV (rAAV) to establish a relationship between intranuclear localization and rAAV transduction. rAAV efficiently entered the cell, and its genome was faintly detectable in a perinuclear distribution and was mobilized to replication centers when the cell was infected with adenovirus. The recruitment of the replication-defective genome into the intranuclear adenovirus domains resulted in enhanced transduction. These studies illustrate the importance of intracellular compartmentalization for such complex interactions as the relationship between AAV and adenovirus. PMID:8627709

  2. Recruitment of wild-type and recombinant adeno-associated virus into adenovirus replication centers.

    PubMed

    Weitzman, M D; Fisher, K J; Wilson, J M

    1996-03-01

    Replication of a human parvovirus, adeno-associated virus (AAV), is facilitated by coinfection with adeno-virus to provide essential helper functions. We have used the techniques of in situ hybridization and immunocytochemistry to characterize the localization of AAV replication within infected cells, Previous studies have shown that adenovirus establishes foci called replication centers within the nucleus, where adenoviral replication and transcription occur. Our studies indicate that AAV is colocalized with the adenovirus replication centers, where it may utilize adenovirus and cellular proteins for its own replication. Expression of the AAV Rep protein inhibits the normal maturation of the adenovirus centers. Similar experiments were performed with recombinant AAV (rAAV) to establish a relationship between intranuclear localization and rAAV transduction. rAAV efficiently entered the cell, and its genome was faintly detectable in a perinuclear distribution and was mobilized to replication centers when the cell was infected with adenovirus. The recruitment of the replication-defective genome into the intranuclear adenovirus domains resulted in enhanced transduction. These studies illustrate the importance of intracellular compartmentalization for such complex interactions as the relationship between AAV and adenovirus.

  3. Avian influenza mucosal vaccination in chickens with replication-defective recombinant adenovirus vaccine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We evaluated protection conferred by mucosal vaccination with replication competent adenovirus (RCA)-free recombinant adenovirus expressing a codon-optimized avian influenza (AI) H5 gene (AdTW68.H5ck). Commercial layer-type chicken groups were singly vaccinated ocularly at 5 days of age, or singly v...

  4. Protection of chickens against avian influenza with non-replicating adenovirus-vectored vaccine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protective immunity against avian influenza (AI) virus was elicited in chickens by single-dose vaccination with a replication competent adenovirus (RCA) -free human adenovirus (Ad) vector encoding a H7 hemagglutinin gene from a low pathogenic North American isolate (AdChNY94.H7). Chickens vaccinate...

  5. Immunocompetent syngeneic cotton rat tumor models for the assessment of replication-competent oncolytic adenovirus

    SciTech Connect

    Steel, Jason C.; Morrison, Brian J.; Mannan, Poonam; Abu-Asab, Mones S.; Wildner, Oliver; Miles, Brian K.; Yim, Kevin C.; Ramanan, Vijay; Prince, Gregory A.; Morris, John C.

    2007-12-05

    Oncolytic adenoviruses as a treatment for cancer have demonstrated limited clinical activity. Contributing to this may be the relevance of preclinical animal models used to study these agents. Syngeneic mouse tumor models are generally non-permissive for adenoviral replication, whereas human tumor xenograft models exhibit attenuated immune responses to the vector. The cotton rat (Sigmodon hispidus) is susceptible to human adenovirus infection, permissive for viral replication and exhibits similar inflammatory pathology to humans with adenovirus replicating in the lungs, respiratory passages and cornea. We evaluated three transplantable tumorigenic cotton rat cell lines, CCRT, LCRT and VCRT as models for the study of oncolytic adenoviruses. All three cells lines were readily infected with adenovirus type-5-based vectors and exhibited high levels of transgene expression. The cell lines supported viral replication demonstrated by the induction of cytopathogenic effect (CPE) in tissue culture, increase in virus particle numbers and assembly of virions seen on transmission electron microscopy. In vivo, LCRT and VCRT tumors demonstrated delayed growth after injection with replicating adenovirus. No in vivo antitumor activity was seen in CCRT tumors despite in vitro oncolysis. Adenovirus was also rapidly cleared from the CCRT tumors compared to LCRT and VCRT tumors. The effect observed with the different cotton rat tumor cell lines mimics the variable results of human clinical trials highlighting the potential relevance of this model for assessing the activity and toxicity of oncolytic adenoviruses.

  6. Adenovirus-based vaccines against avian-origin H5N1 influenza viruses.

    PubMed

    He, Biao; Zheng, Bo-jian; Wang, Qian; Du, Lanying; Jiang, Shibo; Lu, Lu

    2015-02-01

    Since 1997, human infection with avian H5N1, having about 60% mortality, has posed a threat to public health. In this review, we describe the epidemiology of H5N1 transmission, advantages and disadvantages of different influenza vaccine types, and characteristics of adenovirus, finally summarizing advances in adenovirus-based H5N1 systemic and mucosal vaccines.

  7. Comparison of throat swab and nasopharyngeal aspirate specimens for rapid detection of adenovirus.

    PubMed

    Hara, Michimaru; Takao, Shinichi; Shimazu, Yukie

    2015-06-01

    Nasopharyngeal aspirate (NPA) and throat swab (TS) specimens from individual patients were compared with regard to usefulness for adenovirus detection. In 153 adenovirus-infected patients, rapid test sensitivities with NPAs (90.8%) were nearly equivalent to those with TSs (91.5%) based on real-time polymerase chain reaction standards, indicating that NPAs are equally useful.

  8. Adenovirus Type 7 Pneumonia in Children Who Died from Measles-Associated Pneumonia, Hanoi, Vietnam, 2014.

    PubMed

    Hai, Le Thanh; Thach, Hoang Ngoc; Tuan, Ta Anh; Nam, Dao Huu; Dien, Tran Minh; Sato, Yuko; Kumasaka, Toshio; Suzuki, Tadaki; Hanaoka, Nozomu; Fujimoto, Tsuguto; Katano, Harutaka; Hasegawa, Hideki; Kawachi, Shoji; Nakajima, Noriko

    2016-04-01

    During a 2014 measles outbreak in Vietnam, postmortem pathologic examination of hospitalized children who died showed that adenovirus type 7 pneumonia was a contributory cause of death in children with measles-associated immune suppression. Adenovirus type 7 pneumonia should be recognized as a major cause of secondary infection after measles.

  9. Location of the Origin of DNA Replication in Adenovirus Type 2

    PubMed Central

    Horwitz, Marshall S.

    1974-01-01

    Utilizing the isolated left and right halves of both adenovirus type 2 and the nondefective adenovirus simian virus 40 hybrid (Ad2+ND1), studies were undertaken to find the site on the DNA molecules at which replication begins. The data are consistent with several models which include an initiation event at both ends and bidirectional growth. PMID:4363250

  10. Subgenomic viral DNA species synthesized in simian cells by human and simian adenoviruses.

    PubMed Central

    Daniell, E

    1981-01-01

    DNA synthesized after infection of simian tissue culture cells (BSC-1 or CV-1) with human adenovirus type 2 or 5 or with simian adenovirus 7 was characterized. It was demonstrated that as much as 40% of the virus-specific DNA in nuclei of infected monkey cells consists of subgenomic pieces. No subgenomic viral DNA species were detected in the nuclei of human (HeLa) cells infected with these adenovirus types. Restriction analysis showed that these short viral DNA molecules contain normal amounts of the sequences from the ends of the viral genome, whereas internal regions are underrepresented. The production of subgenomic DNAs is not correlated with semipermissive infection. Although adenovirus types 2 and 5 are restricted in monkey cells, these cells are fully permissive for simian adenovirus 7. HR404, an adenovirus type 5 mutant which is not restricted in monkey cells, produced the same percentage of subgenomic DNAs as did its wild type (restricted) parent, and coinfection of monkey cells with adenovirus type 5 DNAs. The array of predominant size classes among the heterogeneously sized short DNAs is serotype specific. Extensive plaque purification and comparison of wild-type adenovirus type 5 with several viral mutants indicated that the distribution of aberrant sizes of DNA is characteristic of the virus and not a result of random replicative errors and then enrichment of particular species. Images PMID:6261009

  11. Optimization and evaluation of a method to detect adenoviruses in river water

    EPA Pesticide Factsheets

    This dataset includes the recoveries of spiked adenovirus through various stages of experimental optimization procedures. This dataset is associated with the following publication:McMinn , B., A. Korajkic, and A. Grimm. Optimization and evaluation of a method to detect adenoviruses in river water. JOURNAL OF VIROLOGICAL METHODS. Elsevier Science Ltd, New York, NY, USA, 231(1): 8-13, (2016).

  12. Studies on the Interaction of Tumor-Derived HD5 Alpha Defensins with Adenoviruses and Implications for Oncolytic Adenovirus Therapy.

    PubMed

    Vragniau, Charles; Hübner, Jens-Martin; Beidler, Peter; Gil, Sucheol; Saydaminova, Kamola; Lu, Zhuo-Zhuang; Yumul, Roma; Wang, Hongjie; Richter, Maximilian; Sova, Pavel; Drescher, Charles; Fender, Pascal; Lieber, André

    2017-03-15

    Defensins are small antimicrobial peptides capable of neutralizing human adenovirus (HAdV) in vitro by binding capsid proteins and blocking endosomal escape of virus. In humans, the alpha defensin HD5 is produced by specialized epithelial cells of the gastrointestinal and genito-urinary tracts. Here, we demonstrate, using patient biopsy specimens, that HD5 is also expressed as an active, secreted peptide by epithelial ovarian and lung cancer cells in situ This finding prompted us to study the role of HD5 in infection and spread of replication-competent, oncolytic HAdV type 3 (HAdV3). HAdV3 produces large amounts of penton-dodecahedra (PtDd), virus-like particles, during replication. We have previously shown that PtDd are involved in opening epithelial junctions, thus facilitating lateral spread of de novo-produced virions. Here, we describe a second function of PtDd, namely, the blocking of HD5. A central tool to prove that viral PtDd neutralize HD5 and support spread of progeny virus was an HAdV3 mutant virus in which formation of PtDd was disabled (mut-Ad3GFP, where GFP is green fluorescent protein). We demonstrated that viral spread of mut-Ad3GFP was blocked by synthetic HD5 whereas that of the wild-type (wt) form (wt-Ad3GFP) was only minimally impacted. In human colon cancer Caco-2 cells, induction of cellular HD5 expression by fibroblast growth factor 9 (FGF9) significantly inhibited viral spread and progeny virus production of mut-Ad3GFP but not of wt-Ad3GFP. Finally, the ectopic expression of HD5 in tumor cells diminished the in vivo oncolytic activity of mut-Ad3GFP but not of wt-Ad3GFP. These data suggest a new mechanism of HAdV3 to overcome innate antiviral host responses. Our study has implications for oncolytic adenovirus therapy.IMPORTANCE Previously, it has been reported that human defensin HD5 inactivates specific human adenoviruses by binding to capsid proteins and blocking endosomal escape of virus. The central new findings described in our

  13. API2-MALT1 oncoprotein promotes lymphomagenesis via unique program of substrate ubiquitination and proteolysis.

    PubMed

    Rosebeck, Shaun; Lim, Megan S; Elenitoba-Johnson, Kojo S J; McAllister-Lucas, Linda M; Lucas, Peter C

    2016-02-26

    Lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is the most common extranodal B cell tumor and accounts for 8% of non-Hodgkin's lymphomas. Gastric MALT lymphoma is the best-studied example and is a prototypical neoplasm that occurs in the setting of chronic inflammation brought on by persistent infection or autoimmune disease. Cytogenetic abnormalities are commonly acquired during the course of disease and the most common is chromosomal translocation t(11;18)(q21;q21), which creates the API2-MALT1 fusion oncoprotein. t(11;18)-positive lymphomas can be clinically aggressive and have a higher rate of dissemination than t(11;18)-negative tumors. Many cancers, including MALT lymphomas, characteristically exhibit deregulated over-activation of cellular survival pathways, such as the nuclear factor-κB (NF-κB) pathway. Molecular characterization of API2-MALT1 has revealed it to be a potent activator of NF-κB, which is required for API2-MALT1-induced cellular transformation, however the mechanisms by which API2-MALT1 exerts these effects are only recently becoming apparent. The API2 moiety of the fusion binds tumor necrosis factor (TNF) receptor associated factor (TRAF) 2 and receptor interacting protein 1 (RIP1), two proteins essential for TNF receptor-induced NF-κB activation. By effectively mimicking ligand-bound TNF receptor, API2-MALT1 promotes TRAF2-dependent ubiquitination of RIP1, which then acts as a scaffold for nucleating and activating the canonical NF-κB machinery. Activation occurs, in part, through MALT1 moiety-dependent recruitment of TRAF6, which can directly modify NF-κB essential modulator, the principal downstream regulator of NF-κB. While the intrinsic MALT1 protease catalytic activity is dispensable for this canonical NF-κB signaling, it is critical for non-canonical NF-κB activation. In this regard, API2-MALT1 recognizes NF-κB inducing kinase (NIK), the essential upstream regulator of non-canonical NF-κB, and cleaves it to

  14. API2-MALT1 oncoprotein promotes lymphomagenesis via unique program of substrate ubiquitination and proteolysis

    PubMed Central

    Rosebeck, Shaun; Lim, Megan S; Elenitoba-Johnson, Kojo S J; McAllister-Lucas, Linda M; Lucas, Peter C

    2016-01-01

    Lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma) is the most common extranodal B cell tumor and accounts for 8% of non-Hodgkin’s lymphomas. Gastric MALT lymphoma is the best-studied example and is a prototypical neoplasm that occurs in the setting of chronic inflammation brought on by persistent infection or autoimmune disease. Cytogenetic abnormalities are commonly acquired during the course of disease and the most common is chromosomal translocation t(11;18)(q21;q21), which creates the API2-MALT1 fusion oncoprotein. t(11;18)-positive lymphomas can be clinically aggressive and have a higher rate of dissemination than t(11;18)-negative tumors. Many cancers, including MALT lymphomas, characteristically exhibit deregulated over-activation of cellular survival pathways, such as the nuclear factor-κB (NF-κB) pathway. Molecular characterization of API2-MALT1 has revealed it to be a potent activator of NF-κB, which is required for API2-MALT1-induced cellular transformation, however the mechanisms by which API2-MALT1 exerts these effects are only recently becoming apparent. The API2 moiety of the fusion binds tumor necrosis factor (TNF) receptor associated factor (TRAF) 2 and receptor interacting protein 1 (RIP1), two proteins essential for TNF receptor-induced NF-κB activation. By effectively mimicking ligand-bound TNF receptor, API2-MALT1 promotes TRAF2-dependent ubiquitination of RIP1, which then acts as a scaffold for nucleating and activating the canonical NF-κB machinery. Activation occurs, in part, through MALT1 moiety-dependent recruitment of TRAF6, which can directly modify NF-κB essential modulator, the principal downstream regulator of NF-κB. While the intrinsic MALT1 protease catalytic activity is dispensable for this canonical NF-κB signaling, it is critical for non-canonical NF-κB activation. In this regard, API2-MALT1 recognizes NF-κB inducing kinase (NIK), the essential upstream regulator of non-canonical NF-κB, and cleaves it to

  15. Human adenoviruses in water: occurrence and health implications: a critical review.

    PubMed

    Jiang, Sunny C

    2006-12-01

    Adenoviruses are important human pathogens that are responsible for both enteric illnesses and respiratory and eye infections. Recently, these viruses have been found to be prevalent in rivers, coastal waters, swimming pool waters, and drinking water supplies worldwide. United Sates Environmental Protection Agency (USEPA) listed adenovirus as one of nine microorganisms on the Contamination Candidate List for drinking water because their survival characteristic during water treatment is not yet fully understood. Adenoviruses have been found to be significantly more stable than fecal indicator bacteria and other enteric viruses during UV treatment. Adenovirus infection may be caused by consumption of contaminated water or inhalation of aerosolized droplets during water recreation. The goal of this review is to summarize the state of technology for adenovirus detection in natural and drinking waters and the human health risk imposed by this emerging pathogen. The occurrence of these viruses in natural and treated waters is summarized from worldwide reports.

  16. Rotavirus and adenovirus prevalence at Tepecik education and research hospital (Turkey).

    PubMed

    Ece, Gulfem; Samlioglu, Pinar; Ulker, Temel; Kose, Sukran; Ersan, Gursel

    2012-06-01

    Diarrhoea affects many people globally. Rotaviruses and enteric adenovirus types 40 and 41 are the most common viruses causing childhood gastroenteritis. The aim of this study was to determine the prevalence of rotavirus and adenovirus from the faecal samples obtained at the Infectious Diseases and Clinical Microbiology Laboratory of Tepecik Education and Research Hospital. The faecal samples were screened for rotavirus, and adenovirus by commercially available immunochromatographic EIA kit (Rotavirus/Adenovirus Combo Rapid Test Device) (San Diego, CA, USA). A total of 1112 stool samples were collected from May 23rd 2008 to May 25th 2010. Of these faecal samples, 201(18.07%) were positive for rotavirus and 14 (1.2 %) for adenovirus antigen. In our study the most common agent detected was rotavirus. Viral antigen analysis in stool specimens is important for diagnosis. Detection of the viral aetiology in gastroenteritis cases will prevent unnecessary antibiotic consumption.

  17. The DEK Oncoprotein Is a Critical Component of the EKLF/KLF1 Enhancer in Erythroid Cells

    PubMed Central

    Lohmann, Felix; Dangeti, Mohan; Soni, Shefali; Chen, Xiaoyong; Planutis, Antanas; Baron, Margaret H.; Choi, Kyunghee

    2015-01-01

    Understanding how transcriptional regulators are themselves controlled is important in attaining a complete picture of the intracellular effects that follow signaling cascades during early development and cell-restricted differentiation. We have addressed this issue by focusing on the regulation of EKLF/KLF1, a zinc finger transcription factor that plays a necessary role in the global regulation of erythroid gene expression. Using biochemical affinity purification, we have identified the DEK oncoprotein as a critical factor that interacts with an essential upstream enhancer element of the EKLF promoter and exerts a positive effect on EKLF levels. This element also binds a core set of erythroid transcription factors, suggesting that DEK is part of a tissue-restricted enhanceosome that contains BMP4-dependent and -independent components. Together with local enrichment of properly coded histones and an open chromatin domain, optimal transcriptional activation of the EKLF locus can be established. PMID:26303528

  18. Human papillomavirus type 16 E7 oncoprotein engages but does not abrogate the mitotic spindle assembly checkpoint

    SciTech Connect

    Yu, Yueyang; Munger, Karl

    2012-10-10

    The mitotic spindle assembly checkpoint (SAC) ensures faithful chromosome segregation during mitosis by censoring kinetochore-microtubule interactions. It is frequently rendered dysfunctional during carcinogenesis causing chromosome missegregation and genomic instability. There are conflicting reports whether the HPV16 E7 oncoprotein drives chromosomal instability by abolishing the SAC. Here we report that degradation of mitotic cyclins is impaired in cells with HPV16 E7 expression. RNAi-mediated depletion of Mad2 or BubR1 indicated the involvement of the SAC, suggesting that HPV16 E7 expression causes sustained SAC engagement. Mutational analyses revealed that HPV16 E7 sequences that are necessary for retinoblastoma tumor suppressor protein binding as well as sequences previously implicated in binding the nuclear and mitotic apparatus (NuMA) protein and in delocalizing dynein from the mitotic spindle contribute to SAC engagement. Importantly, however, HPV16 E7 does not markedly compromise the SAC response to microtubule poisons.

  19. Interaction between the human papillomavirus 16 E7 oncoprotein and gelsolin ignites cancer cell motility and invasiveness.

    PubMed

    Matarrese, Paola; Abbruzzese, Claudia; Mileo, Anna Maria; Vona, Rosa; Ascione, Barbara; Visca, Paolo; Rollo, Francesca; Benevolo, Maria; Malorni, Walter; Paggi, Marco G

    2016-08-09

    The viral oncoprotein E7 from the "high-risk" Human Papillomavirus 16 (HPV16) strain is able, when expressed in human keratinocytes, to physically interact with the actin severing protein gelsolin (GSN). In a previous work it has been suggested that this protein-protein interaction can hinder GSN severing function, thus leading to actin network remodeling. In the present work we investigated the possible implications of this molecular interaction in cancer cell metastatic potential by analyzing two different human CC cell lines characterized by low or high expression levels of HPV16 DNA (SiHa and CaSki, respectively). In addition, a HPV-null CC cell line (C-33A), transfected in order to express the HPV16 E7 oncoprotein as well as two different deletion mutants, was also analyzed. We found that HPV16 E7 expression level was directly related with cervical cancer migration and invasion capabilities and that these HPV16 E7-related features were associated with Epithelial to Mesenchymal Transition (EMT) processes. These effects appeared as strictly attributable to the physical interaction of HPV16 E7 with GSN, since HPV16 E7 deletion mutants unable to bind to GSN were also unable to modify microfilament assembly dynamics and, therefore, cell movements and invasiveness. Altogether, these data profile the importance of the physical interaction between HPV16 E7 and GSN in the acquisition of the metastatic phenotype by CC cells, underscoring the role of HPV16 intracellular load as a risk factor in cancer.

  20. Interaction between the human papillomavirus 16 E7 oncoprotein and gelsolin ignites cancer cell motility and invasiveness

    PubMed Central

    Vona, Rosa; Ascione, Barbara; Visca, Paolo; Rollo, Francesca; Benevolo, Maria; Malorni, Walter; Paggi, Marco G.

    2016-01-01

    The viral oncoprotein E7 from the “high-risk” Human Papillomavirus 16 (HPV16) strain is able, when expressed in human keratinocytes, to physically interact with the actin severing protein gelsolin (GSN). In a previous work it has been suggested that this protein-protein interaction can hinder GSN severing function, thus leading to actin network remodeling. In the present work we investigated the possible implications of this molecular interaction in cancer cell metastatic potential by analyzing two different human CC cell lines characterized by low or high expression levels of HPV16 DNA (SiHa and CaSki, respectively). In addition, a HPV-null CC cell line (C-33A), transfected in order to express the HPV16 E7 oncoprotein as well as two different deletion mutants, was also analyzed. We found that HPV16 E7 expression level was directly related with cervical cancer migration and invasion capabilities and that these HPV16 E7-related features were associated with Epithelial to Mesenchymal Transition (EMT) processes. These effects appeared as strictly attributable to the physical interaction of HPV16 E7 with GSN, since HPV16 E7 deletion mutants unable to bind to GSN were also unable to modify microfilament assembly dynamics and, therefore, cell movements and invasiveness. Altogether, these data profile the importance of the physical interaction between HPV16 E7 and GSN in the acquisition of the metastatic phenotype by CC cells, underscoring the role of HPV16 intracellular load as a risk factor in cancer. PMID:27072581

  1. Interaction of the Human Papillomavirus E6 Oncoprotein with Sorting Nexin 27 Modulates Endocytic Cargo Transport Pathways

    PubMed Central

    Ganti, Ketaki; Massimi, Paola; Manzo-Merino, Joaquin; Tomaić, Vjekoslav; Pim, David; Playford, Martin P.; Lizano, Marcela; Roberts, Sally; Kranjec, Christian; Doorbar, John; Banks, Lawrence

    2016-01-01

    A subset of high-risk Human Papillomaviruses (HPVs) are the causative agents of a large number of human cancers, of which cervical is the most common. Two viral oncoproteins, E6 and E7, contribute directly towards the development and maintenance of malignancy. A characteristic feature of the E6 oncoproteins from cancer-causing HPV types is the presence of a PDZ binding motif (PBM) at its C-terminus, which confers interaction with cellular proteins harbouring PDZ domains. Here we show that this motif allows E6 interaction with Sorting Nexin 27 (SNX27), an essential component of endosomal recycling pathways. This interaction is highly conserved across E6 proteins from multiple high-risk HPV types and is mediated by a classical PBM-PDZ interaction but unlike many E6 targets, SNX27 is not targeted for degradation by E6. Rather, in HPV-18 positive cell lines the association of SNX27 with components of the retromer complex and the endocytic transport machinery is altered in an E6 PBM-dependent manner. Analysis of a SNX27 cargo, the glucose transporter GLUT1, reveals an E6-dependent maintenance of GLUT1 expression and alteration in its association with components of the endocytic transport machinery. Furthermore, knockdown of E6 in HPV-18 positive cervical cancer cells phenocopies the loss of SNX27, both in terms of GLUT1 expression levels and its vesicular localization, with a concomitant marked reduction in glucose uptake, whilst loss of SNX27 results in slower cell proliferation in low nutrient conditions. These results demonstrate that E6 interaction with SNX27 can alter the recycling of cargo molecules, one consequence of which is modulation of nutrient availability in HPV transformed tumour cells. PMID:27649450

  2. Polycation-π interactions are a driving force for molecular recognition by an intrinsically disordered oncoprotein family.

    PubMed

    Song, Jianhui; Ng, Sheung Chun; Tompa, Peter; Lee, Kevin A W; Chan, Hue Sun

    2013-01-01

    Molecular recognition by intrinsically disordered proteins (IDPs) commonly involves specific localized contacts and target-induced disorder to order transitions. However, some IDPs remain disordered in the bound state, a phenomenon coined "fuzziness", often characterized by IDP polyvalency, sequence-insensitivity and a dynamic ensemble of disordered bound-state conformations. Besides the above general features, specific biophysical models for fuzzy interactions are mostly lacking. The transcriptional activation domain of the Ewing's Sarcoma oncoprotein family (EAD) is an IDP that exhibits many features of fuzziness, with multiple EAD aromatic side chains driving molecular recognition. Considering the prevalent role of cation-π interactions at various protein-protein interfaces, we hypothesized that EAD-target binding involves polycation- π contacts between a disordered EAD and basic residues on the target. Herein we evaluated the polycation-π hypothesis via functional and theoretical interrogation of EAD variants. The experimental effects of a range of EAD sequence variations, including aromatic number, aromatic density and charge perturbations, all support the cation-π model. Moreover, the activity trends observed are well captured by a coarse-grained EAD chain model and a corresponding analytical model based on interaction between EAD aromatics and surface cations of a generic globular target. EAD-target binding, in the context of pathological Ewing's Sarcoma oncoproteins, is thus seen to be driven by a balance between EAD conformational entropy and favorable EAD-target cation-π contacts. Such a highly versatile mode of molecular recognition offers a general conceptual framework for promiscuous target recognition by polyvalent IDPs.

  3. Phylogenetic and functional analysis of sequence variation of human papillomavirus type 31 E6 and E7 oncoproteins.

    PubMed

    Ferenczi, Annamária; Gyöngyösi, Eszter; Szalmás, Anita; László, Brigitta; Kónya, József; Veress, György

    2016-09-01

    High-risk human papillomaviruses (HPV) are the causative agents of cervical and other anogenital cancers as well as a subset of head and neck cancers. The E6 and E7 oncoproteins of HPV contribute to oncogenesis by associating with the tumour suppressor protein p53 and pRb, respectively. For HPV types 16 and 18, intratypic sequence variation was shown to have biological and clinical significance. The functional significance of sequence variation among HPV 31 variants was studied less intensively. HPV 31 variants belonging to different variant lineages were found to have differences in persistence and in the ability to cause high grade cervical intraepithelial neoplasia. In the present study, we started to explore the functional effects of natural sequence variation of HPV 31 E6 and E7 oncoproteins. The E6 variants were tested for their effects on p53 protein stability and transcriptional activity, while the E7 variants were tested for their effects on pRb protein level and also on the transcriptional activity of E2F transcription factors. HPV 31 E7 variants displayed uniform effects on pRb stability and also on the activity of E2F transcription factors. HPV 31 E6 variants had remarkable differences in the ability to inhibit the trans-activation function of p53 but not in the ability to induce the in vivo degradation of p53. Our results indicate that natural sequence variation of the HPV 31 E6 protein may be involved in the observed differences in the oncogenic potential between HPV 31 variants.

  4. EWS/FLI-1 oncoprotein subtypes impose different requirements for transformation and metastatic activity in a murine model.

    PubMed

    González, Iranzu; Vicent, Silvestre; de Alava, Enrique; Lecanda, Fernando

    2007-09-01

    Ewing sarcoma/primitive neuroectodermal tumors (EWS/PNET) are characterized by specific chromosomal translocations most often generating a chimeric EWS/FLI-1 gene. Depending on the number of juxtaposed exons assembled, several fusion types have been described with different incidences and prognoses. To assess the impact of each fusion type on the specific phenotypic, tumorigenic, and metastatic features of EWS/PNET, we developed an amenable system using a murine mesenchymal multipotent C3H10T1/2 cell line. Upon transduction of EWS/FLI-1, cells acquired dramatic morphological changes in vitro, including a smaller size and "neurite-like" membrane elongations. Chimeric fusion proteins conferred oncogenic properties in vitro, including anchorage-independent growth and an increased rate of proliferation. Furthermore, EWS/FLI-1 expression blocked mineralization, with concomitant repression of osteoblastic genes, and induced a dramatic repression of the adipocytic differentiation program. Moreover, EWS/FLI-1 promoted an aberrant neural phenotype by the de novo expression of specific neural genes. The intramuscular injection of transduced cells led to tumor development and the induction of overt osteolytic lesions. Analogously, to what was observed in human tumors, type 2 EWS/FLI-1 cells formed primary tumors in immunodeficient mice with a higher incidence and a lower latency than cells bearing types 1 and 3 fusions. By contrast, cells expressing types 2 and 3 fusions showed specific metastatic activity with a higher number of macroscopic metastases in soft tissues and osteolytic lesions in the limbs as compared to type-1-expressing cells. Therefore, the structure of each oncoprotein strongly influenced its tumorigenicity and metastagenicity. Thus, this model provides a basis for understanding the genetic determinants involved in Ewing tumor development and metastatic activity and represents a cellular system to analyze other oncoproteins involved in human sarcomagenesis.

  5. Piwil2 is reactivated by HPV oncoproteins and initiates cell reprogramming via epigenetic regulation during cervical cancer tumorigenesis

    PubMed Central

    Feng, Dingqing; Yan, Keqin; Zhou, Ying; Liang, Haiyan; Liang, Jing; Zhao, Weidong; Dong, Zhongjun; Ling, Bin

    2016-01-01

    The human papillomavirus (HPV) oncoproteins E6 and E7 are risk factors that are primarily responsible for the initiation and progression of cervical cancer, and they play a key role in immortalization and transformation by reprogramming differentiating host epithelial cells. It is unclear how cervical epithelial cells transform into tumor-initiating cells (TICs). Here, we observed that the germ stem cell protein Piwil2 is expressed in pre-cancerous and malignant lesions of the cervix and cervical cancer cell lines with the exception of the non-HPV-infected C33a cell line. Knockdown of Piwil2 by shRNA led to a marked reduction in proliferation and colony formation, in vivo tumorigenicity, chemo-resistance, and the proportion of cancer stem-like cells. In contrast, Piwil2 overexpression induced malignant transformation of HaCaT cells and the acquisition of tumor-initiating capabilities. Gene-set enrichment analysis revealed embryonic stem cell (ESC) identity, malignant biological behavior, and specifically, activation targets of the cell reprogramming factors c-Myc, Klf4, Nanog, Oct4, and Sox2 in Piwil2-overexpressing HaCaT cells. We further confirmed that E6 and E7 reactivated Piwil2 and that E6 and E7 overexpression resulted in a similar gene-set enrichment pattern as Piwil2 overexpression in HaCaT cells. Moreover, Piwil2 overexpression or E6 and E7 activation induced H3K9 acetylation but reduced H3K9 trimethylation, which contributed to the epigenetic reprogramming and ESC signature maintenance, as predicted previously. Our study demonstrates that Piwil2, reactivated by the HPV oncoproteins E6 and E7, plays an essential role in the transformation of cervical epithelial cells to TICs via epigenetics-based cell reprogramming. PMID:27602489

  6. Genetic Induction of Cytolytic Susceptibility in Breast Cancer Cells

    DTIC Science & Technology

    2001-07-01

    papillomaviruses (HPV) and adenoviruses (Ad) both transform cells by expressing functionally related oncogenes (Ad-ElA/E1B; HPV-E7/E6), only HPV are... papillomaviruses ; NK cells; E1A; E7. INTRODUCTION ing et al., 1980). The ElA and E7 oncoproteins appear to Human pa pillomavi ruses (HPV) and adenoviruses... papillomaviruses and in transgenic mice expressing the E7 and E6 oncogenes of human carcinomas. Adv Virus Res. 37, 125-171. papillomavirus type 16. J. Viral. 71

  7. Estramustine phosphate reversibly inhibits an early stage during adenovirus replication.

    PubMed

    Everitt, E; Ekstrand, H; Boberg, B; Hartley-Asp, B

    1990-01-01

    Estramustine phosphate, an estradiol-mustard conjugate, was shown to reversibly inhibit a stage during the first hour of productive adenovirus 2 infection of HeLa cells. This drug, employed in the therapy of advanced prostatic cancer, specifically interacts with microtubule-associated proteins (MAPs) of the cytoskeleton. The results obtained under physiological conditions in vivo suggest a MAPs-interference with the microtubule-mediated vectorial migration of the virus inoculum to the nucleus. Virus attachment, uncoating kinetics and the appearance of established uncoating intermediates were not affected.

  8. Boosting oncolytic adenovirus potency with magnetic nanoparticles and magnetic force.

    PubMed

    Tresilwised, Nittaya; Pithayanukul, Pimolpan; Mykhaylyk, Olga; Holm, Per Sonne; Holzmüller, Regina; Anton, Martina; Thalhammer, Stefan; Adigüzel, Denis; Döblinger, Markus; Plank, Christian

    2010-08-02

    Oncolytic adenoviruses rank among the most promising innovative agents in cancer therapy. We examined the potential of boosting the efficacy of the oncolytic adenovirus dl520 by associating it with magnetic nanoparticles and magnetic-field-guided infection in multidrug-resistant (MDR) cancer cells in vitro and upon intratumoral injection in vivo. The virus was complexed by self-assembly with core-shell nanoparticles having a magnetite core of about 10 nm and stabilized by a shell containing 68 mass % lithium 3-[2-(perfluoroalkyl)ethylthio]propionate) and 32 mass % 25 kDa branched polyethylenimine. Optimized virus binding, sufficiently stable in 50% fetal calf serum, was found at nanoparticle-to-virus ratios of 5 fg of Fe per physical virus particle (VP) and above. As estimated from magnetophoretic mobility measurements, 3,600 to 4,500 magnetite nanocrystallites were associated per virus particle. Ultrastructural analysis by electron and atomic force microscopy showed structurally intact viruses surrounded by magnetic particles that occasionally bridged several virus particles. Viral uptake into cells at a given virus dose was enhanced 10-fold compared to nonmagnetic virus when infections were carried out under the influence of a magnetic field. Increased virus internalization resulted in a 10-fold enhancement of the oncolytic potency in terms of the dose required for killing 50% of the target cells (IC(50) value) and an enhancement of 4 orders of magnitude in virus progeny formation at equal input virus doses compared to nonmagnetic viruses. Furthermore, the full oncolytic effect developed within two days postinfection compared with six days in a nonmagnetic virus as a reference. Plotting target cell viability versus internalized virus particles for magnetic and nonmagnetic virus showed that the inherent oncolytic productivity of the virus remained unchanged upon association with magnetic nanoparticles. Hence, we conclude that the mechanism of boosting the

  9. Novel adenovirus detected in kowari (Dasyuroides byrnei) with pneumonia.

    PubMed

    Gál, János; Mándoki, Míra; Sós, Endre; Kertész, Péter; Koroknai, Viktória; Bányai, Krisztián; Farkas, Szilvia L

    2017-02-15

    A male kowari (Dasyuroides byrnei) originating from a zoo facility was delivered for post mortem evaluation in Hungary. Acute lobar pneumonia with histopathologic changes resembling an adenovirus (AdV) infection was detected by light microscopic examination. The presence of an AdV was confirmed by obtaining partial sequence data from the adenoviral DNA-dependent DNA-polymerase. Although the exact taxonomic position of this novel marsupial origin virus could not be determined, pairwise identity analyses and phylogenetic calculations revealed that it is distantly related to other members in the family Adenoviridae.

  10. Packaging capacity and stability of human adenovirus type 5 vectors.

    PubMed Central

    Bett, A J; Prevec, L; Graham, F L

    1993-01-01

    Adenovirus vectors are extensively used for high-level expression of proteins in mammalian cells and are receiving increasing attention for their potential use as live recombinant vaccines and as transducing viruses for use in gene therapy. Although it is commonly argued that one of the chief advantages of adenovirus vectors is their relative stability, this has not been thoroughly investigated. To examine the genetic stability of adenovirus type 5 vectors and in particular to examine the relationship between genetic stability and genome size, adenovirus vectors were constructed with inserts of 4.88 (herpes simplex virus type 1 gB), 4.10 (herpes simplex virus type 1 gB), or 3.82 (LacZ) kb combined with a 1.88-kb E3 deletion or with a newly generated 2.69-kb E3 deletion. The net excess of DNA over the wild-type (wt) genome size ranged from 1.13 to 3.00 kb or 3.1 to 8.3%. Analysis of these vectors during serial passage in tissue culture revealed that when the size exceeded 105% of the wt genome length by approximately 1.2 kb (4.88-kb insert combined with a 1.88-kb deletion), the resulting vector grew very poorly and underwent rapid rearrangement, resulting in loss of the insert after only a few passages. In contrast, vectors with inserts resulting in viral DNA close to or less than a net genome size of 105% of that of the wt grew well and were relatively stable. In general, viruses with genomes only slightly above 105% of that of the wt were unstable and the rapidity with which rearrangement occurred correlated with the size of the insert. These findings suggest that there is a relatively tight constraint on the amount of DNA which can be packaged into virions and that exceeding the limit results in a sharply decreased rate of virus growth. The resultant strong selection for variants which have undergone rearrangement, generating smaller genomes, is manifested as genetic instability of the virus population. Images PMID:8371349

  11. Canine adenovirus type 1 in a fennec fox (Vulpes zerda).

    PubMed

    Choi, Jeong-Won; Lee, Hyun-Kyoung; Kim, Seong-Hee; Kim, Yeon-Hee; Lee, Kyoung-Ki; Lee, Myoung-Heon; Oem, Jae-Ku

    2014-12-01

    A 10-mo-old female fennec fox (Vulpes zerda) with drooling suddenly died and was examined postmortem. Histologic examination of different tissue samples was performed. Vacuolar degeneration and diffuse fatty change were observed in the liver. Several diagnostic methods were used to screen for canine parvovirus, canine distemper virus, canine influenza virus, canine coronavirus, canine parainfluenza virus, and canine adenovirus (CAdV). Only CAdV type 1 (CAdV-1) was detected in several organs (liver, lung, brain, kidney, spleen, and heart), and other viruses were not found. CAdV-1 was confirmed by virus isolation and nucleotide sequencing.

  12. The Length of the Terminal Repetition in Adenovirus-2 DNA

    PubMed Central

    Roberts, Richard J.; Arrand, John R.; Keller, Walter

    1974-01-01

    Adenovirus-2 DNA was end-labeled by partial digestion with Escherichia coli exonuclease III and resynthesis with the DNA polymerase from avian myeloblastosis virus and α-32P-labeled deoxyribonucleoside triphosphates. This end-labeled DNA was cleaved with several specific endonucleases and the terminal fragments were characterized by gel electrophoresis and pyrimidine tract analysis. Two endonucleases gave identical fragments from both ends, presumably from cleavage within the inverted terminal repetition, while all other endonucleases gave dissimilar fragments from the two ends. From the sizes of these fragments it is estimated that the inverted terminal repetition is between 100 and 140 nucleotide pairs long. Images PMID:4139703

  13. Noninvasive visualization of adenovirus replication with a fluorescent reporter in the E3 region.

    PubMed

    Ono, Hidetaka A; Le, Long P; Davydova, Julia G; Gavrikova, Tatyana; Yamamoto, Masato

    2005-11-15

    To overcome the inefficacy and undesirable side effects of current cancer treatment strategies, conditionally replicative adenoviruses have been developed to exploit the unique mechanism of oncolysis afforded by tumor-specific viral replication. Despite rapid translation into clinical trials and the established safety of oncolytic adenoviruses, the in vivo function of these agents is not well understood due to lack of a noninvasive detection system for adenovirus replication. To address this issue, we propose the expression of a reporter from the adenovirus E3 region as a means to monitor replication. Adenovirus replication reporter vectors were constructed with the enhanced green fluorescent protein (EGFP) gene placed in the deleted E3 region under the control of the adenoviral major late promoter while retaining expression of the adenovirus death protein to conserve the native oncolytic capability of the virus. Strong EGFP fluorescence was detected from these vectors in a replication-dependent manner, which correlated with viral DNA replication. Fluorescence imaging in vivo confirmed the ability to noninvasively detect fluorescent signal during replication, which generally corresponded with the underlying level of viral DNA replication. EGFP representation of viral replication was further confirmed by Western blot comparison with the viral DNA content in the tumors. Imaging reporter expression controlled by the adenoviral major late promoter provides a viable approach to noninvasively monitor adenovirus replication in preclinical studies and has the potential for human application with clinically relevant imaging reporters.

  14. Phylogenetic and pathogenic characterization of novel adenoviruses from long-tailed ducks (Clangula hyemalis)

    USGS Publications Warehouse

    Counihan, Katrina; Skerratt, Lee; Franson, J. Christian; Hollmen, Tuula E.

    2015-01-01

    Novel adenoviruses were isolated from a long-tailed duck (Clangula hyemalis) mortality event near Prudhoe Bay, Alaska in 2000. The long-tailed duck adenovirus genome was approximately 27 kb. A 907 bp hexon gene segment was used to design primers specific for the long-tailed duck adenovirus. Nineteen isolates were phylogenetically characterized based on portions of their hexon gene and 12 were most closely related to Goose adenovirus A. The remaining 7 shared no hexon sequences with any known adenoviruses. Experimental infections of mallards with a long-tailed duck reference adenovirus caused mild lymphoid infiltration of the intestine and paint brush hemorrhages of the mucosa and dilation of the intestine. This study shows novel adenoviruses from long-tailed ducks are diverse and provides further evidence that they should be considered in cases of morbidity and mortality in sea ducks. Conserved and specific primers have been developed that will help screen sea ducks for adenoviral infections.

  15. Adenovirus with DNA Packaging Gene Mutations Increased Virus Release

    PubMed Central

    Wechman, Stephen L.; Rao, Xiao-Mei; McMasters, Kelly M.; Zhou, Heshan Sam

    2016-01-01

    Adenoviruses (Ads) have been extensively manipulated for the development of cancer selective replication, leading to cancer cell death or oncolysis. Clinical studies using E1-modified oncolytic Ads have shown that this therapeutic platform was safe, but with limited efficacy, indicating the necessity of targeting other viral genes for manipulation. To improve the therapeutic efficacy of oncolytic Ads, we treated the entire Ad genome repeatedly with UV-light and have isolated AdUV which efficiently lyses cancer cells as reported previously (Wechman, S. L. et al. Development of an Oncolytic Adenovirus with Enhanced Spread Ability through Repeated UV Irradiation and Cancer Selection. Viruses 2016, 8, 6). In this report, we show that no mutations were observed in the early genes (E1 or E4) of AdUV while several mutations were observed within the Ad late genes which have structural or viral DNA packaging functions. This study also reported the increased release of AdUV from cancer cells. In this study, we found that AdUV inhibits tumor growth following intratumoral injection. These results indicate the potentially significant role of the viral late genes, in particular the DNA packaging genes, to enhance Ad oncolysis. PMID:27999391

  16. Fluctuating expression of microRNAs in adenovirus infected cells.

    PubMed

    Zhao, Hongxing; Chen, Maoshan; Tellgren-Roth, Christian; Pettersson, Ulf

    2015-04-01

    The changes in cellular microRNA (miRNA) expression during the course of an adenovirus type 2 infection in human lung fibroblast were studied by deep RNA sequencing. Expressions of 175 miRNAs with over 100 transcripts per million nucleotides were changed more than 1.5-fold. The expression patterns of these miRNAs changed dramatically during the course of the infection, from upregulation of the miRNAs known as tumor suppressors (such as miR-22, miR-320, let-7, miR-181b, and miR-155) and down-regulation of oncogenic miRNAs (such as miR-21 and miR-31) early to downregulation of tumor suppressor miRNAs (such as let-7 family, mir-30 family, 23/27 cluster) and upregulation of oncogenic miRNAs (include miR-125, miR-27, miR-191) late after infection. The switch in miRNA expression pattern occurred when adenovirus DNA replication started. Furthermore, deregulation of cellular miRNA expression was a step-wise and special sets of miRNAs were deregulated in different phases of infection.

  17. Chimpanzee Adenovirus Vaccine Provides Multispecies Protection against Rift Valley Fever

    PubMed Central

    Warimwe, George M.; Gesharisha, Joseph; Carr, B. Veronica; Otieno, Simeon; Otingah, Kennedy; Wright, Danny; Charleston, Bryan; Okoth, Edward; Elena, Lopez-Gil; Lorenzo, Gema; Ayman, El-Behiry; Alharbi, Naif K.; Al-dubaib, Musaad A.; Brun, Alejandro; Gilbert, Sarah C.; Nene, Vishvanath; Hill, Adrian V. S.

    2016-01-01

    Rift Valley Fever virus (RVFV) causes recurrent outbreaks of acute life-threatening human and livestock illness in Africa and the Arabian Peninsula. No licensed vaccines are currently available for humans and those widely used in livestock have major safety concerns. A ‘One Health’ vaccine development approach, in which the same vaccine is co-developed for multiple susceptible species, is an attractive strategy for RVFV. Here, we utilized a replication-deficient chimpanzee adenovirus vaccine platform with an established human and livestock safety profile, ChAdOx1, to develop a vaccine for use against RVFV in both livestock and humans. We show that single-dose immunization with ChAdOx1-GnGc vaccine, encoding RVFV envelope glycoproteins, elicits high-titre RVFV-neutralizing antibody and provides solid protection against RVFV challenge in the most susceptible natural target species of the virus-sheep, goats and cattle. In addition we demonstrate induction of RVFV-neutralizing antibody by ChAdOx1-GnGc vaccination in dromedary camels, further illustrating the potency of replication-deficient chimpanzee adenovirus vaccine platforms. Thus, ChAdOx1-GnGc warrants evaluation in human clinical trials and could potentially address the unmet human and livestock vaccine needs. PMID:26847478

  18. A novel adenovirus of Western lowland gorillas (Gorilla gorilla gorilla).

    PubMed

    Wevers, Diana; Leendertz, Fabian H; Scuda, Nelly; Boesch, Christophe; Robbins, Martha M; Head, Josephine; Ludwig, Carsten; Kühn, Joachim; Ehlers, Bernhard

    2010-11-05

    Adenoviruses (AdV) broadly infect vertebrate hosts including a variety of primates. We identified a novel AdV in the feces of captive gorillas by isolation in cell culture, electron microscopy and PCR. From the supernatants of infected cultures we amplified DNA polymerase (DPOL), preterminal protein (pTP) and hexon gene sequences with generic pan primate AdV PCR assays. The sequences in-between were amplified by long-distance PCRs of 2-10 kb length, resulting in a final sequence of 15.6 kb. Phylogenetic analysis placed the novel gorilla AdV into a cluster of primate AdVs belonging to the species Human adenovirus B (HAdV-B). Depending on the analyzed gene, its position within the cluster was variable. To further elucidate its origin, feces samples of wild gorillas were analyzed. AdV hexon sequences were detected which are indicative for three distinct and novel gorilla HAdV-B viruses, among them a virus nearly identical to the novel AdV isolated from captive gorillas. This shows that the discovered virus is a member of a group of HAdV-B viruses that naturally infect gorillas. The mixed phylogenetic clusters of gorilla, chimpanzee, bonobo and human AdVs within the HAdV-B species indicate that host switches may have been a component of the evolution of human and non-human primate HAdV-B viruses.

  19. Investigation of the mechanical and interfacial properties of adenovirus

    NASA Astrophysics Data System (ADS)

    Matthews, William Garrett

    The ability to investigate materials at the single molecule and macromolecule level became a reality with the introduction of the atomic force microscope (AFM) by Bennig et al. in 1986. Presented in this dissertation is a modification to the AFM that facilitates imaging of delicate samples under liquids. The instrument was subsequently used to investigate a variety of material and interfacial properties of adenovirus. Firstly, the elasticity of adenovirus particles in air and in water was measured. The virus was found to be some fifty fold more compliant in water than in air, with the measured elastic modulus changing from 15 MPa to 770 MPa. Individual viruses also were translated across a variety of surfaces, and the force required to do these manipulations was recorded. A variety of behaviors were observed, including evidence for rolling of the particles. This measurement constitutes one of the first direct observations of rolling behavior on this length scale. Finally, viruses were observed to shed their capsid proteins when deposited on positively charged magnesium intercalated mica. The resulting core structure was investigated, and the uncoating process was captured in a series of images. These images represent one of the first molecular level processes to be observed at the single molecule level.

  20. Chimpanzee Adenovirus Vaccine Provides Multispecies Protection against Rift Valley Fever.

    PubMed

    Warimwe, George M; Gesharisha, Joseph; Carr, B Veronica; Otieno, Simeon; Otingah, Kennedy; Wright, Danny; Charleston, Bryan; Okoth, Edward; Elena, Lopez-Gil; Lorenzo, Gema; Ayman, El-Behiry; Alharbi, Naif K; Al-dubaib, Musaad A; Brun, Alejandro; Gilbert, Sarah C; Nene, Vishvanath; Hill, Adrian V S

    2016-02-05

    Rift Valley Fever virus (RVFV) causes recurrent outbreaks of acute life-threatening human and livestock illness in Africa and the Arabian Peninsula. No licensed vaccines are currently available for humans and those widely used in livestock have major safety concerns. A 'One Health' vaccine development approach, in which the same vaccine is co-developed for multiple susceptible species, is an attractive strategy for RVFV. Here, we utilized a replication-deficient chimpanzee adenovirus vaccine platform with an established human and livestock safety profile, ChAdOx1, to develop a vaccine for use against RVFV in both livestock and humans. We show that single-dose immunization with ChAdOx1-GnGc vaccine, encoding RVFV envelope glycoproteins, elicits high-titre RVFV-neutralizing antibody and provides solid protection against RVFV challenge in the most susceptible natural target species of the virus-sheep, goats and cattle. In addition we demonstrate induction of RVFV-neutralizing antibody by ChAdOx1-GnGc vaccination in dromedary camels, further illustrating the potency of replication-deficient chimpanzee adenovirus vaccine platforms. Thus, ChAdOx1-GnGc warrants evaluation in human clinical trials and could potentially address the unmet human and livestock vaccine needs.

  1. Mucosal vaccination by adenoviruses displaying reovirus sigma 1

    SciTech Connect

    Weaver, Eric A.; Camacho, Zenaido T.; Hillestad, Matthew L.; Crosby, Catherine M.; Turner, Mallory A.; Guenzel, Adam J.; Fadel, Hind J.; Mercier, George T.; Barry, Michael A.

    2015-08-15

    We developed adenovirus serotype 5 (Ad5) vectors displaying the sigma 1 protein from reovirus as mucosal vaccines. Ad5-sigma retargets to JAM-1 and sialic acid, but has 40-fold reduced gene delivery when compared to Ad5. While weaker at transduction, Ad5-sigma generates stronger T cell responses than Ad5 when used for mucosal immunization. In this work, new Ad5-fiber-sigma vectors were generated by varying the number of fiber β-spiral shaft repeats (R) between the fiber tail and sigma. Increasing chimera length led to decreasing insertion of these proteinsAd5 virions. Ad-R3 and R14 vectors effectively targeted JAM-1 in vitro while R20 did not. When wereused to immunize mice by the intranasal route, Ad5-R3-sigma produced higher serum and vaginal antibody responses than Ad5. These data suggest optimized Ad-sigma vectors may be useful vectors for mucosal vaccination. - Highlights: • Constructed adenoviruses (Ads) displaying different reovirus sigma 1 fusion proteins. • Progressively longer chimeras were more poorly encapsidated onto Ad virions. • Ad5-R3-sigma mediated better systemic and mucosal immune responses than Ad5.

  2. Directed adenovirus evolution using engineered mutator viral polymerases

    PubMed Central

    Uil, Taco G.; Vellinga, Jort; de Vrij, Jeroen; van den Hengel, Sanne K.; Rabelink, Martijn J. W. E.; Cramer, Steve J.; Eekels, Julia J. M.; Ariyurek, Yavuz; van Galen, Michiel; Hoeben, Rob C.

    2011-01-01

    Adenoviruses (Ads) are the most frequently used viruses for oncolytic and gene therapy purposes. Most Ad-based vectors have been generated through rational design. Although this led to significant vector improvements, it is often hampered by an insufficient understanding of Ad’s intricate functions and interactions. Here, to evade this issue, we adopted a novel, mutator Ad polymerase-based, ‘accelerated-evolution’ approach that can serve as general method to generate or optimize adenoviral vectors. First, we site specifically substituted Ad polymerase residues located in either the nucleotide binding pocket or the exonuclease domain. This yielded several polymerase mutants that, while fully supportive of viral replication, increased Ad’s intrinsic mutation rate. Mutator activities of these mutants were revealed by performing deep sequencing on pools of replicated viruses. The strongest identified mutators carried replacements of residues implicated in ssDNA binding at the exonuclease active site. Next, we exploited these mutators to generate the genetic diversity required for directed Ad evolution. Using this new forward genetics approach, we isolated viral mutants with improved cytolytic activity. These mutants revealed a common mutation in a splice acceptor site preceding the gene for the adenovirus death protein (ADP). Accordingly, the isolated viruses showed high and untimely expression of ADP, correlating with a severe deregulation of E3 transcript splicing. PMID:21138963

  3. Recombinant adenovirus vectors for gene therapy and clinical trials.

    PubMed

    Nász, I; Adám, E

    2001-01-01

    In the last decade adenovirus (AdV) vectors have emerged as promising technology in gene therapy. They have been used for genetic modification of a variety of somatic cells in vitro and in vivo. They have been widely used as gene delivery vectors in experiments both with curative and preventive purposes. AdV vectors have been used in the experimental and in some extent in the clinical gene therapy of a variety of cancers. The combination of recombinant AdV technology with chemotherapy (pro drug system) seems to be promising, too. AdV vectors offer several advantages over other vectors. Replication defective vectors can be produced in very high titers (10(11) pfu/ml) thus allowing a substantially greater efficiency of direct gene transfer; they have the capacity to infect both replicating and nonreplicating (quiescent) cells from a variety of tissues and species. Several important limitations of adenovirus mediated gene transfer are also known, such as the relatively short-term (transient) expression of foreign genes, induction of the host humoral and cellular immune response to viral proteins and viral infected cells, which may substantially inhibit the effect of repeated treatment with AdV vectors, the limited cloning capacity and the lack of target cell specificity. However, the well-understood structure, molecular biology and host cell interactions of AdV-s offer some potential solutions to these limitations.

  4. Inactivation kinetics of adenovirus serotype 2 with monochloramine.

    PubMed

    Sirikanchana, Kwanrawee; Shisler, Joanna L; Mariñas, Benito J

    2008-03-01

    The effect of pH (6-10), temperature (10-30 degrees C), disinfectant concentration (1-11mg/l as Cl(2)), and ammonia nitrogen-to-chlorine molar ratio (1.3-52) on the inactivation kinetics of adenovirus serotype 2 with monochloramine was investigated by performing batch-reactor experiments with synthetic 0.01M buffer (phosphate or borate) solutions. The inactivation kinetics was independent of monochloramine concentration and ammonia nitrogen-to-chlorine molar ratio but had strong pH dependence, with the rate of inactivation decreasing with increasing pH. The kinetics at pH 6 and 8 were consistent with pseudo-first-order kinetics, while curves at pH 10 were characterized by a lag phase followed by a pseudo-first-order phase. The rate of inactivation increased with increasing temperature-activation energies of 56.5kJ/mole (pH 8) and 72.6kJ/mole (pH 10). The results obtained in this study revealed that monochloramine disinfection might not generally provide adequate control of adenoviruses in drinking water at high pH and low temperature.

  5. Gene Transcript Abundance Profiles Distinguish Kawasaki Disease from Adenovirus Infection

    PubMed Central

    Popper, Stephen J.; Watson, Virginia E.; Shimizu, Chisato; Kanegaye, John T.; Burns, Jane C.; Relman, David A.

    2010-01-01

    Background Acute Kawasaki disease (KD) is difficult to distinguish from other illnesses that involve acute rash or fever, in part because the etiologic agent(s) and pathophysiology remain poorly characterized. As a result, diagnosis and critical therapies may be delayed. Methods We used DNA microarrays to identify possible diagnostic features of KD. We compared gene expression patterns in the blood of 23 children with acute KD and 18 age-matched febrile children with 3 illnesses that resemble KD. Results Genes associated with platelet and neutrophil activation were expressed at higher levels in patients with KD than in patients with acute adenovirus infections or systemic adverse drug reactions, but levels in patients with KD were not higher than those in patients with scarlet fever. Genes associated with B cell activation were also expressed at higher levels in patients with KD than in control subjects. A striking absence of interferon-stimulated gene expression in patients with KD was confirmed in an independent cohort of patients with KD. Using a set of 38 gene transcripts, we successfully predicted the diagnosis for 21 of 23 patients with KD and 7 of 8 patients with adenovirus infection. Conclusions These findings provide insight into the molecular features that distinguish KD from other febrile illnesses and support the feasibility of developing novel diagnostic reagents for KD based on the host response. PMID:19583510

  6. Construction and characterization of recombinant adenovirus carrying a mouse TIGIT-GFP gene.

    PubMed

    Zheng, J M; Cui, J L; He, W T; Yu, D W; Gao, Y; Wang, L; Chen, Z K; Zhou, H M

    2015-12-29

    Recombinant adenovirus vector systems have been used extensively in protein research and gene therapy. However, the construction and characterization of recombinant adenovirus is a tedious and time-consuming process. TIGIT is a recently discovered immunosuppressive molecule that plays an important role in maintaining immunological balance. The construction of recombinant adenovirus mediating TIGIT expression must be simplified to facilitate its use in the study of TIGIT. In this study, the TIGIT gene was combined with green fluorescent protein (GFP); the TIGIT-GFP gene was inserted into a gateway plasmid to construct a TIGIT-GFP adenovirus. HEK 293A cells were infected with the adenovirus, which was then purified and subjected to virus titering. TIGIT-GFP adenovirus was characterized by flow cytometry and immunofluorescence, and its expression in mouse liver was detected by infection through caudal vein injection. The results showed the successful construction of the TIGIT-GFP adenovirus (5 x 10(10) PFU/mL). Co-expression of TIGIT and GFP was identified in 293A and liver cells; synthesis and positioning of TIGIT-GFP was viewed under a fluorescence microscope. TIGIT-GFP was highly expressed on liver cells 1 day (25.53%) after infection and faded 3 days (11.36%) after injection. In conclusion, the fusion of TIGIT with GFP allows easy, rapid, and uncomplicated detection of TIGIT translation. The construction of a TIGIT-GFP adenovirus, mediating TIGIT expression in vitro and in vivo, lays the foundation for further research into TIGIT function and gene therapy. Moreover, the TIGIT-GFP adenovirus is a helpful tool for studying other proteins (which could replace the TIGIT gene).

  7. Infectivity and expression of the early adenovirus proteins are important regulators of wild-type and DeltaE1B adenovirus replication in human cells.

    PubMed

    Steegenga, W T; Riteco, N; Bos, J L

    1999-09-09

    An adenovirus mutant lacking the expression of the large E1B protein (DeltaE1B) has been reported to replicate selectively in cells lacking the expression of functionally wild-type (wt) p53. Based on these results the DeltaE1B or ONYX-015 virus has been proposed to be an oncolytic virus which might be useful to treat p53-deficient tumors. Recently however, contradictory results have been published indicating that p53-dependent cell death is required for productive adenovirus infection. Since there is an urgent need for new methods to treat aggressive, mutant p53-expressing primary tumors and their metastases we carefully examined adenovirus replication in human cells to determine whether or not the DeltaE1B virus can be used for tumor therapy. The results we present here show that not all human tumor cell lines take up adenovirus efficiently. In addition, we observed inhibition of the expression of adenovirus early proteins in tumor cells. We present evidence that these two factors rather than the p53 status of the cell determine whether adenovirus infection results in lytic cell death. Furthermore, the results we obtained by infecting a panel of different tumor cell lines show that viral spread of the DeltaE1B is strongly inhibited in almost all p53-proficient and -deficient cell lines compared to the wt virus. We conclude that the efficiency of the DeltaE1B virus to replicate efficiently in tumor cells is determined by the ability to infect cells and to express the early adenovirus proteins rather than the status of p53.

  8. Molecular characterization of adenovirus circulating in Central and South America during the 2006–2008 period

    PubMed Central

    García, Josefina; Sovero, Merly; Laguna‐Torres, Victor Alberto; Gomez, Jorge; Chicaiza, Wilson; Barrantes, Melvin; Sanchez, Felix; Jimenez, Mirna; Comach, Guillermo; De Rivera, Ivette L.; Agudo, Roberto; Arango, Ana E.; Barboza, Alma; Aguayo, Nicolas; Kochel, Tadeusz J.

    2009-01-01

    Background  Human Adenoviruses are recognized pathogens, causing a broad spectrum of diseases. Serotype identification is critical for epidemiological surveillance, detection of new strains and understanding of HAdvs pathogenesis. Little data is available about HAdvs subtypes in Latin America. Methods  In this study, we have molecularly characterized 213 adenoviruses collected from ILI presenting patients, during 2006‐08, in Central and South America. Results  Our results indicate that 161(76%) adenoviruses belong to subgroup C, 45 (21%) to subgroup B and 7 (3%) to subtype E4. PMID:19903214

  9. Molecular detection of two adenoviruses associated with disease in Australian lizards.

    PubMed

    Hyndman, T; Shilton, C M

    2011-06-01

    We give the first published description of the pathology and molecular findings associated with adenovirus infection in lizards in Australia. A central netted dragon (Ctenophorus nuchalis) exhibited severe necrotising hepatitis with abundant intranuclear inclusion bodies within hepatocytes and rarely within intestinal epithelial cells. Polymerase chain reaction (PCR) using pooled tissues yielded an amplicon that shared strong nucleotide identity with an agamid adenovirus (EU914203). PCR on the liver of a bearded dragon (Pogona minor minor) with illthrift, coccidiosis, nematodiasis and hepatic lipidosis yielded an amplicon with strong nucleotide identity to a helodermatid adenovirus (EU914207).

  10. Aptamer-hybrid nanoparticle bioconjugate efficiently delivers miRNA-29b to non-small-cell lung cancer cells and inhibits growth by downregulating essential oncoproteins

    PubMed Central

    Perepelyuk, Maryna; Maher, Christina; Lakshmikuttyamma, Ashakumary; Shoyele, Sunday A

    2016-01-01

    MicroRNAs (miRNAs) are potentially attractive candidates for cancer therapy. However, their therapeutic application is limited by lack of availability of an efficient delivery system to stably deliver these potent molecules intracellularly to cancer cells while avoiding healthy cells. We developed a novel aptamer-hybrid nanoparticle bioconjugate delivery system to selectively deliver miRNA-29b to MUC1-expressing cancer cells. Significant downregulation of oncoproteins DNMT3b and MCL1 was demonstrated by these MUC1 aptamer-functionalized hybrid nanoparticles in A549 cells. Furthermore, downregulation of these oncoproteins led to antiproliferative effect and induction of apoptosis in a superior version when compared with Lipofectamine 2000. This novel aptamer-hybrid nanoparticle bioconjugate delivery system could potentially serve as a platform for intracellular delivery of miRNAs to cancer cells, hence improving the therapeutic outcome of lung cancer. PMID:27555773

  11. Upregulation of the matrix metalloproteinase-1 gene by the Ewing's sarcoma associated EWS-ER81 and EWS-Fli-1 oncoproteins, c-Jun and p300.

    PubMed

    Fuchs, Bruno; Inwards, Carrie Y; Janknecht, Ralf

    2003-10-09

    The mechanisms of action of Ewing's sarcoma (EWS) associated EWS-ETS oncoproteins have largely remained unresolved. Here, we analyzed how two EWS-ETS proteins, EWS-ER81 and EWS-Fli-1, in vitro activate the matrix metalloproteinase (MMP)-1 promoter that is upregulated in a subset of EWSs. EWS-ER81 and EWS-Fli-1 interact with and thereby activate the MMP-1 promoter, which is potentiated by the cofactor p300 and the proto-oncoprotein c-Jun. Further, EWS-ER81 binds to c-Jun in vitro and in vivo. The interaction between c-Jun, p300 and EWS-ER81 or EWS-Fli-1 may also be relevant to the regulation of other yet-to-be-identified genes that are responsible for EWS formation.

  12. Molecular Characterization of a Lizard Adenovirus Reveals the First Atadenovirus with Two Fiber Genes and the First Adenovirus with Either One Short or Three Long Fibers per Penton

    PubMed Central

    Pénzes, Judit J.; Menéndez-Conejero, Rosa; Condezo, Gabriela N.; Ball, Inna; Papp, Tibor; Doszpoly, Andor; Paradela, Alberto; Pérez-Berná, Ana J.; López-Sanz, María; Nguyen, Thanh H.; van Raaij, Mark J.; Marschang, Rachel E.; Harrach, Balázs; Benkő, Mária

    2014-01-01

    ABSTRACT Although adenoviruses (AdVs) have been found in a wide variety of reptiles, including numerous squamate species, turtles, and crocodiles, the number of reptilian adenovirus isolates is still scarce. The only fully sequenced reptilian adenovirus, snake adenovirus 1 (SnAdV-1), belongs to the Atadenovirus genus. Recently, two new atadenoviruses were isolated from a captive Gila monster (Heloderma suspectum) and Mexican beaded lizards (Heloderma horridum). Here we report the full genomic and proteomic characterization of the latter, designated lizard adenovirus 2 (LAdV-2). The double-stranded DNA (dsDNA) genome of LAdV-2 is 32,965 bp long, with an average G+C content of 44.16%. The overall arrangement and gene content of the LAdV-2 genome were largely concordant with those in other atadenoviruses, except for four novel open reading frames (ORFs) at the right end of the genome. Phylogeny reconstructions and plesiomorphic traits shared with SnAdV-1 further supported the assignment of LAdV-2 to the Atadenovirus genus. Surprisingly, two fiber genes were found for the first time in an atadenovirus. After optimizing the production of LAdV-2 in cell culture, we determined the protein compositions of the virions. The two fiber genes produce two fiber proteins of different sizes that are incorporated into the viral particles. Interestingly, the two different fiber proteins assemble as either one short or three long fiber projections per vertex. Stoichiometry estimations indicate that the long fiber triplet is present at only one or two vertices per virion. Neither triple fibers nor a mixed number of fibers per vertex had previously been reported for adenoviruses or any other virus. IMPORTANCE Here we show that a lizard adenovirus, LAdV-2, has a penton architecture never observed before. LAdV-2 expresses two fiber proteins—one short and one long. In the virion, most vertices have one short fiber, but a few of them have three long fibers attached to the same penton

  13. Nuclear export of cutaneous HPV8 E7 oncoprotein is mediated by a leucine-rich nuclear export signal via a CRM1 pathway

    SciTech Connect

    Onder, Zeynep; Chang, Vivian; Moroianu, Junona

    2015-01-01

    We recently determined that the nuclear import of cutaneous beta genus HPV8 E7 oncoprotein it is mediated by its zinc-binding domain via direct hydrophobic interactions with the FG nucleoporins Nup62 and Nup153 (Onder and Moroianu, 2014). Here we investigated the nuclear export of HPV8 E7 oncoprotein using confocal microscopy after transfections of HeLa cells with EGFP–8cE7 and mutant plasmids and treatment with Ratjadone A nuclear export inhibitor. We determined that HPV8 E7 contains a leucine-rich nuclear export signal (NES), {sub 76}IRTFQELLF{sub 84}, within its zinc-binding domain that mediates its nuclear export via a CRM1 pathway. We found that HPV8 E7 interacts with CRM1 and that the hydrophobic amino acid residues I76, F79 and L82 of the NES are essential for this interaction and for nuclear export of HPV8 E7 oncoprotein. - Highlights: • HPV8 E7 has a leucine-rich NES within its zinc-binding domain that mediates its nuclear export. • CRM1 nuclear export receptor interacts with HPV8 E7 and mediates its export. • Identification of the critical hydrophobic amino acids of the NES of HPV8 E7.

  14. Molecular Detection of Adenoviruses, Rhabdoviruses, and Paramyxoviruses in Bats from Kenya

    PubMed Central

    Conrardy, Christina; Tao, Ying; Kuzmin, Ivan V.; Niezgoda, Michael; Agwanda, Bernard; Breiman, Robert F.; Anderson, Larry J.; Rupprecht, Charles E.; Tong, Suxiang

    2014-01-01

    We screened 217 bats of at least 20 species from 17 locations in Kenya during July and August of 2006 for the presence of adenovirus, rhabdovirus, and paramyxovirus nucleic acids using generic reverse transcription polymerase chain reaction (RT-PCR) and PCR assays. Of 217 bat fecal swabs examined, 4 bats were adenovirus DNA-positive, 11 bats were paramyxovirus RNA-positive, and 2 bats were rhabdovirus RNA-positive. Three bats were coinfected by two different viruses. By sequence comparison and phylogenetic analysis, the Kenya bat paramyxoviruses and rhabdoviruses from this study may represent novel viral lineages within their respective families; the Kenya bat adenoviruses could not be confirmed as novel, because the same region sequences from other known bat adenovirus genomes for comparison were lacking. Our study adds to previous evidence that bats carry diverse, potentially zoonotic viruses and may be coinfected with more than one virus. PMID:24865685

  15. Fatal pulmonary edema in white-tailed deer (Odocoileus virginianus) associated with adenovirus infection.

    PubMed

    Sorden, S D; Woods, L W; Lehmkuhl, H D

    2000-07-01

    Sporadic sudden deaths in adult white-tailed deer occurred from November 1997 through August 1998 on an Iowa game farm. Three of the 4 deer necropsied had severe pulmonary edema, widespread mild lymphocytic vasculitis, and amphophilic intranuclear inclusion bodies in scattered endothelial cells in blood vessels in the lung and abdominal viscera. Immunohistochemistry with bovine adenovirus 5 antisera and transmission electron microscopy demonstrated adenoviral antigen and nucleocapsids, respectively, within endothelial cells. Adenovirus was isolated in cell culture from 1 of the affected deer. The isolate was neutralized by California black-tailed deer adenovirus antiserum. These findings indicate that adenovirus should be considered in the differential diagnosis of both black-tailed and white-tailed deer with pulmonary edema and/or hemorrhagic enteropathy.

  16. Adenovirus fiber disrupts CAR-mediated intercellular adhesion allowing virus escape.

    PubMed

    Walters, Robert W; Freimuth, Paul; Moninger, Thomas O; Ganske, Ingrid; Zabner, Joseph; Welsh, Michael J

    2002-09-20

    Adenovirus binds its receptor (CAR), enters cells, and replicates. It must then escape to the environment to infect a new host. We found that following infection, human airway epithelia first released adenovirus to the basolateral surface. Virus then traveled between epithelial cells to emerge on the apical surface. Adenovirus fiber protein, which is produced during viral replication, facilitated apical escape. Fiber binds CAR, which sits on the basolateral membrane where it maintains tight junction integrity. When fiber bound CAR, it disrupted junctional integrity, allowing virus to filter between the cells and emerge apically. Thus, adenovirus exploits its receptor for two important but distinct steps in its life cycle: entry into host cells and escape across epithelial barriers to the environment.

  17. Liposomal enhancement of the immunogenicity of adenovirus type 5 hexon and fiber vaccines.

    PubMed Central

    Kramp, W J; Six, H R; Drake, S; Kasel, J A

    1979-01-01

    Immunogenicity of adenovirus capsid proteins carried in liposomes was comparable to that with equivalent doses administered in Freund adjuvant, and both forms were more potent than aqueous vaccines. PMID:489132

  18. Adenovirus type 2 expresses fiber in monkey-human hybrids and reconstructed cells

    SciTech Connect

    Zorn, G.A.; Anderson, C.W.

    1981-02-01

    Adenovirus type 2 protein expression was measured by indirect immunofluorescence in monkey-human hybrids and in cells reconstructed from monkey and human cell karyoplasts and cytoplasts. Monkey-human hybrid clones infected with adenovirus type 2 expressed fiber protein, whereas infected monkey cells alone did not. Hybrids constructed after the parental monkey cells were infected with adenovirus type 2 demonstrated that fiber synthesis in these cells could be rescued by fusion to uninfected human cells. Thus, human cells contain a dominant factor that acts in trans and overcomes the inability of monkey cells to synthesize fiber. These results are consistent with the hypothesis that the block to adenovirus replication in monkey cells involves a nuclear event that prevents the formation of functional mRNA for some late viral proteins including fiber polypeptide.

  19. Identification and purification of a protein encoded by the human adenovirus type 2 transforming region.

    PubMed Central

    Green, M; Brackmann, K H; Cartas, M A; Matsuo, T

    1982-01-01

    The human adenovirus type 2 (Ad2) transforming genes are located in early regions E1a (map position 1.3 to 4.5) and E1b (map position 4.6 to 11.2). We have identified and purified to near homogeneity a major 20,000-molecular-weight (20K) protein and have shown that it is coded by E1b. Using an Ad2-transformed cell antiserum which contained antibody to E1b-coded proteins, we immunoprecipitated 53K and 19K proteins from the nucleoplasm and 53K, 19K, and 20K proteins from the cytoplasmic S-100 fraction of Ad2 productively infected and Ad2-transformed cells. The 19K protein was present in both the nucleoplasm and the cytoplasm, whereas the 20K protein was found only in the cytoplasm. The 53K and 19K proteins are known Ad2 E1b-coded proteins. The 20K protein was purified to near homogeneity in 20 to 50% yields by sequential DEAE-Sephacel chromatography and reverse-phase high-performance liquid chromatography. Purified 20K protein shares most of its methionine-labeled tryptic peptides with E1b-53K, as shown by reverse-phase high-performance liquid chromatography, and therefore is closely related to the 53K protein. The 19K protein does not appear to share tryptic peptides with either 20K or 53K protein. To provide more direct evidence that 20K protein is virus-coded, we translated E1b-specific mRNA in vitro. Both immunoprecipitation analysis and high-performance liquid chromatography purification of the translated product identified a 20K protein that has the same tryptic peptides as the 20K protein isolated from infected and from transformed cells. These findings suggest that the Ad2 20K protein is a primary translation product of an Ad2 E1b mRNA. Images PMID:7045392

  20. Inhibition of Adenovirus In Vitro DNA Replication by Vesicular Stomatitis Virus Leader RNA

    DTIC Science & Technology

    1986-08-18

    19 Inhibition of Macromolecular Synthesis •••••••••••••••••••••••••••• 22 Adenovirus Structure and Life Cycle...be possible to determine the affects of VSV and VSV leader RNA on eukaryotic DNA synthesis. ; . 1 .· 29 Adenovirus Structure and Life Cycle

  1. Ganciclovir Inhibits Human Adenovirus Replication and Pathogenicity in Permissive Immunosuppressed Syrian Hamsters

    PubMed Central

    Ying, Baoling; Tollefson, Ann E.; Spencer, Jacqueline F.; Balakrishnan, Lata; Dewhurst, Stephen; Capella, Cristina; Buller, R. Mark L.; Wold, William S. M.

    2014-01-01

    Adenovirus infections of immunocompromised patients can develop into deadly multiorgan or systemic disease. The virus is especially threatening for pediatric allogeneic hematopoietic stem cell transplant recipients; according to some studies, 10% or more of these patients succumb to disease resulting from adenovirus infection. At present, there is no drug approved for the treatment or prevention of adenovirus infections. Compounds that are approved to treat other virus infections are used off-label to combat adenovirus, but only anecdotal evidence of the efficacy of these drugs exists. Ganciclovir, a drug approved for the treatment of herpesvirus infection, was previously reported to be effective against human adenoviruses in vitro. To model adenovirus infections in immunocompromised humans, we examined ganciclovir's efficacy in immunosuppressed Syrian hamsters intravenously infected with type 5 human adenovirus (Ad5). This animal model is permissive for Ad5 replication, and the animals develop symptoms similar to those seen in humans. We demonstrate that ganciclovir suppresses Ad5 replication in the liver of infected hamsters and that it mitigates the consequences of Ad5 infections in these animals when administered prophylactically or therapeutically. We show that ganciclovir inhibits Ad5 DNA synthesis and late gene expression. The mechanism of action for the drug is not clear; preliminary data suggest that it exerts its antiadenoviral effect by directly inhibiting the adenoviral DNA polymerase. While more extensive studies are required, we believe that ganciclovir is a promising drug candidate to treat adenovirus infections. Brincidofovir, a drug with proven activity against Ad5, was used as a positive control in the prophylactic experiment. PMID:25224011

  2. Ganciclovir inhibits human adenovirus replication and pathogenicity in permissive immunosuppressed Syrian hamsters.

    PubMed

    Ying, Baoling; Tollefson, Ann E; Spencer, Jacqueline F; Balakrishnan, Lata; Dewhurst, Stephen; Capella, Cristina; Buller, R Mark L; Toth, Karoly; Wold, William S M

    2014-12-01

    Adenovirus infections of immunocompromised patients can develop into deadly multiorgan or systemic disease. The virus is especially threatening for pediatric allogeneic hematopoietic stem cell transplant recipients; according to some studies, 10% or more of these patients succumb to disease resulting from adenovirus infection. At present, there is no drug approved for the treatment or prevention of adenovirus infections. Compounds that are approved to treat other virus infections are used off-label to combat adenovirus, but only anecdotal evidence of the efficacy of these drugs exists. Ganciclovir, a drug approved for the treatment of herpesvirus infection, was previously reported to be effective against human adenoviruses in vitro. To model adenovirus infections in immunocompromised humans, we examined ganciclovir's efficacy in immunosuppressed Syrian hamsters intravenously infected with type 5 human adenovirus (Ad5). This animal model is permissive for Ad5 replication, and the animals develop symptoms similar to those seen in humans. We demonstrate that ganciclovir suppresses Ad5 replication in the liver of infected hamsters and that it mitigates the consequences of Ad5 infections in these animals when administered prophylactically or therapeutically. We show that ganciclovir inhibits Ad5 DNA synthesis and late gene expression. The mechanism of action for the drug is not clear; preliminary data suggest that it exerts its antiadenoviral effect by directly inhibiting the adenoviral DNA polymerase. While more extensive studies are required, we believe that ganciclovir is a promising drug candidate to treat adenovirus infections. Brincidofovir, a drug with proven activity against Ad5, was used as a positive control in the prophylactic experiment.

  3. Unambiguous typing of canine adenovirus isolates by deoxyribonucleic acid restriction-endonuclease analysis.

    PubMed Central

    Assaf, R; Marsolais, G; Yelle, J; Hamelin, C

    1983-01-01

    Viral deoxyribonucleic acid extracted from a limited number of cells infected with canine adenovirus type 1 or type 2 was cleaved with several restriction endonucleases. Agarose gel electrophoresis of the limit digests showed stable differences between the canine adenovirus type 1 and type 2 cleavage patterns. Rapid and accurate typing of large numbers of clinical isolates may thus be done by deoxyribonucleic acid restriction-endonuclease analysis. Images Fig. 1. Fig. 2. PMID:6321002

  4. Adenovirus type 2 nuclear RNA accumulating during productive infection.

    PubMed Central

    Bachenheimer, S L

    1977-01-01

    The viral-specific nuclear RNA which accumulates early and late during productive infection of HeLa cells by adenovirus-type 2 (Ad2) has been characterized with respect to its size and stability after denaturation by Me2SO. Early nuclear transcripts, under nondenaturing conditions, sediment in the range 28 to 45S, but treatment with Me2SO prior to sedimentation results in a shift to about 20S. Later nuclear RNA accumulates as a composite of two populations of molecules: one with a broad size distribution centering on 45S under nondenaturing conditions and less than 32S after denaturation and a second having a narrow size distribution around 35S which is quite stable to Me2SO. Analysis of late RNA by hybridization to Sma fragments of Ad2 DNA suggests that the 35S RNA species is derived from a limited portion of the left half of the viral genome. PMID:864839

  5. Going viral: a review of replication-selective oncolytic adenoviruses

    PubMed Central

    Larson, Christopher; Oronsky, Bryan; Scicinski, Jan; Fanger, Gary R.; Stirn, Meaghan; Oronsky, Arnold; Reid, Tony R.

    2015-01-01

    Oncolytic viruses have had a tumultuous course, from the initial anecdotal reports of patients having antineoplastic effects after natural viral infections a century ago to the development of current cutting-edge therapies in clinical trials. Adenoviruses have long been the workhorse of virotherapy, and we review both the scientific and the not-so-scientific forces that have shaped the development of these therapeutics from wild-type viral pathogens, turning an old foe into a new friend. After a brief review of the mechanics of viral replication and how it has been modified to engineer tumor selectivity, we give particular attention to ONYX-015, the forerunner of virotherapy with extensive clinical testing that pioneered the field. The findings from those as well as other oncolytic trials have shaped how we now view these viruses, which our immune system has evolved to vigorously attack, as promising immunotherapy agents. PMID:26280277

  6. Coxsackievirus and adenovirus receptor is essential for cardiomyocyte development.

    PubMed

    Asher, Damon R; Cerny, Anna M; Weiler, Sarah R; Horner, James W; Keeler, Marilyn L; Neptune, Mychell A; Jones, Stephen N; Bronson, Roderick T; Depinho, Ronald A; Finberg, Robert W

    2005-06-01

    The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein that is known to be a site of viral attachment and entry, but its physiologic functions are undefined. CAR expression is maximal in neonates and wanes rapidly after birth in organs such as heart, muscle, and brain, suggesting that CAR plays a role in the development of these tissues. Here, we show that CAR deficiency resulted in an embryonic lethal condition associated with cardiac defects. Specifically, commencing approximately 10.5 days postconception (dpc), CAR-/- cardiomyocytes exhibited regional apoptosis evidenced by both histopathologic features of cell death and positive staining for the apoptotic marker cleaved caspase 3. CAR-/- fetuses invariably suffered from degeneration of the myocardial wall and thoracic hemorrhaging, leading to death by 11.5 dpc. These findings are consistent with the view that CAR provides positive survival signals to cardiomyocytes that are essential for normal heart development.

  7. Oncolytic adenoviruses: A thorny path to glioma cure

    PubMed Central

    Ulasov, I.V.; Borovjagin, A.V.; Schroeder, B.A.; Baryshnikov, A.Y.

    2014-01-01

    Glioblastoma Multiforme (GBM) is a rapidly progressing brain tumor. Despite the relatively low percentage of cancer patients with glioma diagnoses, recent statistics indicate that the number of glioma patients may have increased over the past decade. Current therapeutic options for glioma patients include tumor resection, chemotherapy, and concomitant radiation therapy with an average survival of approximately 16 months. The rapid progression of gliomas has spurred the development of novel treatment options, such as cancer gene therapy and oncolytic virotherapy. Preclinical testing of oncolytic adenoviruses using glioma models revealed both positive and negative sides of the virotherapy approach. Here we present a detailed overview of the glioma virotherapy field and discuss auxiliary therapeutic strategies with the potential for augmenting clinical efficacy of GBM virotherapy treatment. PMID:25685829

  8. Adenovirus Membrane Penetration: Tickling the Tail of a Sleeping Dragon

    PubMed Central

    Wiethoff, Christopher M.; Nemerow, Glen R.

    2015-01-01

    As is the case for nearly every viral pathogen, non-enveloped viruses (NEV) must maintain their integrity under potentially harsh environmental conditions while retaining the ability to undergo rapid disassembly at the right time and right place inside host cells. NEVs generally exist in this metastable state until they encounter key cellular stimuli such as membrane receptors, decreased intracellular pH, digestion by cellular proteases, or a combination of these factors. These stimuli trigger conformational changes in the viral capsid that exposes a sequestered membrane-perturbing protein. This protein subsequently modifies the cell membrane in such a way as to allow passage of the virion and accompanying nucleic acid payload into the cell cytoplasm. Different NEVs employ variations of this general pathway for cell entry (1), however this review will focus on significant new knowledge obtained on cell entry by human adenovirus(HAdV). PMID:25798531

  9. Partial characterization of new adenoviruses found in lizards.

    PubMed

    Ball, Inna; Behncke, Helge; Schmidt, Volker; Geflügel, F T A; Papp, Tibor; Stöhr, Anke C; Marschang, Rachel E

    2014-06-01

    In the years 2011-2012, a consensus nested polymerase chain reaction was used for the detection of adenovirus (AdV) infection in reptiles. During this screening, three new AdVs were detected. One of these viruses was detected in three lizards from a group of green striped tree dragons (Japalura splendida). Another was detected in a green anole (Anolis carolinensis). A third virus was detected in a Jackson's chameleon (Chamaeleo jacksonii). Analysis of a portion of the DNA-dependent DNA polymerase genes of each of these viruses revealed that they all were different from one another and from all previously described reptilian AdVs. Phylogenetic analysis of the partial DNA polymerase gene sequence showed that all newly detected viruses clustered within the genus Atadenovirus. This is the first description of AdVs in these lizard species.

  10. Mucosal vaccination by adenoviruses displaying reovirus sigma 1.

    PubMed

    Weaver, Eric A; Camacho, Zenaido T; Hillestad, Matthew L; Crosby, Catherine M; Turner, Mallory A; Guenzel, Adam J; Fadel, Hind J; Mercier, George T; Barry, Michael A

    2015-08-01

    We developed adenovirus serotype 5 (Ad5) vectors displaying the sigma 1 protein from reovirus as mucosal vaccines. Ad5-sigma retargets to JAM-1 and sialic acid, but has 40-fold reduced gene delivery when compared to Ad5. While weaker at transduction, Ad5-sigma generates stronger T cell responses than Ad5 when used for mucosal immunization. In this work, new Ad5-fiber-sigma vectors were generated by varying the number of fiber β-spiral shaft repeats (R) between the fiber tail and sigma. Increasing chimera length led to decreasing insertion of these proteinsAd5 virions. Ad-R3 and R14 vectors effectively targeted JAM-1 in vitro while R20 did not. When wereused to immunize mice by the intranasal route, Ad5-R3-sigma produced higher serum and vaginal antibody responses than Ad5. These data suggest optimized Ad-sigma vectors may be useful vectors for mucosal vaccination.

  11. Adenovirus receptors and their implications in gene delivery

    PubMed Central

    Sharma, Anurag; Li, Xiaoxin; Bangari, Dinesh S.; Mittal, Suresh K.

    2010-01-01

    Adenoviruses (Ads) have gained popularity as gene delivery vectors for therapeutic and prophylactic applications. Ad entry into host cells involves specific interactions between cell surface receptors and viral capsid proteins. Several cell surface molecules have been identified as receptors for Ad attachment and entry. Tissue tropism of Ad vectors is greatly influenced by their receptor usage. A variety of strategies have been investigated to modify Ad vector tropism by manipulating the receptor-interacting moieties. Many such strategies are aimed at targeting and/or detargeting of Ad vectors. In this review, we discuss the various cell surface molecules that are implicated as receptors for virus attachment and internalization. Special emphasis is given to Ad types that are utilized as gene delivery vectors. Various strategies to modify Ad tropism using the knowledge of Ad receptors are also discussed. PMID:19647886

  12. Adenovirus-mediated gene transfer to tumor cells.

    PubMed

    Cascalló, Manel; Alemany, Ramon

    2004-01-01

    Cell transduction in vitro is only the first step toward proving that a genetherapy vector can be useful to treat tumors. However, tumor targeting in vivo is now the milestone for gene therapy to succeed against disseminated cancer. Therefore, most valuable information is obtained from studies of vector biodistribution. Owing to the hepatotropism of adenoviral vectors, a particularly important parameter is the tumor/liver ratio. This ratio can be given at the level of gene expression if the amount of transgene expression is measured. To optimize the targeting, however, the levels of viral particles that reach the tumor compared to other organs must be studied. Most of this chapter deals with methods to quantify the virus fate in tumor-bearing animals. We present a radioactive labeling method that can be used to study biodistribution. After a small section dealing with tumor models, we describe methods to quantify different parameters related to adenovirus-mediated tumor targeting.

  13. Role of preterminal protein processing in adenovirus replication.

    PubMed Central

    Webster, A; Leith, I R; Nicholson, J; Hounsell, J; Hay, R T

    1997-01-01

    Preterminal protein (pTP), the protein primer for adenovirus DNA replication, is processed at two sites by the virus-encoded protease to yield mature terminal protein (TP). Here we demonstrate that processing to TP, via an intermediate (iTP), is conserved in all serotypes sequenced to date; and in determining the sites cleaved in Ad4 pTP, we extend the previously published substrate specificity of human adenovirus proteases to include a glutamine residue at P4. Furthermore, using monoclonal antibodies raised against pTP, we show that processing to iTP and TP are temporally separated in the infectious cycle, with processing to iTP taking place outside the virus particles. In vitro and in vivo studies of viral DNA replication reveal that iTP can act as a template for initiation and elongation and argue against a role for virus-encoded protease in switching off DNA replication. Virus DNA with TP attached to its 5' end (TP-DNA) has been studied extensively in in vitro DNA replication assays. Given that in vivo pTP-DNA, not TP-DNA, is the template for all but the first round of replication, the two templates were compared in vitro and shown to have different properties. Immunofluorescence studies suggest that a region spanning the TP cleavage site is involved in defining the subnuclear localization of pTP. Therefore, a likely role for the processing of pTP-DNA is to create a distinct template for early transcription (TP-DNA), while the terminal protein moiety, be it TP or pTP, serves to guide the template to the appropriate subcellular location through the course of infection. PMID:9261355

  14. Role of preterminal protein processing in adenovirus replication.

    PubMed

    Webster, A; Leith, I R; Nicholson, J; Hounsell, J; Hay, R T

    1997-09-01

    Preterminal protein (pTP), the protein primer for adenovirus DNA replication, is processed at two sites by the virus-encoded protease to yield mature terminal protein (TP). Here we demonstrate that processing to TP, via an intermediate (iTP), is conserved in all serotypes sequenced to date; and in determining the sites cleaved in Ad4 pTP, we extend the previously published substrate specificity of human adenovirus proteases to include a glutamine residue at P4. Furthermore, using monoclonal antibodies raised against pTP, we show that processing to iTP and TP are temporally separated in the infectious cycle, with processing to iTP taking place outside the virus particles. In vitro and in vivo studies of viral DNA replication reveal that iTP can act as a template for initiation and elongation and argue against a role for virus-encoded protease in switching off DNA replication. Virus DNA with TP attached to its 5' end (TP-DNA) has been studied extensively in in vitro DNA replication assays. Given that in vivo pTP-DNA, not TP-DNA, is the template for all but the first round of replication, the two templates were compared in vitro and shown to have different properties. Immunofluorescence studies suggest that a region spanning the TP cleavage site is involved in defining the subnuclear localization of pTP. Therefore, a likely role for the processing of pTP-DNA is to create a distinct template for early transcription (TP-DNA), while the terminal protein moiety, be it TP or pTP, serves to guide the template to the appropriate subcellular location through the course of infection.

  15. Adenoviruses in Lymphocytes of the Human Gastro-Intestinal Tract

    PubMed Central

    Roy, Soumitra; Calcedo, Roberto; Medina-Jaszek, Angelica; Keough, Martin; Peng, Hui; Wilson, James M.

    2011-01-01

    Objective Persistent adenoviral shedding in stools is known to occur past convalescence following acute adenoviral infections. We wished to establish the frequency with which adenoviruses may colonize the gut in normal human subjects. Methods The presence of adenoviral DNA in intestinal specimens obtained at surgery or autopsy was tested using a nested PCR method. The amplified adenoviral DNA sequences were compared to each other and to known adenoviral species. Lamina propria lymphocytes (LPLs) were isolated from the specimens and the adenoviral copy numbers in the CD4+ and CD8+ fractions were determined by quantitative PCR. Adenoviral gene expression was tested by amplification of adenoviral mRNA. Results Intestinal tissue from 21 of 58 donors and LPLs from 21 of 24 donors were positive for the presence of adenoviral DNA. The majority of the sequences could be assigned to adenoviral species E, although species B and C sequences were also common. Multiple sequences were often present in the same sample. Forty-one non-identical sequences were identified from 39 different tissue donors. Quantitative PCR for adenoviral DNA in CD4+ and CD8+ fractions of LPLs showed adenoviral DNA to be present in both cell types and ranged from a few hundred to several million copies per million cells on average. Active adenoviral gene expression as evidenced by the presence of adenoviral messenger RNA in intestinal lymphocytes was demonstrated in 9 of the 11 donors tested. Conclusion Adenoviral DNA is highly prevalent in lymphocytes from the gastro-intestinal tract indicating that adenoviruses may be part of the normal gut flora. PMID:21980361

  16. Use of cidofovir in pediatric patients with adenovirus infection

    PubMed Central

    Ganapathi, Lakshmi; Arnold, Alana; Jones, Sarah; Patterson, Al; Graham, Dionne; Harper, Marvin; Levy, Ofer

    2016-01-01

    Background: Adenoviruses contribute to morbidity and mortality among immunocompromised pediatric patients including stem cell and solid organ transplant recipients. Cidofovir (CDV), an antiviral compound approved by the FDA in 1996, is used for treatment of adenoviral (ADV) infections in immunocompromised patients despite concern of potential nephrotoxicity.   Methods: We conducted a retrospective 5-year review at Boston Children’s Hospital of 16 patients (mean age = 6.5 years) receiving 19 courses of CDV. During therapy all pertinent data elements were reviewed to characterize potential response to therapy and incidence of renal dysfunction.   Results: Of the 19 CDV courses prescribed, 16 courses (84%) were in patients who had a positive blood ADV Polymerase chain reaction (PCR) alone or in combination with positive ADV PCR/ Direct Immunofluorescence Assay (DFA) at another site. Respiratory symptoms with or without pneumonia were the most common presentation (10/19, 53%). In the majority of blood positive courses (10/16, 63%), viral clearance was also accompanied by clinical response. This was not the case in four courses where patients expired despite viral clearance, including one in which death was directly attributable to adenovirus. There was reversible renal dysfunction observed during the use of CDV. Conclusions:  CDV appeared safe and reasonably tolerated for treatment of ADV in this pediatric population and was associated with viral response and clinical improvement in the majority of patients but reversible renal dysfunction was a side effect. Further studies of the efficacy of CDV for immunocompromised children with ADV infection are warranted. PMID:27239277

  17. Adenovirus Vectors Targeting Distinct Cell Types in the Retina

    PubMed Central

    Sweigard, J. Harry; Cashman, Siobhan M.

    2010-01-01

    Purpose. Gene therapy for a number of retinal diseases necessitates efficient transduction of photoreceptor cells. Whereas adenovirus (Ad) serotype 5 (Ad5) does not transduce photoreceptors efficiently, previous studies have demonstrated improved photoreceptor transduction by Ad5 pseudotyped with Ad35 (Ad5/F35) or Ad37 (Ad5/F37) fiber or by the deletion of the RGD domain in the Ad5 penton base (Ad5ΔRGD). However, each of these constructs contained a different transgene cassette, preventing the evaluation of the relative performance of these vectors, an important consideration before the use of these vectors in the clinic. The aim of this study was to evaluate these vectors in the retina and to attempt photoreceptor-specific transgene expression. Methods. Three Ad5-based vectors containing the same expression cassette were generated and injected into the subretinal space of adult mice. Eyes were analyzed for green fluorescence protein expression in flat-mounts, cross-sections, quantitative RT-PCR, and a modified stereological technique. A 257-bp fragment derived from the mouse opsin promoter was analyzed in the context of photoreceptor-specific transgene expression. Results. Each virus tested efficiently transduced the retinal pigment epithelium. The authors found no evidence that Ad5/F35 or Ad5/F37 transduced photoreceptors. Instead, they found that Ad5/F37 transduced Müller cells. Robust photoreceptor transduction by Ad5ΔRGD was detected. Photoreceptor-specific transgene expression from the 257-bp mouse opsin promoter in the context of Ad5ΔRGD vectors was found. Conclusions. Adenovirus vectors may be designed with tropism to distinct cell populations. Robust photoreceptor-specific transgene expression can be achieved in the context of Ad5ΔRGD vectors. PMID:19892875

  18. Replication-competent human adenovirus 11p vectors can propagate in Vero cells

    SciTech Connect

    Gokumakulapalle, Madhuri; Mei, Ya-Fang

    2016-08-15

    The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus 11p (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were as capable as carcinoma cells at propagating full-length replication-competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields. - Highlights: • Africa green monkey cell lines were monitored for human adenovirus 11p GFP vector infection. • Human CD46 molecules were detectable in these monkey cell lines. • Adenovirus 11p GFP vector can be propagated in Vero cells increases the safety of Ad11p-based vectors for clinical trials. • To use Vero cells for preparation of Ad11p vector avoids the potential inclusion of oncogenes from tumor cells.

  19. Isolation of a novel adenovirus from California sea lions Zalophus californianus.

    PubMed

    Goldstein, T; Colegrove, K M; Hanson, M; Gulland, F M D

    2011-05-09

    Viral hepatitis associated with adenoviral infection has been reported in California sea lions Zalophus californianus admitted to rehabilitation centers along the California coast since the 1970s. Canine adenovirus 1 (CAdV-1) causes viral hepatitis in dogs and infects a number of wildlife species. Attempts to isolate the virus from previous sea lion hepatitis cases were unsuccessful, but as the hepatitis had morphologic features resembling canine infectious hepatitis, and since the virus has a wide host range, it was thought that perhaps the etiologic agent was CAdV-1. Here, we identify a novel adenovirus in 2 stranded California sea lions and associate the infection with viral hepatitis and endothelial cell infection. Phylogenetic analysis confirmed the classification of the sea lion adenovirus in the Mastadenovirus genus with the most similarity to tree shrew adenovirus 1 (TSAdV-1, 77%). However, as the sea lion adenovirus appeared to be equally distant from the other Mastadenovirus species based on phylogenetic analysis, results indicate that it represents an independent lineage and species. Although sequences from this novel virus, otarine adenovirus 1 (OtAdV-1), show some similarity to CAdV-1 and 2, it is clearly distinct and likely the cause of the viral hepatitis in the stranded California sea lions.

  20. Replication of adenovirus type 4 DNA by a purified fraction from infected cells.

    PubMed Central

    Temperley, S M; Hay, R T

    1991-01-01

    An extract from Adenovirus type 4 infected HeLa cells was fractionated by ion-exchange and DNA affinity chromatography. One fraction, which bound tightly to single stranded DNA, contained predominantly a protein of apparent molecular weight 65,000 and three less abundant proteins. Immunological cross-reactivity with adenovirus type 2 proteins confirmed the presence of preterminal protein and indicated that the abundant species was the virus coded DNA binding protein. This fraction contained an aphidicolin resistant DNA polymerase activity and in the presence of a linearised plasmid containing the adenovirus type 4 origin of DNA replication efficient transfer of dCMP onto preterminal protein, indicative of initiation, was observed. Furthermore, addition of all four deoxyribonucleotide triphosphates and an ATP regenerating system resulted in the elongation of initiated molecules to generate plasmid molecules covalently attached to preterminal protein. Adenovirus type 4 DNA binding protein was extensively purified from crude adenovirus-4 infected HeLa extract by immunoaffinity chromatography using a monoclonal antibody raised against adenovirus type 2 DNA binding protein. A low level of initiation of DNA replication was detected in the fraction depleted of DNA binding protein but activity was restored by addition of purified DNA binding protein. DNA binding protein therefore plays an important role in the initiation of Ad4 DNA replication. Images PMID:1829516

  1. TheQ1 Influence of Innate and Pre-Existing Immunity on Adenovirus Therapy

    PubMed Central

    Zaiss, Anne K.; Machado, Hidevaldo B.; Herschman, Harvey R.

    2010-01-01

    Recombinant adenovirus serotype 5 (Ad5) vectors have been studied extensively in preclinical gene therapy models and in a range of clinical trials. However, innate immune responses to adenovirus vectors limit effectiveness of Ad5 based therapies. Moreover, extensive pre-existing Ad5 immunity in human populations will likely limit the clinical utility of adenovirus vectors, unless methods to circumvent neutralizing antibodies that bind virus and block target cell transduction can be developed; Furthermore, memory T cell and humoral responses to Ad5 are associated with increased toxicity, raising safety concerns for therapeutic adenovirus vectors in immunized hosts. Most preclinical studies have been performed in naïve animals; although pre-existing immunity is among the greatest hurdles for adenovirus therapies, it is also one of the most neglected experimentally. Here we summarize findings using adenovirus vectors in naïve animals, in Ad-immunized animals and in clinical trials, and review strategies proposed to overcome innate immune responses and pre-existing immunity. PMID:19711370

  2. Direct selection of targeted adenovirus vectors by random peptide display on the fiber knob.

    PubMed

    Miura, Y; Yoshida, K; Nishimoto, T; Hatanaka, K; Ohnami, S; Asaka, M; Douglas, J T; Curiel, D T; Yoshida, T; Aoki, K

    2007-10-01

    Targeting of gene transfer at the level of cell entry is one of the most attractive challenges in vector development. However, attempts to redirect adenovirus vectors to alternative receptors by engineering the capsid-coding region have shown limited success because proper targeting ligand-receptor systems on the cells of interest are generally unknown. Systematic approaches to generate adenovirus vectors targeting any given cell type need to be developed to achieve this goal. Here, we constructed an adenovirus library that was generated by a Cre-lox-mediated in vitro recombination between an adenoviral fiber-modified plasmid library and genomic DNA to display random peptides on a fiber knob. As proof of concept, we screened the adenovirus display library on a glioma cell line and observed selection of several particular peptide sequences. The targeted vector carrying the most frequently isolated peptide significantly enhanced gene transduction in the glioma cell line but not in many other cell lines. Because the insertion of a pre-selected peptide into a fiber knob often fails to generate an adenovirus vector, the selection of targeting peptides is highly useful in the context of the adenoviral capsid. This vector-screening system can facilitate the development of a targeted adenovirus vector for a variety of applications in medicine.

  3. Adenovirus Vectors Target Several Cell Subtypes of Mammalian Inner Ear In Vivo

    PubMed Central

    Li, Wenyan; Shen, Jun

    2016-01-01

    Mammalian inner ear harbors diverse cell types that are essential for hearing and balance. Adenovirus is one of the major vectors to deliver genes into the inner ear for functional studies and hair cell regeneration. To identify adenovirus vectors that target specific cell subtypes in the inner ear, we studied three adenovirus vectors, carrying a reporter gene encoding green fluorescent protein (GFP) from two vendors or with a genome editing gene Cre recombinase (Cre), by injection into postnatal days 0 (P0) and 4 (P4) mouse cochlea through scala media by cochleostomy in vivo. We found three adenovirus vectors transduced mouse inner ear cells with different specificities and expression levels, depending on the type of adenoviral vectors and the age of mice. The most frequently targeted region was the cochlear sensory epithelium, including auditory hair cells and supporting cells. Adenovirus with GFP transduced utricular supporting cells as well. This study shows that adenovirus vectors are capable of efficiently and specifically transducing different cell types in the mammalian inner ear and provides useful tools to study inner ear gene function and to evaluate gene therapy to treat hearing loss and vestibular dysfunction. PMID:28116172

  4. Construction of adenovirus vectors encoding the lumican gene by gateway recombinant cloning technology

    PubMed Central

    Wang, Gui-Fang; Qi, Bing; Tu, Lei-Lei; Liu, Lian; Yu, Guo-Cheng; Zhong, Jing-Xiang

    2016-01-01

    AIM To construct adenovirus vectors of lumican gene by gateway recombinant cloning technology to further understand the role of lumican gene in myopia. METHODS Gateway recombinant cloning technology was used to construct adenovirus vectors. The wild-type (wt) and mutant (mut) forms of the lumican gene were synthesized and amplified by polymerase chain reaction (PCR). The lumican cDNA fragments were purified and ligated into the adenovirus shuttle vector pDown-multiple cloning site (MCS)-/internal ribozyme entry site (IRES)/enhanced green fluorescent protein (EGFP). Then the desired DNA fragments were integrated into the destination vector pAV.Des1d yielding the final expression constructs pAV.Ex1d-cytomegalovirus (CMV)>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES /EGFP, respectively. RESULTS The adenovirus plasmids pAV.Ex1d-CMV>wt-lumican/IRES/EGFP and pAV.Ex1d-CMV>mut-lumican/IRES/EGFP were successfully constructed by gateway recombinant cloning technology. Positive clones identified by PCR and sequencing were selected and packaged into recombinant adenovirus in HEK293 cells. CONCLUSION We construct adenovirus vectors containing the lumican gene by gateway recombinant cloning technology, which provides a basis for investigating the role of lumican gene in the pathogenesis of high myopia. PMID:27672590

  5. Detection of infectious enteroviruses and adenoviruses in tap water in urban areas in Korea.

    PubMed

    Lee, Seung-Hoon; Kim, Sang-Jong

    2002-01-01

    We investigated the viral contamination of tap water at 11 urban sites in Korea between 1997 and 1998 over a period of 11 months. A total of 23 tap water samples were examined for infectious enteroviruses and adenoviruses by a cell culture technique followed by polymerase chain reaction (PCR) amplification. To identify the recovered viruses, sequence analysis of PCR products was performed. Infectious enteroviruses and adenoviruses were detected in 11 (47.8%) and 9 (39.1%) of the samples, respectively. Both enteroviruses and adenoviruses were detected in five samples (21.7%). The level of viral contamination was quite high, ranging from 2 x 10(-3) to 2.9 x 10(-2) Most Probable Number of Infectious Unit L(-1), far above the recommended virus level in drinking water set by the US EPA. Poliovirus type I derived from vaccine was frequently detected and the remainder comprised coxsackievirus B type or echovirus type 6, which were causative agents of aseptic meningitis in Korea in 1997 and 1998, respectively. Several types of adenovirus were detected in tap water samples and some water samples were found to contain adenoviruses which were closely related to enteric adenovirus types 40 and 41.

  6. Translation of adenovirus 2 late mRNAs microinjected into cultured African green monkey kidney cells

    SciTech Connect

    Richardson, W.D.; Anderson, C.W.

    1984-08-01

    Adenovirus 2-infected monkey cells fail to synthesize fiber, a 62,000 M/sub r/ virion polypeptide expressed at late times in productively infected cells. Yet these cells contain fiber mRNA that, after isolation, can be translated in vitro. The reason for the failure of monkey cells to translate fiber mRNA has been approached by microinjecting adenovirus mRNA into the cytoplasm of cultured monkey cells. Late adenovirus 2 mRNA, isolated from infected HeLa cells, was efficiently expressed when microinjected into the African green monkey kidney cell line CV-C. Expressed viral proteins identified by immunoprecipitation included the adenovirus fiber polypeptide. This result demonstrates that the monkey cell translational apparatus is capable of recognizing and expressing functional adenovirus mRNA. Microinjection of late virus mRNA into cells previously infected with wild-type adenovirus 2 failed to increase significantly the yield of infectious virus. 26 references, 2 figures, 1 table.

  7. Structure of the C-terminal head domain of the fowl adenovirus type 1 short fibre

    SciTech Connect

    El Bakkouri, Majida; Seiradake, Elena; Cusack, Stephen; Ruigrok, Rob W.H. Schoehn, Guy

    2008-08-15

    There are more than 100 known adenovirus serotypes, including 50 human serotypes. They can infect all 5 major vertebrate classes but only Aviadenovirus infecting birds and Mastadenovirus infecting mammals have been well studied. CELO (chicken embryo lethal orphan) adenovirus is responsible for mild respiratory pathologies in birds. Most studies on CELO virus have focussed on its genome sequence and organisation whereas the structural work on CELO proteins has only recently started. Contrary to most adenoviruses, the vertices of CELO virus reveal pentons with two fibres of different lengths. The distal parts (or head) of those fibres are involved in cellular receptor binding. Here we have determined the atomic structure of the short-fibre head of CELO (amino acids 201-410) at 2.0 A resolution. Despite low sequence identity, this structure is conserved compared to the other adenovirus fibre heads. We have used the existing CELO long-fibre head structure and the one we show here for a structure-based alignment of 11 known adenovirus fibre heads which was subsequently used for the construction of an evolutionary tree. Both the fibre head sequence and structural alignments suggest that enteric human group F adenovirus 41 (short fibre) is closer to the CELO fibre heads than the canine CAdV-2 fibre head, that lies closer to the human virus fibre heads.

  8. Adenovirus-mediated efficient gene transfer into cultured three-dimensional organoids.

    PubMed

    Wang, Ning; Zhang, Hongyu; Zhang, Bing-Qiang; Liu, Wei; Zhang, Zhonglin; Qiao, Min; Zhang, Hongmei; Deng, Fang; Wu, Ningning; Chen, Xian; Wen, Sheng; Zhang, Junhui; Liao, Zhan; Zhang, Qian; Yan, Zhengjian; Yin, Liangjun; Ye, Jixing; Deng, Youlin; Luu, Hue H; Haydon, Rex C; Liang, Houjie; He, Tong-Chuan

    2014-01-01

    Three-dimensional organoids have been recently established from various tissue-specific progenitors (such as intestinal stem cells), induced pluripotent stem cells, or embryonic stem cells. These cultured self-sustaining stem cell-based organoids may become valuable systems to study the roles of tissue-specific stem cells in tissue genesis and disease development. It is thus conceivable that effective genetic manipulations in such organoids may allow us to reconstruct disease processes and/or develop novel therapeutics. Recombinant adenoviruses are one of the most commonly used viral vectors for in vitro and in vivo gene deliveries. In this study, we investigate if adenoviruses can be used to effectively deliver transgenes into the cultured "mini-gut" organoids derived from intestinal stem cells. Using adenoviral vectors that express fluorescent proteins, we demonstrate that adenoviruses can effectively deliver transgenes into the cultured 3-D "mini-gut" organoids. The transgene expression can last at least 10 days in the cultured organoids. As a proof-of-principle experiment, we demonstrate that adenovirus-mediated noggin expression effectively support the survival and self-renewal of mini-gut organoids, while adenovirus-mediated expression of BMP4 inhibits the self-sustainability and proliferation of the organoids. Thus, our results strongly suggest that adenovirus vectors can be explored as effective gene delivery vehicles to introduce genetic manipulations in 3-D organoids.

  9. The Merlin/NF2 tumor suppressor functions through the YAP oncoprotein to regulate tissue homeostasis in mammals

    PubMed Central

    Zhang, Nailing; Bai, Haibo; David, Karen K.; Dong, Jixin; Zheng, Yonggang; Cai, Jing; Giovannini, Marco; Liu, Pentao; Anders, Robert A.; Pan, Duojia

    2010-01-01

    The conserved Hippo signaling pathway regulates organ size in both Drosophila and mammals. While a core kinase cascade leading from the protein kinase Hippo (Hpo) (Mst1 and Mst2 in mammals) to the transcription coactivator Yorkie (Yki) (YAP in mammals) has been established, upstream regulators of the Hippo kinase cascade are less well defined, especially in mammals. Using liver-specific conditional knockout mice, we demonstrate that the Merlin/NF2 tumor suppressor and the YAP oncoprotein function antagonistically to regulate liver development. While inactivation of Yap led to loss of hepatocytes and biliary epithelial cells, inactivation of Nf2 led to hepatocellular carcinoma and bile duct hamartoma. Strikingly, the Nf2-deficient phenotype was largely suppressed by heterozygous deletion of Yap, suggesting that YAP is a major effector of Merlin/NF2 in growth regulation. Our studies link Merlin/NF2 to mammalian Hippo signaling and implicate YAP activation as a mediator of pathologies relevant to Neurofibromatosis 2. PMID:20643348

  10. Discovery of a small-molecule binder of the oncoprotein gankyrin that modulates gankyrin activity in the cell

    NASA Astrophysics Data System (ADS)

    Chattopadhyay, Anasuya; O’Connor, Cornelius J.; Zhang, Fengzhi; Galvagnion, Celine; Galloway, Warren R. J. D.; Tan, Yaw Sing; Stokes, Jamie E.; Rahman, Taufiq; Verma, Chandra; Spring, David R.; Itzhaki, Laura S.

    2016-04-01

    Gankyrin is an ankyrin-repeat oncoprotein whose overexpression has been implicated in the development of many cancer types. Elevated gankyrin levels are linked to aberrant cellular events including enhanced degradation of tumour suppressor protein p53, and inhibition of gankyrin activity has therefore been identified as an attractive anticancer strategy. Gankyrin interacts with several partner proteins, and a number of these protein-protein interactions (PPIs) are of relevance to cancer. Thus, molecules that bind the PPI interface of gankyrin and interrupt these interactions are of considerable interest. Herein, we report the discovery of a small molecule termed cjoc42 that is capable of binding to gankyrin. Cell-based experiments demonstrate that cjoc42 can inhibit gankyrin activity in a dose-dependent manner: cjoc42 prevents the decrease in p53 protein levels normally associated with high amounts of gankyrin, and it restores p53-dependent transcription and sensitivity to DNA damage. The results represent the first evidence that gankyrin is a “druggable” target with small molecules.

  11. Discovery of a small-molecule binder of the oncoprotein gankyrin that modulates gankyrin activity in the cell

    PubMed Central

    Chattopadhyay, Anasuya; O’Connor, Cornelius J.; Zhang, Fengzhi; Galvagnion, Celine; Galloway, Warren R. J. D.; Tan, Yaw Sing; Stokes, Jamie E.; Rahman, Taufiq; Verma, Chandra; Spring, David R.; Itzhaki, Laura S.

    2016-01-01

    Gankyrin is an ankyrin-repeat oncoprotein whose overexpression has been implicated in the development of many cancer types. Elevated gankyrin levels are linked to aberrant cellular events including enhanced degradation of tumour suppressor protein p53, and inhibition of gankyrin activity has therefore been identified as an attractive anticancer strategy. Gankyrin interacts with several partner proteins, and a number of these protein-protein interactions (PPIs) are of relevance to cancer. Thus, molecules that bind the PPI interface of gankyrin and interrupt these interactions are of considerable interest. Herein, we report the discovery of a small molecule termed cjoc42 that is capable of binding to gankyrin. Cell-based experiments demonstrate that cjoc42 can inhibit gankyrin activity in a dose-dependent manner: cjoc42 prevents the decrease in p53 protein levels normally associated with high amounts of gankyrin, and it restores p53-dependent transcription and sensitivity to DNA damage. The results represent the first evidence that gankyrin is a “druggable” target with small molecules. PMID:27046077

  12. TORC2 regulates germinal center repression of the TCL1 oncoprotein to promote B cell development and inhibit transformation.

    PubMed

    Kuraishy, Ali I; French, Samuel W; Sherman, Mara; Herling, Marco; Jones, Dan; Wall, Randolph; Teitell, Michael A

    2007-06-12

    Aberrant expression of the TCL1 oncoprotein promotes malignant transformation of germinal center (GC) B cells. Repression of TCL1 in GC B cells facilitates FAS-mediated apoptosis and prevents lymphoma formation. However, the mechanism for this repression is unknown. Here we show that the CREB coactivator TORC2 directly regulates TCL1 expression independent of CREB Ser-133 phosphorylation and CBP/p300 recruitment. GC signaling through CD40 or the BCR, which activates pCREB-dependent genes, caused TORC2 phosphorylation, cytosolic emigration, and TCL1 repression. Signaling via cAMP-inducible pathways inhibited TCL1 repression and reduced apoptosis, consistent with a prosurvival role for TCL1 before GC selection and supporting an initiating role for aberrant TCL1 expression during GC lymphomagenesis. Our data indicate that a novel CREB/TORC2 regulatory mode controls the normal program of GC gene activation and repression that promotes B cell development and circumvents oncogenic progression. Our results also reconcile a paradox in which signals that activate pCREB/CBP/p300 genes concurrently repress TCL1 to initiate its silencing.

  13. Interplay Between Oncoproteins and Antioxidant Enzymes in Esophageal Carcinoma Treated Without and With Chemoradiotherapy: A Prospective Study

    SciTech Connect

    Kaur, Tranum; Gupta, Rajesh; Vaiphei, Kim; Kapoor, Rakesh; Gupta, N.M.; Khanduja, K.L.

    2008-02-01

    Purpose: To analyze p53, bcl-2, c-myc, and cyclooxygenase-2 protein expression changes and examine their relationship with various antioxidant enzymes in esophageal carcinoma patients. Methods and Materials: Patients in Group 1 underwent transhiatal esophagectomy and those in Group 2 were administered chemoradiotherapy followed by surgery after 4 weeks of neoadjuvant therapy. Results: The relationship analysis among the various protein markers and antioxidant enzymes showed an inverse correlation between bcl-2 and superoxide dismutase/catalase in tumor tissues, irrespective of the treatment arm followed. An important positive association was observed between bcl-2 and reduced glutathione levels in the tumor tissue of patients receiving neoadjuvant therapy. Another apoptosis-modulating marker, c-myc, in the tumor tissue of Group 2 patients showed similar pattern levels (high and low) as that of superoxide dismutase/catalase. The association of cyclooxygenase-2 and p53 with various antioxidant enzymes showed a significant positive correlation between cyclooxygenase-2 expression and catalase activity and an inverse trend between p53 expression and superoxide dismutase and catalase activity in the tumor tissue of patients given neoadjuvant therapy. In addition, patients with overexpressed p53 protein levels had lower glutathione peroxidase enzyme levels and vice versa in the tumor tissue of patients who had undergone surgery as their main mode of treatment. Conclusion: The results of this study broaden the insight into the relationships shared among oncoproteins and the antioxidant defense system, and this could be helpful in the clinical management of esophageal carcinoma.

  14. Functional Roles of E6 and E7 Oncoproteins in HPV-Induced Malignancies at Diverse Anatomical Sites

    PubMed Central

    Tomaić, Vjekoslav

    2016-01-01

    Approximately 200 human papillomaviruses (HPVs) infect human epithelial cells, of which the alpha and beta types have been the most extensively studied. Alpha HPV types mainly infect mucosal epithelia and a small group of these causes over 600,000 cancers per year worldwide at various anatomical sites, especially anogenital and head-and-neck cancers. Of these the most important is cervical cancer, which is the leading cause of cancer-related death in women in many parts of the world. Beta HPV types infect cutaneous epithelia and may contribute towards the initiation of non-melanoma skin cancers. HPVs encode two oncoproteins, E6 and E7, which are directly responsible for the development of HPV-induced carcinogenesis. They do this cooperatively by targeting diverse cellular pathways involved in the regulation of cell cycle control, of apoptosis and of cell polarity control networks. In this review, the biological consequences of papillomavirus targeting of various cellular substrates at diverse anatomical sites in the development of HPV-induced malignancies are highlighted. PMID:27775564

  15. The oncoprotein HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote the proliferation of breast cancer cells

    SciTech Connect

    Zhang, Yingyi; Zhao, Yu; Li, Leilei; Shen, Yu; Cai, Xiaoli; Zhang, Xiaodong; Ye, Lihong

    2013-05-03

    Highlights: •HBXIP is able to upregulate the expression of PDGFB in breast cancer cells. •HBXIP serves as a coactivator of activating transcription factor Sp1. •HBXIP stimulates the PDGFB promoter via activating transcription factor Sp1. •HBXIP promotes the proliferation of breast cancer cell via upregulating PDGFB. -- Abstract: We have reported that the oncoprotein hepatitis B virus X-interacting protein (HBXIP) acts as a novel transcriptional coactivator to promote proliferation and migration of breast cancer cells. Previously, we showed that HBXIP was able to activate nuclear factor-κB (NF-κB) in breast cancer cells. As an oncogene, the platelet-derived growth factor beta polypeptide (PDGFB) plays crucial roles in carcinogenesis. In the present study, we found that both HBXIP and PDGFB were highly expressed in breast cancer cell lines. Interestingly, HBXIP was able to increase transcriptional activity of NF-κB through PDGFB, suggesting that HBXIP is associated with PDGFB in the cells. Moreover, HBXIP was able to upregulate PDGFB at the levels of mRNA, protein and promoter in the cells. Then, we identified that HBXIP stimulated the promoter of PDGFB through activating transcription factor Sp1. In function, HBXIP enhanced the proliferation of breast cancer cells through PDGFB in vitro. Thus, we conclude that HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote proliferation of breast cancer cells.

  16. A plant alkaloid, veratridine, potentiates cancer chemosensitivity by UBXN2A-dependent inhibition of an oncoprotein, mortalin-2

    PubMed Central

    Abdullah, Ammara; Sane, Sanam; Branick, Kate A.; Freeling, Jessica L.; Wang, Hongmin; Zhang, Dong; Rezvani, Khosrow

    2015-01-01

    Veratridine (VTD), an alkaloid derived from the Liliaceae plant shows anti-tumor effects; however, its molecular targets have not been thoroughly studied. Using a high-throughput drug screen, we found that VTD enhances transactivation of UBXN2A, resulting in upregulation of UBXN2A in the cytoplasm, where UBXN2A binds and inhibits the oncoprotein mortalin-2 (mot-2). VTD-treated cancer cells undergo cell death in UBXN2A- and mot-2-dependent manners. The cytotoxic function of VTD is grade-dependent, and the combined treatment with a sub-optimal dose of the standard chemotherapy, 5-Fluorouracil (5-FU) and etoposide, demonstrated a synergistic effect, resulting in higher therapeutic efficacy. VTD influences the CD44+ stem cells, possibly through UBXN2A-dependent inhibition of mot-2. The VTD-dependent expression of UBXN2A is a potential candidate for designing novel strategies for colon cancer treatment because: 1) In 50% of colon cancer patients, UBXN2A protein levels in tumor tissues are significantly lower than those in the adjacent normal tissues. 2) Cytoplasmic expression of the mot-2 protein is very low in non-cancerous cells; thus, VTD can produce tumor-specific toxicity while normal cells remain intact. 3) Finally, VTD or its modified analogs offer a valuable adjuvant chemotherapy strategy to improve the efficacy of 5-FU-based chemotherapy for colon cancer patients harboring WT-p53. PMID:26188124

  17. The high-risk HPV E6 oncoprotein preferentially targets phosphorylated nuclear forms of hDlg

    SciTech Connect

    Narayan, Nisha; Subbaiah, Vanitha Krishna; Banks, Lawrence

    2009-04-25

    High-risk mucosal HPV E6 oncoproteins target a number of PDZ domain-containing substrates for proteasome mediated degradation. One of these, Discs Large (Dlg), is involved in the regulation of cell polarity and proliferation control. Previous studies had suggested that Dlg when hyperphosphorylated by osmotic shock, or when present in the nucleus could be preferentially targeted by E6. In this study we use phospho-specific antibodies directed against Dlg phosphorylated at residues S158 and S442 to show that these two observations are, in fact, linked. Dlg, when phosphorylated on S158 and S442 by CDK1 or CDK2, shows a preferential nuclear accumulation. However, these forms of Dlg are absent in cells derived from HPV-induced cervical cancers. Upon either proteasome inhibition or siRNA ablation of E6 expression, we see specific rescue of these phosphorylated forms of Dlg. These results demonstrate that nuclear forms of Dlg phosphorylated on its CDK phospho-acceptor sites has enhanced susceptibility to E6-induced degradation and place previous studies on the stress-induced phosphorylation of Dlg into a relevant biological context.

  18. DNA and redox state induced conformational changes in the DNA-binding domain of the Myb oncoprotein.

    PubMed Central

    Myrset, A H; Bostad, A; Jamin, N; Lirsac, P N; Toma, F; Gabrielsen, O S

    1993-01-01

    The DNA-binding domain of the oncoprotein Myb comprises three imperfect repeats, R1, R2 and R3. Only R2 and R3 are required for sequence-specific DNA-binding. Both are assumed to contain helix-turn-helix (HTH)-related motifs, but multidimensional heteronuclear NMR spectroscopy revealed a disordered structure in R2 where the second HTH helix was predicted [Jamin et al. (1993) Eur. J. Biochem., 216, 147-154]. We propose that the disordered region folds into a 'recognition' helix and generates a full HTH-related motif upon binding to DNA. This would move Cys43 into the hydrophobic core of R2. We observed that Cys43 was accessible to N-ethylmaleimide alkylation in the free protein, but inaccessible in the DNA complex. Mutant proteins with charged (C43D) or polar (C43S) side chains in position 43 bound DNA with reduced affinity, while hydrophobic replacements (C43A, C43V and C43I) gave unaltered or improved DNA-binding. Specific DNA-binding enhanced protease resistance dramatically. Fluorescence emission spectra and quenching experiments supported a DNA-induced conformational change. Moreover, reversible oxidation of Cys43 had an effect similar to the inactivating C43D mutation. The highly oxidizable Cys43 could function as a molecular sensor for a redox regulatory mechanism turning specific DNA-binding on or off by controlling the DNA-induced conformational change in R2. Images PMID:8223472

  19. The Catalytic Efficiency of Lipin 1β Increases by Physically Interacting with the Proto-oncoprotein c-Fos*

    PubMed Central

    Cardozo Gizzi, Andres M.; Prucca, Cesar G.; Gaveglio, Virginia L.; Renner, Marianne L.; Pasquaré, Susana J.; Caputto, Beatriz L.

    2015-01-01

    Phosphatidic acid (PA) is a central precursor for membrane phospholipid biosynthesis. The lipin family is a magnesium-dependent type I PA phosphatase involved in de novo synthesis of neutral lipids and phospholipids. The regulation of lipin activity may govern the pathways by which these lipids are synthesized and control the cellular levels of important signaling lipids. Moreover, the proto-oncoprotein c-Fos has an emerging role in glycerolipid synthesis regulation; by interacting with key synthesizing enzymes it is able to increase overall phospho- and glycolipid synthesis. We studied the lipin 1β enzyme activity in a cell-free system using PA/Triton X-100 mixed micelles as substrate, analyzing it in the presence/absence of c-Fos. We found that lipin 1β kcat value increases around 40% in the presence of c-Fos, with no change in the lipin 1β affinity for the PA/Triton X-100 mixed micelles. We also probed a physical interaction between both proteins. Although the c-Fos domain involved in lipin activation is its basic domain, the interaction domain is mapped to the N-terminal c-Fos. In conclusion, we provide evidence for a novel positive regulator of lipin 1β PA phosphatase activity that is not achieved via altering its subcellular localization or affinity for membranes but rather through directly increasing its catalytic efficiency. PMID:26475860

  20. ALK oncoproteins in atypical inflammatory myofibroblastic tumours: novel RRBP1-ALK fusions in epithelioid inflammatory myofibroblastic sarcoma.

    PubMed

    Lee, Jen-Chieh; Li, Chien-Feng; Huang, Hsuan-Ying; Zhu, Mei-Jun; Mariño-Enríquez, Adrián; Lee, Chung-Ta; Ou, Wen-Bin; Hornick, Jason L; Fletcher, Jonathan A

    2017-02-01

    ALK oncogenic activation mechanisms were characterized in four conventional spindle-cell inflammatory myofibroblastic tumours (IMT) and five atypical IMT, each of which had ALK genomic perturbations. Constitutively activated ALK oncoproteins were purified by ALK immunoprecipitation and electrophoresis, and were characterized by mass spectrometry. The four conventional IMT had TPM3/4-ALK fusions (two cases) or DCTN1-ALK fusions (two cases), whereas two atypical spindle-cell IMT had TFG-ALK and TPM3-ALK fusion in one case each, and three epithelioid inflammatory myofibroblastic sarcomas had RANBP2-ALK fusions in two cases, and a novel RRBP1-ALK fusion in one case. The epithelioid inflammatory myofibroblastic sarcoma with RRBP1-ALK fusion had cytoplasmic ALK expression with perinuclear accentuation, different from the nuclear membranous ALK localization in epithelioid inflammatory myofibroblastic sarcomas with RANBP2-ALK fusions. Evaluation of three additional uncharacterized epithelioid inflammatory myofibroblastic sarcomas with ALK cytoplasmic/perinuclear- accentuation expression demonstrated RRBP1-ALK fusion in two cases. These studies show that atypical spindle-cell IMT can utilize the same ALK fusion mechanisms described previously in conventional IMT, whereas in clinically aggressive epithelioid inflammatory myofibroblastic sarcoma we identify a novel recurrent ALK oncogenic mechanism, resulting from fusion with the RRBP1 gene. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  1. Ubiquitination and degradation of the hominoid-specific oncoprotein TBC1D3 is regulated by protein palmitoylation

    SciTech Connect

    Kong, Chen; Lange, Jeffrey J.; Samovski, Dmitri; Su, Xiong; Liu, Jialiu; Sundaresan, Sinju; Stahl, Philip D.

    2013-05-03

    Highlights: •Hominoid-specific oncogene TBC1D3 is targeted to plasma membrane by palmitoylation. •TBC1D3 is palmitoylated on two cysteine residues: 318 and 325. •TBC1D3 palmitoylation governs growth factors-induced TBC1D3 degradation. •Post-translational modifications may regulate oncogenic properties of TBC1D3. -- Abstract: Expression of the hominoid-specific oncoprotein TBC1D3 promotes enhanced cell growth and proliferation by increased activation of signal transduction through several growth factors. Recently we documented the role of CUL7 E3 ligase in growth factors-induced ubiquitination and degradation of TBC1D3. Here we expanded our study to discover additional molecular mechanisms that control TBC1D3 protein turnover. We report that TBC1D3 is palmitoylated on two cysteine residues: 318 and 325. The expression of double palmitoylation mutant TBC1D3:C318/325S resulted in protein mislocalization and enhanced growth factors-induced TBC1D3 degradation. Moreover, ubiquitination of TBC1D3 via CUL7 E3 ligase complex was increased by mutating the palmitoylation sites, suggesting that depalmitoylation of TBC1D3 makes the protein more available for ubiquitination and degradation. The results reported here provide novel insights into the molecular mechanisms that govern TBC1D3 protein degradation. Dysregulation of these mechanisms in vivo could potentially result in aberrant TBC1D3 expression and promote oncogenesis.

  2. A plant alkaloid, veratridine, potentiates cancer chemosensitivity by UBXN2A-dependent inhibition of an oncoprotein, mortalin-2.

    PubMed

    Abdullah, Ammara; Sane, Sanam; Branick, Kate A; Freeling, Jessica L; Wang, Hongmin; Zhang, Dong; Rezvani, Khosrow

    2015-09-15

    Veratridine (VTD), an alkaloid derived from the Liliaceae plant shows anti-tumor effects; however, its molecular targets have not been thoroughly studied. Using a high-throughput drug screen, we found that VTD enhances transactivation of UBXN2A, resulting in upregulation of UBXN2A in the cytoplasm, where UBXN2A binds and inhibits the oncoprotein mortalin-2 (mot-2). VTD-treated cancer cells undergo cell death in UBXN2A- and mot-2-dependent manners. The cytotoxic function of VTD is grade-dependent, and the combined treatment with a sub-optimal dose of the standard chemotherapy, 5-Fluorouracil (5-FU) and etoposide, demonstrated a synergistic effect, resulting in higher therapeutic efficacy. VTD influences the CD44+ stem cells, possibly through UBXN2A-dependent inhibition of mot-2. The VTD-dependent expression of UBXN2A is a potential candidate for designing novel strategies for colon cancer treatment because: 1) In 50% of colon cancer patients, UBXN2A protein levels in tumor tissues are significantly lower than those in the adjacent normal tissues. 2) Cytoplasmic expression of the mot-2 protein is very low in non-cancerous cells; thus, VTD can produce tumor-specific toxicity while normal cells remain intact. 3) Finally, VTD or its modified analogs offer a valuable adjuvant chemotherapy strategy to improve the efficacy of 5-FU-based chemotherapy for colon cancer patients harboring WT-p53.

  3. Oncogenic nexus of cancerous inhibitor of protein phosphatase 2A (CIP2A): An oncoprotein with many hands

    PubMed Central

    De, Pradip; Carlson, Jennifer; Leyland-Jones, Brian; Dey, Nandini

    2014-01-01

    Oncoprotein CIP2A a Cancerous Inhibitor of PP2A forms an “oncogenic nexus” by virtue of its control on PP2A and MYC stabilization in cancer cells. The expression and prognostic function of CIP2A in different solid tumors including colorectal carcinoma, head & neck cancers, gastric cancers, lung carcinoma, cholangiocarcinoma, esophageal cancers, pancreatic carcinoma, brain cancers, breast carcinoma, bladder cancers, ovarian carcinoma, renal cell carcinomas, tongue cancers, cervical carcinoma, prostate cancers, and oral carcinoma as well as a number of hematological malignancies are just beginning to emerge. Herein, we reviewed the recent progress in our understanding of (1) how an “oncogenic nexus” of CIP2A participates in the tumorigenic transformation of cells and (2) how we can prospect/view the clinical relevance of CIP2A in the context of cancer therapy. The review will try to understand the role of CIP2A (a) as a biomarker in cancers and evaluate the prognostic value of CIP2A in different cancers (b) as a therapeutic target in cancers and (c) in drug response and developing chemo-resistance in cancers. PMID:25015035

  4. Molecular role of the PAX5-ETV6 oncoprotein in promoting B-cell acute lymphoblastic leukemia.

    PubMed

    Smeenk, Leonie; Fischer, Maria; Jurado, Sabine; Jaritz, Markus; Azaryan, Anna; Werner, Barbara; Roth, Mareike; Zuber, Johannes; Stanulla, Martin; den Boer, Monique L; Mullighan, Charles G; Strehl, Sabine; Busslinger, Meinrad

    2017-02-20

    PAX5 is a tumor suppressor in B-ALL, while the role of PAX5 fusion proteins in B-ALL development is largely unknown. Here, we studied the function of PAX5-ETV6 and PAX5-FOXP1 in mice expressing these proteins from the Pax5 locus. Both proteins arrested B-lymphopoiesis at the pro-B to pre-B-cell transition and, contrary to their proposed dominant-negative role, did not interfere with the expression of most regulated Pax5 target genes. Pax5-Etv6, but not Pax5-Foxp1, cooperated with loss of the Cdkna2a/b tumor suppressors in promoting B-ALL development. Regulated Pax5-Etv6 target genes identified in these B-ALLs encode proteins implicated in pre-B-cell receptor (BCR) signaling and migration/adhesion, which could contribute to the proliferation, survival, and tissue infiltration of leukemic B cells. Together with similar observations made in human PAX5-ETV6(+) B-ALLs, these data identified PAX5-ETV6 as a potent oncoprotein that drives B-cell leukemia development.

  5. Selective Modification of Adenovirus Replication Can Be Achieved through Rational Mutagenesis of the Adenovirus Type 5 DNA Polymerase

    PubMed Central

    Capella, Cristina; Beltejar, Michael-John; Brown, Caitlin; Fong, Vincent; Daddacha, Waaqo; Kim, Baek

    2012-01-01

    Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2- to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates. PMID:22811532

  6. Selective modification of adenovirus replication can be achieved through rational mutagenesis of the adenovirus type 5 DNA polymerase.

    PubMed

    Capella, Cristina; Beltejar, Michael-John; Brown, Caitlin; Fong, Vincent; Daddacha, Waaqo; Kim, Baek; Dewhurst, Stephen

    2012-10-01

    Mutations that reduce the efficiency of deoxynucleoside (dN) triphosphate (dNTP) substrate utilization by the HIV-1 DNA polymerase prevent viral replication in resting cells, which contain low dNTP concentrations, but not in rapidly dividing cells such as cancer cells, which contain high levels of dNTPs. We therefore tested whether mutations in regions of the adenovirus type 5 (Ad5) DNA polymerase that interact with the dNTP substrate or DNA template could alter virus replication. The majority of the mutations created, including conservative substitutions, were incompatible with virus replication. Five replication-competent mutants were recovered from 293 cells, but four of these mutants failed to replicate in A549 lung carcinoma cells and Wi38 normal lung cells. Purified polymerase proteins from these viruses exhibited only a 2- to 4-fold reduction in their dNTP utilization efficiency but nonetheless could not be rescued, even when intracellular dNTP concentrations were artificially raised by the addition of exogenous dNs to virus-infected A549 cells. The fifth mutation (I664V) reduced biochemical dNTP utilization by the viral polymerase by 2.5-fold. The corresponding virus replicated to wild-type levels in three different cancer cell lines but was significantly impaired in all normal cell lines in which it was tested. Efficient replication and virus-mediated cell killing were rescued by the addition of exogenous dNs to normal lung fibroblasts (MRC5 cells), confirming the dNTP-dependent nature of the polymerase defect. Collectively, these data provide proof-of-concept support for the notion that conditionally replicating, tumor-selective adenovirus vectors can be created by modifying the efficiency with which the viral DNA polymerase utilizes dNTP substrates.

  7. Enhanced therapeutic efficacy of an adenovirus-PEI-bile-acid complex in tumors with low coxsackie and adenovirus receptor expression.

    PubMed

    Lee, Cho-Hee; Kasala, Dayananda; Na, Youjin; Lee, Min Sang; Kim, Sung Wan; Jeong, Ji Hoon; Yun, Chae-Ok

    2014-07-01

    Adenovirus (Ad) is a potential vehicle for cancer gene therapy. However, cells that express low levels of the coxsackie and adenovirus receptor (CAR) demonstrate poor Ad infection efficiency. We developed a bile acid-conjugated poly(ethyleneimine) (DA3)-coated Ad complex (Ad/DA3) to enhance Ad transduction efficiency. The size distribution and zeta potential of Ad/DA3 increased to 324 ± 3.08 nm and 10.13 ± 0.21 mV, respectively, compared with those of naked Ad (108 ± 2.26 nm and -17.7 ± 1.5 mV). The transduction efficiency of Ad/DA3 increased in a DA3 polymer concentration-dependent manner. Enhanced gene transfer by Ad/DA3 was more evident in CAR-moderate and CAR-negative cancer cells. Competition assays with a CAR-specific antibody revealed that internalization of Ad/DA3 was not mediated primarily by CAR but involved clathrin-, caveolae-, and macropinocytosis-mediated endocytosis. Cancer cell death was significantly increased when oncolytic Ad and DA3 were complexed (RdB-KOX/DA3) compared to that of naked oncolytic Ad and was inversely proportional to CAR levels. Importantly, RdB-KOX/DA3 significantly enhanced apoptosis, reduced angiogenesis, reduced proliferation, and increased active viral replication in human tumor xenografts compared to that of naked Ad. These results demonstrate that a hybrid vector system can increase the efficacy of oncolytic Ad virotherapy, particularly in CAR-limited tumors.

  8. Retargeted oncolytic adenovirus displaying a single variable domain of camelid heavy-chain-only antibody in a fiber protein.

    PubMed

    van Erp, Elisabeth A; Kaliberova, Lyudmila N; Kaliberov, Sergey A; Curiel, David T

    2015-01-01

    Conditionally replicative adenoviruses are promising agents for oncolytic virotherapy. Various approaches have been attempted to retarget adenoviruses to tumor-specific antigens to circumvent deficiency of receptor for adenoviral binding and to provide an additional level of tumor specificity. Functional incorporation of highly specific targeting molecules into the viral capsid can potentially retarget adenoviral infection. However, conventional antibodies are not compatible with the cytoplasmic adenovirus capsid synthesis. The goal of this study was to evaluate the utility of single variable domains derived from heavy chain camelid antibodies for retargeting of adenovirus infection. We have combined transcriptional targeting using a tumor-specific promoter with transductional targeting through viral capsid incorporation of antihuman carcinoembryonic antigen single variable domains. Obtained data demonstrated that employment of a single variable domain genetically incorporated into an adenovirus fiber increased specificity of infection and efficacy of replication of single variable domain-targeted oncolytic adenovirus. The double targeting, both transcriptional through the C-X-C chemokine receptor type 4 promoter and transductional using the single variable domain, is a promising means to improve the therapeutic index for these advanced generation conditionally replicative adenoviruses. A successful strategy to transductional retargeting of oncolytic adenovirus infection has not been shown before and therefore we believe this is the first employment of transductional targeting using single variable domains derived from heavy chain camelid antibodies to enhance specificity of conditionally replicative adenoviruses.

  9. Molecular epidemiology and surveillance of circulating rotavirus and adenovirus in Congolese children with gastroenteritis.

    PubMed

    Mayindou, Gontran; Ngokana, Berge; Sidibé, Anissa; Moundélé, Victoire; Koukouikila-Koussounda, Felix; Christevy Vouvoungui, Jeannhey; Kwedi Nolna, Sylvie; Velavan, Thirumalaisamy P; Ntoumi, Francine

    2016-04-01

    Infectious Diarrhea caused by rotavirus and adenovirus, is a leading cause of death in children in sub-Sahara Africa but there is limited published data on the diverse rotavirus genotypes and adenovirus serotypes circulating in the Republic of Congo. In this study, we investigated the prevalence of severe diarrhea caused by rotavirus A (RVA) and Adenovirus serotype 40 and 41 in Congolese children hospitalized with severe gastroenteritis. Stool samples were collected from 655 Congolese children less than 60 months of age hospitalized with acute gastroenteritis between June 2012 and June 2013. Rotavirus and adenovirus antigens were tested using commercially available ELISA kits and the RVA G- and P- genotypes were identified by seminested multiplex RT-PCR. Three hundred and four (46.4%) children were tested positive for RVA. Adenovirus infection was found in 5.5% of the 564 tested children. Rotavirus infection was frequently observed in children between 6-12 months (55.9%). The dry season months recorded increased RVA infection while no seasonality of adenovirus infection was demonstrated. The most common RVA genotypes were G1 (57.5%), G2 (6.4%), G1G2 mixture (15.5%), P[8] (58%), P[6] (13.2%), and P[8]P[6] mixture (26%). Additionally, the genotype G12P[6] was significantly associated with increased vomiting. This first study on Congolese children demonstrates a high prevalence and clinical significance of existing rotavirus genotypes. Adenovirus prevalence is similar to that of other Central African countries. This baseline epidemiology and molecular characterization study will contribute significantly to the RVA surveillance after vaccine implementation in the country.

  10. A replicating adenovirus capsid display recombinant elicits antibodies against Plasmodium falciparum sporozoites in Aotus nancymaae monkeys.

    PubMed

    Karen, Kasey A; Deal, Cailin; Adams, Robert J; Nielsen, Carolyn; Ward, Cameron; Espinosa, Diego A; Xie, Jane; Zavala, Fidel; Ketner, Gary

    2015-01-01

    Decades of success with live adenovirus vaccines suggest that replication-competent recombinant adenoviruses (rAds) could serve as effective vectors for immunization against other pathogens. To explore the potential of a live rAd vaccine against malaria, we prepared a viable adenovirus 5 (Ad5) recombinant that displays a B-cell epitope from the circumsporozoite protein (CSP) of Plasmodium falciparum on the virion surface. The recombinant induced P. falciparum sporozoite-neutralizing antibodies in mice. Human adenoviruses do not replicate in mice. Therefore, to examine immunogenicity in a system in which, as in humans, the recombinant replicates, we constructed a similar recombinant in an adenovirus mutant that replicates in monkey cells and immunized four Aotus nancymaae monkeys. The recombinant replicated in the monkeys after intratracheal instillation, the first demonstration of replication of human adenoviruses in New World monkeys. Immunization elicited antibodies both to the Plasmodium epitope and the Ad5 vector. Antibodies from all four monkeys recognized CSP on intact parasites, and plasma from one monkey neutralized sporozoites in vitro and conferred partial protection against P. falciparum sporozoite infection after passive transfer to mice. Prior enteric inoculation of two animals with antigenically wild-type adenovirus primed a response to the subsequent intratracheal inoculation, suggesting a route to optimizing performance. A vaccine is not yet available against P. falciparum, which induces the deadliest form of malaria and kills approximately one million children each year. The live capsid display recombinant described here may constitute an early step in a critically needed novel approach to malaria immunization.

  11. An adenovirus linked to mortality and disease in long-tailed ducks (Clangula hyemalis) in Alaska

    USGS Publications Warehouse

    Hollmén, Tuula E.; Franson, J.C.; Flint, P.L.; Grand, J.B.; Lanctot, Richard B.; Docherty, D.E.; Wilson, H.M.

    2003-01-01

    An adenovirus was isolated from intestinal samples of two long-tailed ducks (Clangula hyemalis) collected during a die-off in the Beaufort Sea off the north coast of Alaska in 2000. The virus was not neutralized by reference antiserum against known group I, II, or III avian adenoviruses and may represent a new serotype. The prevalence of the virus was determined in live-trapped long-tailed ducks at the mortality site and at a reference site 100 km away where no mortality was observed. Prevalence of adenovirus antibodies in serum samples at the mortality site was 86% compared to 10% at the reference site. Furthermore, 50% of cloacal swabs collected at the mortality site and only 7% of swabs from the reference site were positive for adenoviruses. In 2001, no mortality was observed at either of the study areas, and virus prevalence in both serum and cloacal samples was low, providing further evidence that the adenovirus was linked to the mortality event in 2000. The virus was used to infect long-tailed ducks under experimental conditions and resulted in lesions previously described for avian adenovirus infections and similar to those observed in long-tailed duck carcasses from the Beaufort Sea. The status of long-tailed ducks has recently become a concern in Alaska due to precipitous declines in breeding populations there since the mid-1970s. Our findings suggest that the newly isolated adenovirus is a disease agent and source of mortality in long-tailed ducks, and thus could be a contributing factor in population declines.

  12. An adenovirus linked to mortality and disease in long-tailed ducks (Clangula hyemalis) in Alaska.

    PubMed

    Hollmén, Tuula E; Franson, J Christian; Flint, Paul L; Grand, James B; Lanctot, Richard B; Docherty, Douglas E; Wilson, Heather M

    2003-01-01

    An adenovirus was isolated from intestinal samples of two long-tailed ducks (Clangula hyemalis) collected during a die-off in the Beaufort Sea off the north coast of Alaska in 2000. The virus was not neutralized by reference antiserum against known group I, II, or III avian adenoviruses and may represent a new serotype. The prevalence of the virus was determined in live-trapped long-tailed ducks at the mortality site and at a reference site 100 km away where no mortality was observed. Prevalence of adenovirus antibodies in serum samples at the mortality site was 86% compared to 10% at the reference site. Furthermore, 50% of cloacal swabs collected at the mortality site and only 7% of swabs from the reference site were positive for adenoviruses. In 2001, no mortality was observed at either of the study areas, and virus prevalence in both serum and cloacal samples was low, providing further evidence that the adenovirus was linked to the mortality event in 2000. The virus was used to infect long-tailed ducks under experimental conditions and resulted in lesions previously described for avian adenovirus infections and similar to those observed in long-tailed duck carcasses from the Beaufort Sea. The status of long-tailed ducks has recently become a concern in Alaska due to precipitous declines in breeding populations there since the mid-1970s. Our findings suggest that the newly isolated adenovirus is a disease agent and source of mortality in long-tailed ducks, and thus could be a contributing factor in population declines.

  13. Impact of preexisting adenovirus vector immunity on immunogenicity and protection conferred with an adenovirus-based H5N1 influenza vaccine.

    PubMed

    Pandey, Aseem; Singh, Neetu; Vemula, Sai V; Couëtil, Laurent; Katz, Jacqueline M; Donis, Ruben; Sambhara, Suryaprakash; Mittal, Suresh K

    2012-01-01

    The prevalence of preexisting immunity to adenoviruses in the majority of the human population might adversely impact the development of adaptive immune responses against adenovirus vector-based vaccines. To address this issue, we primed BALB/c mice either intranasally (i.n.) or intramuscularly (i.m.) with varying doses of wild type (WT) human adenovirus subtype 5 (HAd5). Following the development of immunity against HAd5, we immunized animals via the i.n. or i.m. route of inoculation with a HAd vector (HAd-HA-NP) expressing the hemagglutinin (HA) and nucleoprotein (NP) of A/Vietnam/1203/04 (H5N1) influenza virus. The immunogenicity and protection results suggest that low levels of vector immunity (<520 virus-neutralization titer) induced by priming mice with up to 10(7) plaque forming units (p.f.u.) of HAd-WT did not adversely impact the protective efficacy of the vaccine. Furthermore, high levels of vector immunity (approximately 1500 virus-neutralization titer) induced by priming mice with 10(8) p.f.u. of HAd-WT were overcome by either increasing the vaccine dose or using alternate routes of vaccination. A further increase in the priming dose to 10(9) p.f.u. allowed only partial protection. These results suggest possible strategies to overcome the variable levels of human immunity against adenoviruses, leading to better utilization of HAd vector-based vaccines.

  14. Genetic and Molecular Epidemiological Characterization of a Novel Adenovirus in Antarctic Penguins Collected between 2008 and 2013

    PubMed Central

    Lee, Sook-Young; Kim, Jeong-Hoon; Seo, Tae-Kun; No, Jin Sun; Kim, Hankyeom; Kim, Won-keun; Choi, Han-Gu; Kang, Sung-Ho; Song, Jin-Won

    2016-01-01

    Antarctica is considered a relatively uncontaminated region with regard to the infectious diseases because of its extreme environment, and isolated geography. For the genetic characterization and molecular epidemiology of the newly found penguin adenovirus in Antarctica, entire genome sequencing and annual survey of penguin adenovirus were conducted. The entire genome sequences of penguin adenoviruses were completed for two Chinstrap penguins (Pygoscelis antarctica) and two Gentoo penguins (Pygoscelis papua). The whole genome lengths and G+C content of penguin adenoviruses were found to be 24,630–24,662 bp and 35.5–35.6%, respectively. Notably, the presence of putative sialidase gene was not identified in penguin adenoviruses by Rapid Amplification of cDNA Ends (RACE-PCR) as well as consensus specific PCR. The penguin adenoviruses were demonstrated to be a new species within the genus Siadenovirus, with a distance of 29.9–39.3% (amino acid, 32.1–47.9%) in DNA polymerase gene, and showed the closest relationship with turkey adenovirus 3 (TAdV-3) in phylogenetic analysis. During the 2008–2013 study period, the penguin adenoviruses were annually detected in 22 of 78 penguins (28.2%), and the molecular epidemiological study of the penguin adenovirus indicates a predominant infection in Chinstrap penguin population (12/30, 40%). Interestingly, the genome of penguin adenovirus could be detected in several internal samples, except the lymph node and brain. In conclusion, an analysis of the entire adenoviral genomes from Antarctic penguins was conducted, and the penguin adenoviruses, containing unique genetic character, were identified as a new species within the genus Siadenovirus. Moreover, it was annually detected in Antarctic penguins, suggesting its circulation within the penguin population. PMID:27309961

  15. ET-05INTRANASAL DELIVERY OF NEURAL STEM CELLS LOADED WITH ONCOLYTIC ADENOVIRUS EXTENDS SURVIVAL OF MICE WITH INTRACRANIAL GLIOMA

    PubMed Central

    Balyasnikova, Irina; Dey, Mahua; Tobias, Alex; Kanojia, Deepak; Lee, Gina; Han, Yu; Zhang, Lingjiao; Ahmed, Atique; Aboody, Karen; Lesniak, Maciej

    2014-01-01

    Glioblastoma multiforme (GBM), the most common adult primary brain tumor, is in need of better and effective therapeutic options. In this study, we hypothesized that neural stem cells (NSCs) loaded with CRAd-S-pK7 oncolytic adenovirus (OV) can be successfully delivered via nasal application (IN) and will impart therapeutic efficacy in a murine model of human glioma. Recently, we demonstrated that radiation therapy increases the expression of CXCL12 in glioma xenografts, thus contributing to the enhanced migration of stem cells to the tumor site. We now show that hypoxic preconditioning of NSCs for 24h (1% oxygen) results in the two-fold increase of the CXCR4 expression on the surface of NSCs and doubles the rate of their migration towards glioma cells (p < 0.001) compared to untreated group. Hypoxic preconditioning of NSCs did not alter the OV replication as measured by E1A copy number. For in vivo studies, a patient derived GBM43 glioma cells were intracranially inoculated in nude mice. One week later, mice were treated with fractioned dose of irradiation at 2Gy for 5 days. NSCs (5x105) cultured under normoxic and hypoxic conditions were loaded with OV at 50 viral particles per cell, and IN inoculated 24h following the completion of the radiation. Kaplan-Meier survival analysis demonstrated that irradiation resulted in 44% improvement (p < 0.001) in median survival versus non-treated group. Survival of irradiated mice was not affected by either NSC's or OV-loaded NSCs, but was extended (p < 0.05) another 25% in the group of animals treated with OV-loaded NSCs cultured under hypoxic conditions. Thus, our data further validate the IN delivery as a novel route for the stem cell-based anti-glioma therapy, and suggest that modification of NSCs by enhancing their ability to migrate might significantly increase their therapeutic potential for IN delivery to target GBM.

  16. Interaction of human adenoviruses and coliphages with kaolinite and bentonite.

    PubMed

    Bellou, Maria I; Syngouna, Vasiliki I; Tselepi, Maria A; Kokkinos, Petros A; Paparrodopoulos, Spyros C; Vantarakis, Apostolos; Chrysikopoulos, Constantinos V

    2015-06-01

    Human adenoviruses (hAdVs) are pathogenic viruses responsible for public health problems worldwide. They have also been used as viral indicators in environmental systems. Coliphages (e.g., MS2, ΦX174) have also been studied as indicators of viral pollution in fecally contaminated water. Our objective was to evaluate the distribution of three viral fecal indicators (hAdVs, MS2, and ΦΧ174), between two different phyllosilicate clays (kaolinite and bentonite) and the aqueous phase. A series of static and dynamic experiments were conducted under two different temperatures (4, 25°C) for a time period of seven days. HAdV adsorption was examined in DNase I reaction buffer (pH=7.6, and ionic strength (IS)=1.4mM), whereas coliphage adsorption in phosphate buffered saline solution (pH=7, IS=2mM). Moreover, the effect of IS on hAdV adsorption under static conditions was evaluated. The adsorption of hAdV was assessed by real-time PCR and its infectivity was tested by cultivation methods. The coliphages MS2 and ΦΧ174 were assayed by the double-layer overlay method. The experimental results have shown that coliphage adsorption onto both kaolinite and bentonite was higher for the dynamic than the static experiments; whereas hAdV adsorption was lower under dynamic conditions. The adsorption of hAdV increased with decreasing temperature, contrary to the results obtained for the coliphages. This study examines the combined effect of temperature, agitation, clay type, and IS on hAdV adsorption onto clays. The results provide useful new information on the effective removal of viral fecal indicators (MS2, ΦX174 and hAdV) from dilute aqueous solutions by adsorption onto kaolinite and bentonite. Factors enabling enteric viruses to penetrate soils, groundwater and travel long distances within aquifers are important public health issues. Because the observed adsorption behavior of surrogate coliphages MS2 and ΦΧ174 is substantially different to that of hAdV, neither MS2 nor

  17. Cross-talk between Human Papillomavirus Oncoproteins and Hedgehog Signaling Synergistically Promotes Stemness in Cervical Cancer Cells

    PubMed Central

    Vishnoi, Kanchan; Mahata, Sutapa; Tyagi, Abhishek; Pandey, Arvind; Verma, Gaurav; Jadli, Mohit; Singh, Tejveer; Singh, Sukh Mahendra; Bharti, Alok C.

    2016-01-01

    Viral oncoproteins E6/E7 play key oncogenic role in human papillomavirus (HPV)-mediated cervical carcinogenesis in conjunction with aberrant activation of cellular signaling events. GLI-signaling has been implicated in metastasis and tumor recurrence of cervical cancer. However, the interaction of GLI-signaling with HPV oncogenes is unknown. We examined this relationship in established HPV-positive and HPV-negative cervical cancer cell lines using specific GLI inhibitor, cyclopamine and HPVE6/E7 siRNAs. Cervical cancer cell lines showed variable expression of GLI-signaling components. HPV16-positive SiHa cells, overexpressed GLI1, Smo and Patch. Inhibition by cyclopamine resulted in dose-dependent reduction of Smo and GLI1 and loss of cell viability with a higher magnitude in HPV-positive cells. Cyclopamine selectively downregulated HPVE6 expression and resulted in p53 accumulation, whereas HPVE7 and pRb level remained unaffected. siRNA-mediated silencing of HPV16E6 demonstrated reduced GLI1 transcripts in SiHa cells. Cervical cancer stem-like cells isolated by side population analysis, displayed retention of E6 and GLI1 expression. Fraction of SP cells was reduced in cyclopamine-treated cultures. When combined with E6-silencing cyclopamine resulted in loss of SP cell’s sphere-forming ability. Co-inhibition of GLI1 and E6 in cervical cancer cells showed additive anti-cancer effects. Overall, our data show existence of a cooperative interaction between GLI signaling and HPVE6. PMID:27678330

  18. Increased expression of PD-L1 by the human papillomavirus 16 E7 oncoprotein inhibits anticancer immunity

    PubMed Central

    Liu, Chaoqi; Lu, Jiao; Tian, Huiqun; Du, Wei; Zhao, Lin; Feng, Jing; Yuan, Ding; Li, Zhiying

    2017-01-01

    Cytotoxic T lymphocyte dysfunction is frequently associated with PD-L1/PD-1 pathway activation, and is a principal obstacle in cancer therapy. In the present study, the mechanisms underlying the human papillomavirus (HPV)-induced evasion of cervical cancer cells to the host immune system via the programmed death ligand 1/programmed death 1 (PD-L1/PD-1) signaling pathway was investigated. A significant increase in the expression of the HPV16E7 viral protein and PD-L1 in cervical tissues was observed when compared with normal cervical tissues. In addition, a positive correlation between HPV16E7 and PD-L1 expression was observed by immunohistochemical staining and reverse transcription-polymerase chain reaction. Overexpressing HPV16E7 oncoprotein in the epithelial carcinoma of PC3 cells increased the expression level of the PD-L1 protein and inhibited peripheral blood mononuclear cell (PBMC) proliferation and cytotoxic T lymphocyte (CTL) activity. Upon knockdown of HPV16E7 in HPV16-associated CaSki cervical cancer cells with a relevant siRNA, a reduction in PD-L1 protein expression was observed, as well as a significant increase in PBMC proliferation and CTL activity. A recombinant plasmid, MSCVPIG-soluble PD-1, was constructed and transfected into the CaSki cell line, and was co-cultured with PBMCs. PBMC proliferation and CTL activity were observed to increase significantly. In conclusion, the results presented in the current study suggest that overexpression of PD-L1, induced by HPV16E7, may be responsible for lymphocyte dysfunction. In addition, soluble PD-1 may restore the function of tumor-infiltrating lymphocytes by inhibiting the PD-L1/PD-1 signaling pathway. These results may provide a novel insight for immunotherapeutic approaches in the treatment of cervical cancer. PMID:28075442

  19. The Human Papillomavirus Type 16 E5 Oncoprotein Inhibits Epidermal Growth Factor Trafficking Independently of Endosome Acidification ▿

    PubMed Central

    Suprynowicz, Frank A.; Krawczyk, Ewa; Hebert, Jess D.; Sudarshan, Sawali R.; Simic, Vera; Kamonjoh, Christopher M.; Schlegel, Richard

    2010-01-01

    The human papillomavirus type 16 E5 oncoprotein (16E5) enhances acute, ligand-dependent activation of the epidermal growth factor receptor (EGFR) and concomitantly alkalinizes endosomes, presumably by binding to the 16-kDa “c” subunit of the V-ATPase proton pump (16K) and inhibiting V-ATPase function. However, the relationship between 16K binding, endosome alkalinization, and altered EGFR signaling remains unclear. Using an antibody that we generated against 16K, we found that 16E5 associated with only a small fraction of endogenous 16K in keratinocytes, suggesting that it was unlikely that E5 could significantly affect V-ATPase function by direct inhibition. Nevertheless, E5 inhibited the acidification of endosomes, as determined by a new assay using a biologically active, pH-sensitive fluorescent EGF conjugate. Since we also found that 16E5 did not alter cell surface EGF binding, the number of EGFRs on the cell surface, or the endocytosis of prebound EGF, we postulated that it might be blocking the fusion of early endosomes with acidified vesicles. Our studies with pH-sensitive and -insensitive fluorescent EGF conjugates and fluorescent dextran confirmed that E5 prevented endosome maturation (acidification and enlargement) by inhibiting endosome fusion. The E5-dependent defect in vesicle fusion was not due to detectable disruption of actin, tubulin, vimentin, or cytokeratin filaments, suggesting that membrane fusion was being directly affected rather than vesicle transport. Perhaps most importantly, while bafilomycin A1 (like E5) binds to 16K and inhibits endosome acidification, it did not mimic the ability of E5 to inhibit endosome enlargement or the trafficking of EGF. Thus, 16E5 alters EGF endocytic trafficking via a pH-independent inhibition of vesicle fusion. PMID:20686024

  20. Regulation of the Abundance of Kaposi’s Sarcoma-Associated Herpesvirus ORF50 Protein by Oncoprotein MDM2

    PubMed Central

    Chang, Tzu-Hsuan; Chen, Lee-Wen; Shih, Ying-Ju; Chang, Li-Kwan; Liu, Shih-Tung; Chang, Pey-Jium

    2016-01-01

    The switch between latency and the lytic cycle of Kaposi’s sarcoma-associated herpesvirus (KSHV) is controlled by the expression of virally encoded ORF50 protein. Thus far, the regulatory mechanism underlying the protein stability of ORF50 is unknown. Our earlier studies have demonstrated that a protein abundance regulatory signal (PARS) at the ORF50 C-terminal region modulates its protein abundance. The PARS region consists of PARS-I (aa 490–535) and PARS-II (aa 590–650), and mutations in either component result in abundant expression of ORF50. Here, we show that ORF50 protein is polyubiquitinated and its abundance is controlled through the proteasomal degradation pathway. The PARS-I motif mainly functions as a nuclear localization signal in the control of ORF50 abundance, whereas the PARS-II motif is required for the binding of ubiquitin enzymes in the nucleus. We find that human oncoprotein MDM2, an ubiquitin E3 ligase, is capable of interacting with ORF50 and promoting ORF50 degradation in cells. The interaction domains between both proteins are mapped to the PARS region of ORF50 and the N-terminal 220-aa region of MDM2. Additionally, we identify lysine residues at positions 152 and 154 in the N-terminal domain of ORF50 critically involved in MDM2-mediated downregulation of ORF50 levels. Within KSHV-infected cells, the levels of MDM2 were greatly reduced during viral lytic cycle and genetic knockdown of MDM2 in these cells favored the enhancement of ORF50 expression, supporting that MDM2 is a negative regulator of ORF50 expression. Collectively, the study elucidates the regulatory mechanism of ORF50 stability and implicates that MDM2 may have a significant role in the maintenance of viral latency by lowering basal level of ORF50. PMID:27698494

  1. Regulation of the Abundance of Kaposi's Sarcoma-Associated Herpesvirus ORF50 Protein by Oncoprotein MDM2.

    PubMed

    Chang, Tzu-Hsuan; Wang, Shie-Shan; Chen, Lee-Wen; Shih, Ying-Ju; Chang, Li-Kwan; Liu, Shih-Tung; Chang, Pey-Jium

    2016-10-01

    The switch between latency and the lytic cycle of Kaposi's sarcoma-associated herpesvirus (KSHV) is controlled by the expression of virally encoded ORF50 protein. Thus far, the regulatory mechanism underlying the protein stability of ORF50 is unknown. Our earlier studies have demonstrated that a protein abundance regulatory signal (PARS) at the ORF50 C-terminal region modulates its protein abundance. The PARS region consists of PARS-I (aa 490-535) and PARS-II (aa 590-650), and mutations in either component result in abundant expression of ORF50. Here, we show that ORF50 protein is polyubiquitinated and its abundance is controlled through the proteasomal degradation pathway. The PARS-I motif mainly functions as a nuclear localization signal in the control of ORF50 abundance, whereas the PARS-II motif is required for the binding of ubiquitin enzymes in the nucleus. We find that human oncoprotein MDM2, an ubiquitin E3 ligase, is capable of interacting with ORF50 and promoting ORF50 degradation in cells. The interaction domains between both proteins are mapped to the PARS region of ORF50 and the N-terminal 220-aa region of MDM2. Additionally, we identify lysine residues at positions 152 and 154 in the N-terminal domain of ORF50 critically involved in MDM2-mediated downregulation of ORF50 levels. Within KSHV-infected cells, the levels of MDM2 were greatly reduced during viral lytic cycle and genetic knockdown of MDM2 in these cells favored the enhancement of ORF50 expression, supporting that MDM2 is a negative regulator of ORF50 expression. Collectively, the study elucidates the regulatory mechanism of ORF50 stability and implicates that MDM2 may have a significant role in the maintenance of viral latency by lowering basal level of ORF50.

  2. Increased expression of PD‑L1 by the human papillomavirus 16 E7 oncoprotein inhibits anticancer immunity.

    PubMed

    Liu, Chaoqi; Lu, Jiao; Tian, Huiqun; Du, Wei; Zhao, Lin; Feng, Jing; Yuan, Ding; Li, Zhiying

    2017-03-01

    Cytotoxic T lymphocyte dysfunction is frequently associated with PD‑L1/PD‑1 pathway activation, and is a principal obstacle in cancer therapy. In the present study, the mechanisms underlying the human papillomavirus (HPV)‑induced evasion of cervical cancer cells to the host immune system via the programmed death ligand  1/programmed death 1 (PD‑L1/PD‑1) signaling pathway was investigated. A significant increase in the expression of the HPV16E7 viral protein and PD‑L1 in cervical tissues was observed when compared with normal cervical tissues. In addition, a positive correlation between HPV16E7 and PD‑L1 expression was observed by immunohistochemical staining and reverse transcription‑polymerase chain reaction. Overexpressing HPV16E7 oncoprotein in the epithelial carcinoma of PC3 cells increased the expression level of the PD‑L1 protein and inhibited peripheral blood mononuclear cell (PBMC) proliferation and cytotoxic T lymphocyte (CTL) activity. Upon knockdown of HPV16E7 in HPV16‑associated CaSki cervical cancer cells with a relevant siRNA, a reduction in PD‑L1 protein expression was observed, as well as a significant increase in PBMC proliferation and CTL activity. A recombinant plasmid, MSCVPIG‑soluble PD‑1, was constructed and transfected into the CaSki cell line, and was co‑cultured with PBMCs. PBMC proliferation and CTL activity were observed to increase significantly. In conclusion, the results presented in the current study suggest that overexpression of PD‑L1, induced by HPV16E7, may be responsible for lymphocyte dysfunction. In addition, soluble PD‑1 may restore the function of tumor‑infiltrating lymphocytes by inhibiting the PD‑L1/PD‑1 signaling pathway. These results may provide a novel insight for immunotherapeutic approaches in the treatment of cervical cancer.

  3. Increased expression of PD-L1 by the human papillomavirus 16 E7 oncoprotein inhibits anticancer immunity.

    PubMed

    Liu, Chaoqi; Lu, Jiao; Tian, Huiqun; Du, Wei; Zhao, Lin; Feng, Jing; Yuan, Ding; Li, Zhiying

    2016-12-23

    Cytotoxic T lymphocyte dysfunction is frequently associated with PD‑L1/PD‑1 pathway activation, and is a principal obstacle in cancer therapy. In the present study, the mechanisms underlying the human papillomavirus (HPV)‑induced evasion of cervical cancer cells to the host immune system via the programmed death ligand  1/programmed death 1 (PD‑L1/PD‑1) signaling pathway was investigated. A significant increase in the expression of the HPV16E7 viral protein and PD‑L1 in cervical tissues was observed when compared with normal cervical tissues. In addition, a positive correlation between HPV16E7 and PD‑L1 expression was observed by immunohistochemical staining and reverse transcription‑polymerase chain reaction. Overexpressing HPV16E7 oncoprotein in the epithelial carcinoma of PC3 cells increased the expression level of the PD‑L1 protein and inhibited peripheral blood mononuclear cell (PBMC) proliferation and cytotoxic T lymphocyte (CTL) activity. Upon knockdown of HPV16E7 in HPV16‑associated CaSki cervical cancer cells with a relevant siRNA, a reduction in PD‑L1 protein expression was observed, as well as a significant increase in PBMC proliferation and CTL activity. A recombinant plasmid, MSCVPIG‑soluble PD‑1, was constructed and transfected into the CaSki cell line, and was co‑cultured with PBMCs. PBMC proliferation and CTL activity were observed to increase significantly. In conclusion, the results presented in the current study suggest that overexpression of PD‑L1, induced by HPV16E7, may be responsible for lymphocyte dysfunction. In addition, soluble PD‑1 may restore the function of tumor‑infiltrating lymphocytes by inhibiting the PD‑L1/PD‑1 signaling pathway. These results may provide a novel insight for immunotherapeutic approaches in the treatment of cervical cancer.

  4. Human papillomavirus oncoproteins differentially modulate epithelial-mesenchymal transition in 5-FU-resistant cervical cancer cells.

    PubMed

    Vishnoi, Kanchan; Mahata, Sutapa; Tyagi, Abhishek; Pandey, Arvind; Verma, Gaurav; Jadli, Mohit; Singh, Tejveer; Singh, Sukh Mahendra; Bharti, Alok C

    2016-10-01

    Etiological role of viral proteins E6 and E7 of high-risk HPV in cervical carcinogenesis is well established. However, their contribution in chemoresistance and epithelial-mesenchymal transition (EMT) that leads to advanced metastatic lesions and chemoresistance is poorly defined. In the present study, contribution of viral oncoproteins in acquisition of EMT character during onset of chemoresistance was assessed. A chemoresistant cell line (SiHaCR) was developed from an established HPV16-positive cervical cancer cell line, SiHa, by escalating selection pressure of 5-fluorouracil (5-FU). Expression of Survivin, ABCG2, Snail, Slug, Twist, and Vimentin was examined in SiHa and SiHaCR cells by reverse transcriptase-PCR (RT-PCR) and immunoblotting assays. Mesenchymal phenotype in SiHaCR cells was confirmed by assessment of migration and invasion potentials. SiHaCR cells displayed elevated level of functional and molecular markers associated with chemoresistance (Survivin, ABCG2) and EMT (Snail, Slug, Twist, Vimentin) and reduced E-cadherin. SiHaCR also showed increased levels of HPV16 E6 and E7 transcripts. Specific silencing of HPV16 E6, but not E7 using corresponding siRNA, demonstrated a differential involvement of HPV oncogenes in manifestation of EMT. HPV16 E6 silencing resulted in reduction of Slug and Twist expression. However, the expression of Snail and Vimentin was only marginally affected. In contrast, there was an increase in the expression of E-cadherin. A reduced migration and invasion capabilities were observed only in E6-silenced SiHaCR cells, which further confirmed functional contribution of HPV16 E6 in manifestation of EMT. Taken together, our study demonstrated an active involvement of HPV16 E6 in regulation of EMT, which promotes chemoresistance in cervical cancer.

  5. Cross-talk between Human Papillomavirus Oncoproteins and Hedgehog Signaling Synergistically Promotes Stemness in Cervical Cancer Cells.

    PubMed

    Vishnoi, Kanchan; Mahata, Sutapa; Tyagi, Abhishek; Pandey, Arvind; Verma, Gaurav; Jadli, Mohit; Singh, Tejveer; Singh, Sukh Mahendra; Bharti, Alok C

    2016-09-28

    Viral oncoproteins E6/E7 play key oncogenic role in human papillomavirus (HPV)-mediated cervical carcinogenesis in conjunction with aberrant activation of cellular signaling events. GLI-signaling has been implicated in metastasis and tumor recurrence of cervical cancer. However, the interaction of GLI-signaling with HPV oncogenes is unknown. We examined this relationship in established HPV-positive and HPV-negative cervical cancer cell lines using specific GLI inhibitor, cyclopamine and HPVE6/E7 siRNAs. Cervical cancer cell lines showed variable expression of GLI-signaling components. HPV16-positive SiHa cells, overexpressed GLI1, Smo and Patch. Inhibition by cyclopamine resulted in dose-dependent reduction of Smo and GLI1 and loss of cell viability with a higher magnitude in HPV-positive cells. Cyclopamine selectively downregulated HPVE6 expression and resulted in p53 accumulation, whereas HPVE7 and pRb level remained unaffected. siRNA-mediated silencing of HPV16E6 demonstrated reduced GLI1 transcripts in SiHa cells. Cervical cancer stem-like cells isolated by side population analysis, displayed retention of E6 and GLI1 expression. Fraction of SP cells was reduced in cyclopamine-treated cultures. When combined with E6-silencing cyclopamine resulted in loss of SP cell's sphere-forming ability. Co-inhibition of GLI1 and E6 in cervical cancer cells showed additive anti-cancer effects. Overall, our data show existence of a cooperative interaction between GLI signaling and HPVE6.

  6. The lncRNA HOTAIRM1 regulates the degradation of PML-RARA oncoprotein and myeloid cell differentiation by enhancing the autophagy pathway

    PubMed Central

    Chen, Zhen-Hua; Wang, Wen-Tao; Huang, Wei; Fang, Ke; Sun, Yu-Meng; Liu, Shu-Rong; Luo, Xue-Qun; Chen, Yue-Qin

    2017-01-01

    Increasing evidence has indicated that long noncoding RNAs (lncRNAs) are of great importance in different cell contexts. However, only a very small number of lncRNAs have been experimentally validated and functionally annotated during human hematopoiesis. Here, we report an lncRNA, HOTAIRM1, which is associated with myeloid differentiation and has pivotal roles in the degradation of oncoprotein PML-RARA and in myeloid cell differentiation by regulating autophagy pathways. We first revealed that HOTAIRM1 has different variants that are expressed at different levels in cells and that the expression pattern of HOTAIRM1 is closely related to that of the PML-RARA oncoprotein in acute promyelocytic leukemia (APL) patients. We further revealed that the downregulation of HOTAIRM1 could inhibit all-trans retinoic acid (ATRA) -induced degradation of PML-RARA in APL cells and repress the process of differentiation from promyelocytic to granulocytic cells. More importantly, we found that HOTAIRM1 regulates autophagy and that autophagosome formation was inhibited when HOTAIRM1 expression was reduced in the cells. Finally, through the use of a dual luciferase activity assay, AGO2 RNA immunoprecipitation and RNA pull-down, HOTAIRM1 was revealed to act as a microRNA sponge in a pathway that included miR-20a/106b, miR-125b and their targets ULK1, E2F1 and DRAM2. We constructed a human APL-ascites SCID mouse model to validate the function of HOTAIRM1 and its regulatory pathway in vivo. This is the first report showing that a lncRNAs regulates autophagy and the degradation of the PML-RARA oncoprotein during the process of myeloid cell differentiation blockade, suggesting that lncRNAs may be the potential therapeutic targets for leukemia. PMID:27740626

  7. E4orf6 Variants with Separate Abilities To Augment Adenovirus Replication and Direct Nuclear Localization of the E1B 55-Kilodalton Protein

    PubMed Central

    Orlando, Joseph S.; Ornelles, David A.

    2002-01-01

    The E4orf6 protein of group C adenovirus is an oncoprotein that, in association with the E1B 55-kDa protein and by E1B-independent means, promotes virus replication. An arginine-faced amphipathic α-helix in the E4orf6 protein is required for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein and to enhance replication of an E4 deletion virus. In this study, E4orf6 protein variants containing arginine substitutions in the amphipathic α-helix were analyzed. Two of the six arginine residues within the α-helix, arginine-241 and arginine-243, were critical for directing nuclear localization of the E1B 55-kDa protein. The four remaining arginine residues appear to provide a net positive charge for the E4orf6 protein to direct nuclear localization of the E1B 55-kDa protein. The molecular determinants of the arginine-faced amphipathic α-helix that were required for the functional interaction between the E4orf6 and E1B 55-kDa proteins seen in the transfected cell differed from those required to support a productive infection. Several E4orf6 protein variants with arginine-to-glutamic acid substitutions that failed to direct nuclear localization of the E1B 55-kDa protein restored replication of an E4 deletion virus. Additionally, a variant containing an arginine-to-alanine substitution at position 243 that directed nuclear localization of the E1B 55-kDa protein failed to enhance virus replication. These results indicate that the ability of the E4orf6 protein to relocalize the E1B 55-kDa protein to the nucleus can be separated from the ability of the E4orf6 protein to support a productive infection. PMID:11773420

  8. Adenovirus disease in six small bowel, kidney and heart transplant recipients; pathology and clinical outcome.

    PubMed

    Mehta, Vikas; Chou, Pauline C; Picken, Maria M

    2015-11-01

    Adenoviruses are emerging as important viral pathogens in hematopoietic stem cell and solid organ transplant recipients, impacting morbidity, graft survival, and even mortality. The risk seems to be highest in allogeneic hematopoietic stem cell transplant recipients as well as heart, lung, and small bowel transplant recipients. Most of the adenovirus diseases develop in the first 6 months after transplantation, particularly in pediatric patients. Among abdominal organ recipients, small bowel grafts are most frequently affected, presumably due to the presence of a virus reservoir in the mucosa-associated lymphoid tissue. Management of these infections may be difficult and includes the reduction of immunosuppression, whenever possible, combined with antiviral therapy, if necessary. Therefore, an awareness of the pathology associated with such infections is important in order to allow early detection and specific treatment. We reviewed six transplant recipients (small bowel, kidney, and heart) with adenovirus graft involvement from two institutions. We sought to compare the diagnostic morphology and the clinical and laboratory findings. The histopathologic features of an adenovirus infection of the renal graft and one native kidney in a heart transplant recipient included a vaguely granulomatous mixed inflammatory infiltrate associated with rare cells showing a cytopathic effect (smudgy nuclei). A lymphocytic infiltrate, simulating T cell rejection, with admixture of eosinophils was also seen. In the small bowel grafts, there was a focal mixed inflammatory infiltrate with associated necrosis in addition to cytopathic effects. In the heart, allograft adenovirus infection was silent with no evidence of inflammatory changes. Immunohistochemical stain for adenovirus was positive in all grafts and in one native kidney. All patients were subsequently cleared of adenovirus infection, as evidenced by follow-up biopsies, with no loss of the grafts. Adenovirus infection can

  9. Lesions and transmission of experimental adenovirus hemorrhagic disease in black-tailed deer fawns.

    PubMed

    Woods, L W; Hanley, R S; Chiu, P H; Lehmkuhl, H D; Nordhausen, R W; Stillian, M H; Swift, P K

    1999-03-01

    Adenovirus infection was the cause of an epizootic of hemorrhagic disease that is believed to have killed thousands of mule deer (Odocoileus hemionus) in California during the latter half of 1993. A systemic vasculitis with pulmonary edema and hemorrhagic enteropathy or a localized vasculitis associated with necrotizing stomatitis/pharyngitis/glossitis or osteomyelitis of the jaw were common necropsy findings in animals that died during this epizootic. To study transmission of adenovirus infection in deer and susceptibility of black-tailed deer (Odocoileus hemionus columbianus) fawns to adenovirus infection, six 3-6-month-old black-tailed fawns were divided into two treatment groups. One group was inoculated intravenously and the other group was inoculated through the mucous membranes of the eyes, nose and mouth with purified adenovirus. Each treatment group also included two additional fawns (four total) that were not inoculated but were exposed to inoculated animals (contact animals). One fawn served as a negative control. Between 4 and 16 days postinoculation, 8/10 fawns developed systemic or localized infection with lesions identical to lesions seen in animals with natural disease that died during the epizootic. Transmission was by direct contact, and the route of inoculation did not affect the incubation period or the distribution of the virus (systemic or the localized infection). Immunohistochemical analysis using polyclonal antiserum against bovine adenovirus type 5 demonstrated staining in endothelial cells of vessels in numerous tissues in animals with systemic infection and endothelial staining only in vessels subtending necrotic foci in the upper alimentary tract in animals with the localized form of the disease. All inoculated or exposed animals had staining in the tonsillar epithelium. Transmission electron microscopic examination of lung and ileum from two fawns with pulmonary edema and hemorrhagic enteropathy demonstrated endothelial necrosis and

  10. Genome Sequence of a Cynomolgus Macaque Adenovirus (CynAdV-1) Isolate from a Primate Colony in the United Kingdom.

    PubMed

    Zeng, Zhiwei; Zhang, Jing; Jing, Shuping; Cheng, Zetao; Bofill-Mas, Silvia; Maluquer de Motes, Carlos; Hundesa, Ayalkibet; Girones, Rosina; Seto, Donald; Zhang, Qiwei

    2016-11-03

    The genome sequence of a simian adenovirus from a cynomolgus macaque, denoted CynAdV-1, is presented here. Phylogenetic analysis supports CynAdV-1 in an independent clade, comprising a new simian adenovirus (SAdV) species. These genome data are critical for understanding the evolution and relationships of primate adenoviruses, including zoonosis and emergent human pathogens.

  11. Delivery of oncolytic adenovirus into the nucleus of tumorigenic cells by tumor microparticles for virotherapy.

    PubMed

    Ran, Li; Tan, Xiaohua; Li, Yanchun; Zhang, Huafeng; Ma, Ruihua; Ji, Tiantian; Dong, Wenqian; Tong, Tong; Liu, Yuying; Chen, Degao; Yin, Xiaonan; Liang, Xiaoyu; Tang, Ke; Ma, Jingwei; Zhang, Yi; Cao, Xuetao; Hu, Zhuowei; Qin, Xiaofeng; Huang, Bo

    2016-05-01

    Oncolytic viruses have been utilized for the treatment of various cancers. However, delivery of the viral particles to tumor cells remains a major challenge. Microparticles (MP) are vesicle forms of plasma membrane fragments of 0.1-1 μm in size that are shed by cells. We have previously shown the delivery of chemotherapeutic drugs using tumor cell-derived MPs (T-MP). Here we report that T-MPs can be utilized as a unique carrier system to deliver oncolytic adenoviruses to human tumors, leading to highly efficient cytolysis of tumor cells needed for in vivo treatment efficacy. This T-MP-mediated oncolytic virotherapy approach holds multiple advantages, including: 1) delivery of oncolytic adenovirus by T-MPs is able to avoid the antiviral effect of host antibodies; 2) delivery of oncolytic adenovirus by T-MPs is not limited by virus-specific receptor that mediates the entry of virus into tumor cells; 3) T-MPs are apt at delivering oncolytic adenoviruses to the nucleus of tumor cells as well as to stem-like tumor-repopulating cells for the desired purpose of killing them. These findings highlight a novel oncolytic adenovirus delivery system with highly promising clinical applications.

  12. Localization of the N-terminus of minor coat protein IIIa in the adenovirus capsid

    PubMed Central

    San Martín, Carmen; Glasgow, Joel N.; Borovjagin, Anton; Beatty, Matthew S.; Kashentseva, Elena A.; T. Curiel, David; Marabini, Roberto; Dmitriev, Igor P.

    2008-01-01

    Summary Minor coat protein IIIa is conserved in all adenoviruses and required for correct viral assembly, but its precise function in capsid organization is unknown. The latest adenovirus capsid model proposes that IIIa is located underneath the vertex region. To obtain experimental evidence on the location of IIIa and further define its role, we engineered the IIIa gene to encode heterologous N-terminal peptide extensions. Recombinant adenovirus variants with IIIa encoding six-histidine tag (6-His), 6-His and FLAG peptides, or 6-His linked to FLAG with a (Gly4Ser)3 linker were rescued and analyzed for virus yield, capsid incorporation of heterologous peptides, and capsid stability. Longer extensions could not be rescued. Western blot analysis confirmed that the modified IIIa proteins were expressed in infected cells and incorporated into virions. In the adenovirus encoding the 6-His-linker-FLAG-IIIa gene, the 6-His tag was present in light particles but not in mature virions. Immuno-electron microscopy of this virus showed that the FLAG epitope is not accessible to antibodies on the viral particles. Three-dimensional electron microscopy (3DEM) and difference mapping located the IIIa N-terminal extension beneath the vertex complex, wedged at the interface between penton base and the peripentonal hexons, therefore supporting the latest proposed model. The position of the IIIa N-terminus and its low tolerance for modification provide new clues for understanding the role of this minor coat protein in adenovirus capsid assembly and disassembly. PMID:18786542

  13. Nitrogen Gas Plasma Generated by a Static Induction Thyristor as a Pulsed Power Supply Inactivates Adenovirus

    PubMed Central

    Sakudo, Akikazu; Toyokawa, Yoichi; Imanishi, Yuichiro

    2016-01-01

    Adenovirus is one of the most important causative agents of iatrogenic infections derived from contaminated medical devices or finger contact. In this study, we investigated whether nitrogen gas plasma, generated by applying a short high-voltage pulse to nitrogen using a static induction thyristor power supply (1.5 kilo pulse per second), exhibited a virucidal effect against adenoviruses. Viral titer was reduced by one log within 0.94 min. Results from detection of viral capsid proteins, hexon and penton, by Western blotting and immunochromatography were unaffected by the plasma treatment. In contrast, analysis using the polymerase chain reaction suggested that plasma treatment damages the viral genomic DNA. Reactive chemical products (hydrogen peroxide, nitrate, and nitrite), ultraviolet light (UV-A) and slight temperature elevations were observed during the operation of the gas plasma device. Viral titer versus intensity of each potential virucidal factor were used to identify the primary mechanism of disinfection of adenovirus. Although exposure to equivalent levels of UV-A or heat treatment did not inactivate adenovirus, treatment with a relatively low concentration of hydrogen peroxide efficiently inactivated the virus. Our results suggest the nitrogen gas plasma generates reactive chemical products that inactivate adenovirus by damaging the viral genomic DNA. PMID:27322066

  14. Correction of a deletion mutant by gene targeting with an adenovirus vector.

    PubMed Central

    Wang, Q; Taylor, M W

    1993-01-01

    The usefulness of adenovirus type 5 as a vector for homologous recombination was examined in CHO cells by using the adenine phosphoribosyltransferase (aprt) gene. Infection of a hemizygous CHO APRT- cell line containing a 3-bp deletion in exon 5 of the aprt gene with a recombinant adenovirus containing the wild-type gene resulted in restoration of the APRT+ phenotype at a frequency of 10(-5) to 10(-6) per infected cell. A relatively high frequency (approximately 6 to 20%) of the transductants appears to result from a homologous recombination event. The mutation on the chromosomal aprt gene is corrected in the homologous recombinants, and APRT expression is restored to a normal hemizygous level. Neither adenovirus nor exogenous promoter sequences are detected in the homologous recombinants. The remaining transductants result from random integration of the aprt gene with the adenovirus sequence. A number of adenovirus vectors containing different promoter sequences linked to the hamster aprt gene were constructed. A possible role for the promoter region in the homologous recombination event was indicated by the lack of homologous recombination in constructs lacking an active promoter. Images PMID:8423811

  15. Isolation of an adenovirus and an adeno-associated virus from goat kids with enteritis.

    PubMed

    Olson, Erik J; Haskell, Scott R R; Frank, Rodney K; Lehmkuhl, Howard D; Hobbs, Lea Ann; Warg, Janet V; Landgraf, John G; Wünschmann, Arno

    2004-09-01

    A dairy goat operation in Minnesota experienced a sudden, markedly increased mortality among its neonatal goats. Approximately 60 of 130 kids (46%) died. The animals had diarrhea and dyspnea of 1-2 days duration before death. Necropsy of 4 goat kids revealed marked, acute, catarrhal enteritis and fibrinous pleuropneumonia. Mannheimia haemolytica was isolated from the lungs. Basophilic inclusion bodies filling the entire nucleus were present in enterocytes of the ileum of 3 goats. Adenoviral particles were detected in the feces by electron microscopy and adenovirus was subsequently isolated from the intestinal content together with a parvo-like virus (dependovirus). Morphology, physicochemical characteristics, and neutralization tests indicated that the adenovirus resembled ovine adenovirus-2 (OAdV-2). However, the PstI restriction endonuclease pattern produced by the goat adenovirus was distinct from that of OAdV-2. This is the first report of enteritis in goats with an adenovirus antigenically related to OAdV-2 and with a parvo-like dependovirus.

  16. Hyperplastic stomatitis and esophagitis in a tortoise (Testudo graeca) associated with an adenovirus infection.

    PubMed

    Garcia-Morante, Beatriz; Pénzes, Judit J; Costa, Taiana; Martorell, Jaime; Martínez, Jorge

    2016-09-01

    A 2-year-old female, spur-thighed tortoise (Testudo graeca) was presented with poor body condition (1/5) and weakness. Fecal analysis revealed large numbers of oxyurid-like eggs, and radiographs were compatible with gastrointestinal obstruction. Despite supportive medical treatment, the animal died. At gross examination, an intestinal obstruction was confirmed. Histopathology revealed severe hyperplastic esophagitis and stomatitis with marked epithelial cytomegaly and enormous basophilic intranuclear inclusion bodies. Electron microscopy examination revealed a large number of 60-80 nm, nonenveloped, icosahedral virions arranged in crystalline arrays within nuclear inclusions of esophageal epithelial cells, morphologically compatible with adenovirus-like particles. PCR for virus identification was performed with DNA extracted from formalin-fixed, paraffin-embedded tissues. A nested, consensus pan-adenovirus PCR and sequencing analysis showed a novel adenovirus. According to phylogenetic calculations, it clustered to genus Atadenovirus in contrast with all other chelonian adenoviruses described to date. The present report details the pathologic findings associated with an adenovirus infection restricted to the upper digestive tract.

  17. A Novel Vaccine Approach for Chagas Disease Using Rare Adenovirus Serotype 48 Vectors

    PubMed Central

    Farrow, Anitra L.; Peng, Binghao J.; Gu, Linlin; Krendelchtchikov, Alexandre; Matthews, Qiana L.

    2016-01-01

    Due to the increasing amount of people afflicted worldwide with Chagas disease and an increasing prevalence in the United States, there is a greater need to develop a safe and effective vaccine for this neglected disease. Adenovirus serotype 5 (Ad5) is the most common adenovirus vector used for gene therapy and vaccine approaches, but its efficacy is limited by preexisting vector immunity in humans resulting from natural infections. Therefore, we have employed rare serotype adenovirus 48 (Ad48) as an alternative choice for adenovirus/Chagas vaccine therapy. In this study, we modified Ad5 and Ad48 vectors to contain T. cruzi’s amastigote surface protein 2 (ASP-2) in the adenoviral early gene. We also modified Ad5 and Ad48 vectors to utilize the “Antigen Capsid-Incorporation” strategy by adding T. cruzi epitopes to protein IX (pIX). Mice that were immunized with the modified vectors were able to elicit T. cruzi-specific humoral and cellular responses. This study indicates that Ad48-modified vectors function comparable to or even premium to Ad5-modified vectors. This study provides novel data demonstrating that Ad48 can be used as a potential adenovirus vaccine vector against Chagas disease. PMID:26978385

  18. Characterization of the knob domain of the adenovirus type 5 fiber protein expressed in Escherichia coli.

    PubMed Central

    Henry, L J; Xia, D; Wilke, M E; Deisenhofer, J; Gerard, R D

    1994-01-01

    The adenovirus fiber protein is used for attachment of the virus to a specific receptor on the cell surface. Structurally, the protein consists of a long, thin shaft that protrudes from the vertex of the virus capsid and terminates in a globular domain termed the knob. To verify that the knob is the domain which interacts with the cellular receptor, we have cloned and expressed the knob from adenovirus type 5 together with a single repeat of the shaft in Escherichia coli. The protein was purified by conventional chromatography and functionally characterized for its interaction with the adenovirus receptor. The recombinant knob domain bound about 4,700 sites per HeLa cell with an affinity of 3 x 10(9) M-1 and blocked adenovirus infection of human cells. Antibodies raised against the knob also blocked virus infection. By gel filtration and X-ray diffraction analysis of protein crystals, the knob was shown to consist of a homotrimer of 21-kDa subunits. The results confirm that the trimeric knob is the ligand for attachment to the adenovirus receptor. Images PMID:8035520

  19. Innate Functions of Immunoglobulin M Lessen Liver Gene Transfer with Helper-Dependent Adenovirus

    PubMed Central

    Unzu, Carmen; Morales-Kastresana, Aizea; Sampedro, Ana; Serrano-Mendioroz, Irantzu; Azpilikueta, Arantza; Ochoa, María Carmen; Dubrot, Juan; Martínez-Ansó, Eduardo

    2014-01-01

    The immune system poses obstacles to viral vectors, even in the first administration to preimmunized hosts. We have observed that the livers of B cell-deficient mice were more effectively transduced by a helper-dependent adenovirus serotype-5 (HDA) vector than those of WT mice. This effect was T-cell independent as shown in athymic mice. Passive transfer of the serum from adenovirus-naïve WT to Rag1KO mice resulted in a reduction in gene transfer that was traced to IgM purified from serum of adenovirus-naïve mice. To ascribe the gene transfer inhibition activity to either adenoviral antigen-specific or antigen-unspecific functions of IgM, we used a monoclonal IgM antibody of unrelated specificity. Both the polyclonal and the irrelevant monoclonal IgM inhibited gene transfer by the HDA vector to either cultured hepatocellular carcinoma cells or to the liver of mice in vivo. Adsorption of polyclonal or monoclonal IgMs to viral capsids was revealed by ELISAs on adenovirus-coated plates. These observations indicate the existence of an inborn IgM mechanism deployed against a prevalent virus to reduce early post-infection viremia. In conclusion, innate IgM binding to adenovirus serotype-5 capsids restrains gene-transfer and offers a mechanism to be targeted for optimization of vector dosage in gene therapy with HDA vectors. PMID:24465560

  20. Heterologous Immunity between Adenoviruses and Hepatitis C Virus: A New Paradigm in HCV Immunity and Vaccines

    PubMed Central

    Singh, Shakti; Vedi, Satish; Samrat, Subodh Kumar; Li, Wen; Kumar, Rakesh; Agrawal, Babita

    2016-01-01

    Adenoviruses (Ad) are commonly used as vectors for gene therapy and/or vaccine delivery. Recombinant Ad vectors are being tested as vaccines for many pathogens. We have made a surprising observation that peptides derived from various hepatitis C virus (HCV) antigens contain extensive regions of homology with multiple adenovirus proteins, and conclusively demonstrate that adenovirus vector can induce robust, heterologous cellular and humoral immune responses against multiple HCV antigens. Intriguingly, the induction of this cross-reactive immunity leads to significant reduction of viral loads in a recombinant vaccinia-HCV virus infected mouse model, supporting their role in antiviral immunity against HCV. Healthy human subjects with Ad-specific pre-existing immunity demonstrated cross-reactive cellular and humoral immune responses against multiple HCV antigens. These findings reveal the potential of a previously uncharacterized property of natural human adenovirus infection to dictate, modulate and/or alter the course of HCV infection upon exposure. This intrinsic property of adenovirus vectors to cross-prime HCV immunity can also be exploited to develop a prophylactic and/or therapeutic vaccine against HCV. PMID:26751211

  1. Adenovirus and mycoplasma infection in an ornate box turtle (Terrapene ornata ornata) in Hungary.

    PubMed

    Farkas, Szilvia L; Gál, János

    2009-07-02

    A female, adult ornate box turtle (Terrapene ornata ornata) with fatty liver was submitted for virologic examination in Hungary. Signs of an adenovirus infection including degeneration of the liver cells, enlarged nuclei and intranuclear inclusion bodies were detected by light microscopic examination. The presence of an adenovirus was later confirmed by obtaining partial sequence data from the adenoviral DNA-dependent DNA-polymerase. Phylogenetic analyses revealed that this novel chelonian adenovirus was distinct from previously described reptilian adenoviruses, not belonging to any of the recognized genera of the family Adenoviridae. As a part of the routine diagnostic procedure for chelonians the detection of herpes-, rana- and iridoviruses together with Mycoplasma spp. was attempted. Amplicons were generated by a general mycoplasma polymerase chain reaction (PCR) targeting the 16S/23S ribosomal RNA (rRNA) intergenic spacer region, as well as, a specific Mycoplasma agassizii PCR targeting the 16S rRNA gene. Based on the analyses of partial sequences of the 16S rRNA gene, the Mycoplasma sp. of the ornate box turtle seemed to be identical with the recently described eastern box turtle (Terrapene carolina carolina) Mycoplasma sp. This is the first report of a novel chelonian adenovirus and a mycoplasma infection in an ornate box turtle (T. ornata ornata) in Europe.

  2. Radioisotopic imaging allows optimization of adenovirus lung deposition for cystic fibrosis gene therapy.

    PubMed

    Lerondel, S; Le Pape, A; Sené, C; Faure, L; Bernard, S; Diot, P; Nicolis, E; Mehtali, M; Lusky, M; Cabrini, G; Pavirani, A

    2001-01-01

    Cystic fibrosis is a common, heriditary disease resulting from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Airway transfer of the CFTR gene is a potential strategy to treat or prevent the lung pathology that is the main cause of morbidity and mortality. Among the vectors used for gene therapy, adenoviruses have shown their ability to transfer the CFTR gene to respiratory epithelial cells, using either instillation or nebulization. Our objective was to characterize the lung deposition of aerosolized adenovirus by quantitative radioisotopic imaging, the only noninvasive technique allowing in vivo quantitation of inhaled drugs. We first labeled an adenovirus expressing human CFTR with the gamma-emitting radioisotope, technetium 99m (99mTc), and determined the best labeling conditions to allow preservation of virus bioactivity. We then administered the radioaerosol to baboons, determined lung regional deposition of 99mTc-labeled adenovirus, and compared the expression of CFTR transcripts 3 and 21 days after inhalation. The expression of vector-encoded mRNA ranged from 4 to 22% with respect to the endogenous CFTR mRNA depending on the lung segments. Moreover, we have developed a model using 99mTc-DTPA (diethylenetriamine pentaacetic acid), which can be used, as an alternative to adenovirus, to determine the profile of lung deposition of the vector. This study demonstrates that scintigraphy is a useful technique to achieve optimization of gene administration to the airways.

  3. Heterologous Immunity between Adenoviruses and Hepatitis C Virus: A New Paradigm in HCV Immunity and Vaccines.

    PubMed

    Singh, Shakti; Vedi, Satish; Samrat, Subodh Kumar; Li, Wen; Kumar, Rakesh; Agrawal, Babita

    2016-01-01

    Adenoviruses (Ad) are commonly used as vectors for gene therapy and/or vaccine delivery. Recombinant Ad vectors are being tested as vaccines for many pathogens. We have made a surprising observation that peptides derived from various hepatitis C virus (HCV) antigens contain extensive regions of homology with multiple adenovirus proteins, and conclusively demonstrate that adenovirus vector can induce robust, heterologous cellular and humoral immune responses against multiple HCV antigens. Intriguingly, the induction of this cross-reactive immunity leads to significant reduction of viral loads in a recombinant vaccinia-HCV virus infected mouse model, supporting their role in antiviral immunity against HCV. Healthy human subjects with Ad-specific pre-existing immunity demonstrated cross-reactive cellular and humoral immune responses against multiple HCV antigens. These findings reveal the potential of a previously uncharacterized property of natural human adenovirus infection to dictate, modulate and/or alter the course of HCV infection upon exposure. This intrinsic property of adenovirus vectors to cross-prime HCV immunity can also be exploited to develop a prophylactic and/or therapeutic vaccine against HCV.

  4. Identification and Application of Neutralizing Epitopes of Human Adenovirus Type 55 Hexon Protein

    PubMed Central

    Tian, Xingui; Ma, Qiang; Jiang, Zaixue; Huang, Junfeng; Liu, Qian; Lu, Xiaomei; Luo, Qingming; Zhou, Rong

    2015-01-01

    Human adenovirus type 55 (HAdV55) is a newly identified re-emergent acute respiratory disease (ARD) pathogen with a proposed recombination of hexon gene between HAdV11 and HAdV14 strains. The identification of the neutralizing epitopes is important for the surveillance and vaccine development against HAdV55 infection. In this study, four type-specific epitope peptides of HAdV55 hexon protein, A55R1 (residues 138 to 152), A55R2 (residues 179 to 187), A55R4 (residues 247 to 259) and A55R7 (residues 429 to 443), were predicted by multiple sequence alignment and homology modeling methods, and then confirmed with synthetic peptides by enzyme-linked immunosorbent assay (ELISA) and neutralization tests (NT). Finally, the A55R2 was incorporated into human adenoviruses 3 (HAdV3) and a chimeric adenovirus rAd3A55R2 was successfully obtained. The chimeric rAd3A55R2 could induce neutralizing antibodies against both HAdV3 and HAdV55. This current study will contribute to the development of novel adenovirus vaccine candidate and adenovirus structural analysis. PMID:26516903

  5. Screening for adenoviruses in haematological neoplasia: High prevalence in mantle cell lymphoma.

    PubMed

    Kosulin, Karin; Rauch, Margit; Ambros, Peter F; Pötschger, Ulrike; Chott, Andreas; Jäger, Ulrich; Drach, Johannes; Nader, Alexander; Lion, Thomas

    2014-02-01

    Human adenoviruses possess oncogenic capacity which is well documented in mammalian animal models, but their possible implication in human malignancy has remained enigmatic. Following primary infection, adenoviruses can persist in a latent state in lymphocytes where the virus is apparently able to evade immune surveillance. In the present study, we have employed a broad-spectrum adenovirus polymerase chain reaction (PCR) assay to systematically screen more than 200 diagnostic specimens of different lymphoid malignancies including acute lymphocytic leukaemia (n=50), chronic lymphocytic leukaemia (n=50), various types of malignant lymphoma (n=100) and multiple myeloma (n=11) for the presence of adenoviral sequences. While most entities analysed revealed negative findings in virtually all specimens tested, adenoviral DNA was detected in 15/36 (42%) mantle cell lymphomas investigated. The most prevalent adenoviral species detected was C, and less commonly B. Adenovirus-positive findings in patients with mantle cell lymphoma were made at different sites including bone marrow (n=7), intestine (n=5), lymph nodes (n=2) and tonsillar tissue (n=1). The presence of adenoviral sequences identified by PCR was confirmed in individual cells by fluorescence in-situ hybridisation (FISH). The frequent observation of adenoviruses in mantle cell lymphoma is intriguings, and raises questions about their possible involvement in the pathogenesis of this lymphoid malignancy.

  6. Adenovirus Core Protein pVII Is Translocated into the Nucleus by Multiple Import Receptor Pathways†

    PubMed Central

    Wodrich, Harald; Cassany, Aurelia; D'Angelo, Maximiliano A.; Guan, Tinglu; Nemerow, Glen; Gerace, Larry

    2006-01-01

    Adenoviruses are nonenveloped viruses with an ∼36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin α, importin β, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome. PMID:16973564

  7. Adenovirus core protein pVII is translocated into the nucleus by multiple import receptor pathways.

    PubMed

    Wodrich, Harald; Cassany, Aurelia; D'Angelo, Maximiliano A; Guan, Tinglu; Nemerow, Glen; Gerace, Larry

    2006-10-01

    Adenoviruses are nonenveloped viruses with an approximately 36-kb double-stranded DNA genome that replicate in the nucleus. Protein VII, an abundant structural component of the adenovirus core that is strongly associated with adenovirus DNA, is imported into the nucleus contemporaneously with the adenovirus genome shortly after virus infection and may promote DNA import. In this study, we evaluated whether protein VII uses specific receptor-mediated mechanisms for import into the nucleus. We found that it contains potent nuclear localization signal (NLS) activity by transfection of cultured cells with protein VII fusion constructs and by microinjection of cells with recombinant protein VII fusions. We identified three NLS-containing regions in protein VII by deletion mapping and determined important NLS residues by site-specific mutagenesis. We found that recombinant protein VII and its NLS-containing domains strongly and specifically bind to importin alpha, importin beta, importin 7, and transportin, which are among the most abundant cellular nuclear import receptors. Moreover, these receptors can mediate the nuclear import of protein VII fusions in vitro in permeabilized cells. Considered together, these data support the hypothesis that protein VII is a major NLS-containing adaptor for receptor-mediated import of adenovirus DNA and that multiple import pathways are utilized to promote efficient nuclear entry of the viral genome.

  8. Innate functions of immunoglobulin M lessen liver gene transfer with helper-dependent adenovirus.

    PubMed

    Unzu, Carmen; Melero, Ignacio; Morales-Kastresana, Aizea; Sampedro, Ana; Serrano-Mendioroz, Irantzu; Azpilikueta, Arantza; Ochoa, María Carmen; Dubrot, Juan; Martínez-Ansó, Eduardo; Fontanellas, Antonio

    2014-01-01

    The immune system poses obstacles to viral vectors, even in the first administration to preimmunized hosts. We have observed that the livers of B cell-deficient mice were more effectively transduced by a helper-dependent adenovirus serotype-5 (HDA) vector than those of WT mice. This effect was T-cell independent as shown in athymic mice. Passive transfer of the serum from adenovirus-naïve WT to Rag1KO mice resulted in a reduction in gene transfer that was traced to IgM purified from serum of adenovirus-naïve mice. To ascribe the gene transfer inhibition activity to either adenoviral antigen-specific or antigen-unspecific functions of IgM, we used a monoclonal IgM antibody of unrelated specificity. Both the polyclonal and the irrelevant monoclonal IgM inhibited gene transfer by the HDA vector to either cultured hepatocellular carcinoma cells or to the liver of mice in vivo. Adsorption of polyclonal or monoclonal IgMs to viral capsids was revealed by ELISAs on adenovirus-coated plates. These observations indicate the existence of an inborn IgM mechanism deployed against a prevalent virus to reduce early post-infection viremia. In conclusion, innate IgM binding to adenovirus serotype-5 capsids restrains gene-transfer and offers a mechanism to be targeted for optimization of vector dosage in gene therapy with HDA vectors.

  9. Prevalence of neutralising antibodies against adenoviruses in lizards and snakes.

    PubMed

    Ball, Inna; Ofner, Sabine; Funk, Richard S; Griffin, Chris; Riedel, Ulf; Möhring, Jens; Marschang, Rachel E

    2014-10-01

    Adenoviruses (AdVs) are relatively common in lizards and snakes, and several genetically distinct AdVs have been isolated in cell culture. The aims of this study were to examine serological relationships among lizard and snake AdVs and to determine the frequency of AdV infections in these species. Isolates from a boa constrictor (Boa constrictor), a corn snake (Pantherophis gutattus) and a central bearded dragon (Pogona vitticeps), and two isolates from helodermatid lizards (Heloderma horridum and H. suspectum) were used in neutralisation tests for the detection of antibodies in plasma from 263 lizards from seven families (including 12 species) and from 141 snakes from four families (including 28 species) from the USA and Europe. Most lizard and snake samples had antibodies against a range of AdV isolates, indicating that AdV infection is common among these squamates. Neutralisation tests with polyclonal antibodies raised in rabbits demonstrated serological cross-reactivity between both helodermatid lizard isolates. However, squamate plasma showed different reactions to each of these lizard isolates in neutralisation tests.

  10. Luciferase imaging for evaluation of oncolytic adenovirus replication in vivo.

    PubMed

    Guse, K; Dias, J D; Bauerschmitz, G J; Hakkarainen, T; Aavik, E; Ranki, T; Pisto, T; Särkioja, M; Desmond, R A; Kanerva, A; Hemminki, A

    2007-06-01

    Oncolytic viruses kill cancer cells by tumor-selective replication. Clinical data have established the safety of the approach but also the need of improvements in potency. Efficacy of oncolysis is linked to effective infection of target cells and subsequent productive replication. Other variables include intratumoral barriers, access to target cells, uptake by non-target organs and immune response. Each of these aspects relates to the location and degree of virus replication. Unfortunately, detection of in vivo replication has been difficult, labor intensive and costly and therefore not much studied. We hypothesized that by coinfection of a luciferase expressing E1-deleted virus with an oncolytic virus, both viruses would replicate when present in the same cell. Photon emission due to conversion of D-Luciferin is sensitive and penetrates tissues well. Importantly, killing of animals is not required and each animal can be imaged repeatedly. Two different murine xenograft models were used and intratumoral coinjections of luciferase encoding virus were performed with eight different oncolytic adenoviruses. In both models, we found significant correlation between photon emission and infectious virus production. This suggests that the system can be used for non-invasive quantitation of the amplitude, persistence and dynamics of oncolytic virus replication in vivo, which could be helpful for the development of more effective and safe agents.

  11. Phylogenomic characterization of California sea lion adenovirus-1.

    PubMed

    Cortés-Hinojosa, Galaxia; Gulland, Frances M D; Goldstein, Tracey; Venn-Watson, Stephanie; Rivera, Rebecca; Waltzek, Thomas B; Salemi, Marco; Wellehan, James F X

    2015-04-01

    Significant adenoviral diversity has been found in humans, but in domestic and wild animals the number of identified viruses is lower. Here we present the complete genome of a recently discovered mastadenovirus, California sea lion adenovirus 1 (CSLAdV-1) isolated from California sea lions (Zalophus californianus), an important pathogen associated with hepatitis in pinnipeds. The genome of this virus has the typical mastadenoviral structure with some notable differences at the carboxy-terminal end, including a dUTPase that does not cluster with other mastadenoviral dUTPases, and a fiber that shows similarity to a trans-sialidase of Trypanosoma cruzi and choline-binding protein A (CbpA) of Streptococcus pneumoniae. The GC content is low (36%), and phylogenetic analyses placed the virus near the root of the clade infecting laurasiatherian hosts in the genus Mastadenovirus. These findings support the hypothesis that CSLAdV-1 in California sea lions represents a host jump from an unknown mammalian host in which it is endemic.

  12. The composed antigenic structure of the adenovirus hexon protein.

    PubMed

    Nász, I

    1988-01-01

    Three panels of MAbs were prepared against AV1, AV35, and BAV2 hexons, respectively. It was shown, that in all cases, antibodies are developed against the genus specific determinant of the adenovirus hexon. The other MAbs specified numerous identical or overlapping epitopes on the hexon types studied. The epitopes could be characterized as intertype-, intersubgenus-, subgenus- and type-specific ones beside the genus-specific determinant based on the RPs of the MAbs from indirect ELISA and passive HA results. The identical epitopes are present in more than one copy on the trimeric form of the hexon capsomer. The epitopes on the hexon molecule could be separated into three antigenic sites, of which one antigenic site is characterized by seven epitope clusters (antigenic site II). Monoclonal antibodies were able to precipitate different hexon types in gel diffusion tests by which the differentiation of the distinct epitopes seemed to be possible. With the help of monoclonal antibodies to AV1 hexon, 17 hours after the infection, hexons (or epitopes) were detected in the cytoplasm and in the nucleus of the infected cells showing different distribution patterns in indirect immunofluorescence assay.

  13. Adenovirus-mediated interleukin (IL)-24 immunotherapy for cancer.

    PubMed

    Ramesh, Rajagopal; Ioannides, Constantine G; Roth, Jack A; Chada, Sunil

    2010-01-01

    Interleukin-24 (IL-24) is a member of the IL-10 cytokine family. IL-24, also known as melanoma differentiation associated gene 7 (mda-7), is a unique cytokine in that it has cytokine properties and functions as a novel tumor suppressor gene. Studies by us and other investigators using viral and non-viral vectors have demonstrated IL-24 overexpression in human cancer cells inhibited tumor growth both in vitro and in vivo. A majority of these studies using immunodeficient animal models have focused on demonstrating the direct anticancer properties of IL-24. Very few studies have focused on studying the immunotherapeutic properties of IL-24 despite it being reported to function as a Th1 cytokine. A phase I clinical trial using an adenovirus vector expressing IL-24 (Ad-IL24/INGN241) reported Ad-IL24 treatment of cancer patients resulted in changes in cytokines and T cells. However, well-designed and detailed preclinical studies to support the clinical findings are warranted. Demonstrating immune modulation by IL-24 will provide a rationale for developing IL-24-based immunotherapeutic approaches for cancer treatment.In the present chapter, we provide experimental details for conducting IL-24-based immunotherapy studies. As it is not possible for the authors to cover all of the information the authors recommend reading other immunology-based literature and procedures for a better understanding of conducting preclinical studies.

  14. Oncolytic Replication of E1b-Deleted Adenoviruses

    PubMed Central

    Cheng, Pei-Hsin; Wechman, Stephen L.; McMasters, Kelly M.; Zhou, Heshan Sam

    2015-01-01

    Various viruses have been studied and developed for oncolytic virotherapies. In virotherapy, a relatively small amount of viruses used in an intratumoral injection preferentially replicate in and lyse cancer cells, leading to the release of amplified viral particles that spread the infection to the surrounding tumor cells and reduce the tumor mass. Adenoviruses (Ads) are most commonly used for oncolytic virotherapy due to their infection efficacy, high titer production, safety, easy genetic modification, and well-studied replication characteristics. Ads with deletion of E1b55K preferentially replicate in and destroy cancer cells and have been used in multiple clinical trials. H101, one of the E1b55K-deleted Ads, has been used for the treatment of late-stage cancers as the first approved virotherapy agent. However, the mechanism of selective replication of E1b-deleted Ads in cancer cells is still not well characterized. This review will focus on three potential molecular mechanisms of oncolytic replication of E1b55K-deleted Ads. These mechanisms are based upon the functions of the viral E1B55K protein that are associated with p53 inhibition, late viral mRNA export, and cell cycle disruption. PMID:26561828

  15. Modification of Antigen Impacts on Memory Quality after Adenovirus Vaccination.

    PubMed

    Colston, Julia M; Bolinger, Beatrice; Cottingham, Matthew G; Gilbert, Sarah; Klenerman, Paul

    2016-04-15

    The establishment of robust T cell memory is critical for the development of novel vaccines for infections and cancers. Classical memory generated by CD8(+)T cells is characterized by contracted populations homing to lymphoid organs. T cell memory inflation, as seen for example after CMV infection, is the maintenance of expanded, functional, tissue-associated effector memory cell pools. Such memory pools may also be induced after adenovirus vaccination, and we recently defined common transcriptional and phenotypic features of these populations in mice and humans. However, the rules that govern which epitopes drive memory inflation compared with classical memory are not fully defined, and thus it is not currently possible to direct this process. We used our adenoviral model of memory inflation to first investigate the role of the promoter and then the role of the epitope context in determining memory formation. Specifically, we tested the hypothesis that conventional memory could be converted to inflationary memory by simple presentation of the Ag in the form of minigene vectors. When epitopes from LacZ and murine CMV that normally induce classical memory responses were presented as minigenes, they induced clear memory inflation. These data demonstrate that, regardless of the transgene promoter, the polypeptide context of a CD8(+)T cell epitope may determine whether classical or inflating memory responses are induced. The ability to direct this process by the use of minigenes is relevant to the design of vaccines and understanding of immune responses to pathogens.

  16. Adenovirus as a gene therapy vector for hematopoietic cells.

    PubMed

    Marini, F C; Yu, Q; Wickham, T; Kovesdi, I; Andreeff, M

    2000-06-01

    Adenovirus (Adv)-mediated gene transfer has recently gained new attention as a means to deliver genes for hematopoietic stem cell (HSC) or progenitor cell gene therapy. In the past, HSCs have been regarded as poor Adv targets, mainly because they lack the specific Adv receptors required for efficient and productive Adv infection. In addition, the nonintegrating nature of Adv has prevented its application to HSC and bone marrow transduction protocols where long-term expression is required. There is even controversy as to whether Adv can infect hematopoietic cells at all. In fact, the ability of Adv to infect epithelium-based targets and its inability to effectively transfect HSCs have been used in the development of eradication schemes that use Adv to preferentially infect and "purge" tumor cell-contaminating HSC grafts. However, there are data supporting the existence of productive Adv infections into HSCs. Such protocols involve the application of cytokine mixtures, high multiplicities of infection, long incubation periods, and more recently, immunological and genetic modifications to Adv itself to enable it to efficiently transfer genes into HSCs. This is a rapidly growing field, both in terms of techniques and applications. This review examines the two sides of the Adv/CD34 controversy as well as the current developments in this field.

  17. Epidemiology of enteric adenovirus infection in prospectively monitored Argentine families.

    PubMed Central

    Mistchenko, A. S.; Huberman, K. H.; Gomez, J. A.; Grinstein, S.

    1992-01-01

    To examine the role of enteric adenoviruses (EAV) in an urban area of Buenos Aires (Argentina), we prospectively studied faecal samples from 49 families of newborns. These were monitored weekly for diarrhoea for 2 years. A total of 180 samples from cases of diarrhoea and 766 samples obtained during diarrhoea-free periods were studied by dot-blot hybridization with an EAV-specific DNA probe. EAV were found in 6/180 (3.3%) cases of diarrhoea and 6/766 (0.8%) asymptomatic samples (P < 0.015). Incidence of EAV was 3.9 cases per 100 person-years in children < 60 months old. EAV-related diarrhoeas were slight and of short duration. In addition, 129 faeces from hospital out-patients, 1-30 months old, were also studied. EAV was identified in 7/129 cases (5.4%). These cases were 9.5 +/- 3.5 months old and the diarrhoea was mild or severe, of 3 +/- 1.5 days of duration. We suggest that EAV are low-risk causes of diarrhoea under natural conditions, although a few children may develop more severe diarrhoea. The diagnosis of EAV needs to be considered in these patients. PMID:1468536

  18. Magnesium-Dependent Interaction of PKR with Adenovirus VAI

    SciTech Connect

    K Launer -Felty; C Wong; A Wahid; G Conn; J Cole

    2011-12-31

    Protein kinase R (PKR) is an interferon-induced kinase that plays a pivotal role in the innate immunity pathway for defense against viral infection. PKR is activated to undergo autophosphorylation upon binding to RNAs that contain duplex regions. Activated PKR phosphorylates the {alpha}-subunit of eukaryotic initiation factor 2, thereby inhibiting protein synthesis in virus-infected cells. Viruses have evolved diverse PKR-inhibitory strategies to evade the antiviral response. Adenovirus encodes virus-associated RNA I (VAI), a highly structured RNA inhibitor that binds PKR but fails to activate. We have characterized the stoichiometry and affinity of PKR binding to define the mechanism of PKR inhibition by VAI. Sedimentation velocity and isothermal titration calorimetry measurements indicate that PKR interactions with VAI are modulated by Mg{sup 2+}. Two PKR monomers bind in the absence of Mg{sup 2+}, but a single monomer binds in the presence of divalent ion. Known RNA activators of PKR are capable of binding multiple PKR monomers to allow the kinase domains to come into close proximity and thus enhance dimerization. We propose that VAI acts as an inhibitor of PKR because it binds and sequesters a single PKR in the presence of divalent cation.

  19. How I treat adenovirus in hematopoietic stem cell transplant recipients

    PubMed Central

    Lindemans, Caroline A.; Leen, Ann M.

    2010-01-01

    Adenovirus (AdV) infections are very common in the general pediatric population. The delayed clearance in young persons imposes a threat to immunocompromised patients after hematopoietic stem cell transplantation (HSCT), who can reactivate the virus, resulting in life-threatening disseminated disease. Although a definitive cure requires adequate immune reconstitution, 2 approaches appear to be feasible and effective to improve the outcomes of AdV infections. Strict monitoring with AdV quantitative polymerase chain reaction followed by preemptive treatment with low-dose (1 mg/kg) cidofovir 3 times a week, is effective in most cases to bridge the severely immunocompromised period shortly after HSCT, with acceptable toxicity rates. For centers who have the access, AdV-specific cytotoxic T cells can be the other important cornerstone of anti-AdV therapy with promising results so far. Methods to positively influence the reconstitution of the immune system after HSCT and optimizing new and currently available cellular immunotherapies will make HSCT safer against the threat of AdV infection/reactivation and associated disease. PMID:20837781

  20. Taxonomy proposal for Old World monkey adenoviruses: characterisation of several non-human, non-ape primate adenovirus lineages.

    PubMed

    Pantó, Laura; Podgorski, Iva I; Jánoska, Máté; Márkó, Orsolya; Harrach, Balázs

    2015-12-01

    A species classification regarding Old World monkey adenoviruses is proposed. We determined the nucleotide sequences of PCR-amplified fragments from the genes of the IVa2, DNA-dependent DNA polymerase, penton base, and hexon proteins from every simian adenovirus (SAdV) serotype that originated from Old World monkeys for which the full genome sequence had not yet been published. We confirmed that the majority of Old Word monkey SAdVs belong to two previously established species. Interestingly, one is the most recently established human AdV species, Human mastadenovirus G, which includes a single human virus, HAdV-52, as well as SAdV-1, -2, -7, -11, -12, and -15. The other approved species, Simian mastadenovirus A includes SAdV-3, -4, -6, -9, -10, -14, and -48. Several SAdVs (SAdV-5, -8, -49, -50) together with baboon AdV-1 and rhesus monkey AdV strains A1139, A1163, A1173, A1258, A1285, A1296, A1312, A1327 and A1335 have been proposed to be classified into an additional species, Simian mastadenovirus B. Another proposed species, Simian mastadenovirus C has been described for SAdV-19, baboon AdV-2/4 and -3. Our study revealed the existence of four additional AdV lineages. The corresponding new candidate species are Simian mastadenovirus D (for SAdV-13), Simian mastadenovirus E (for SAdV-16), Simian mastadenovirus F (for SAdV-17 and -18), and Simian mastadenovirus G (for SAdV-20). Several biological and genomic properties, such as the host origin, haemagglutination profile, number of fibre genes, and G+C content of the genome, strongly support this classification. Three SAdV strains originating from the American Type Culture Collection turned out to be mixtures of at least two virus types, either of the same species (SAdV-12 and -15 types from Human mastadenovirus G) or of two different species (SAdV-5 types from Simian mastadenovirus B and Human mastadenovirus G).

  1. Development and Pre-Clinical Evaluation of a Novel Prostate-Restricted Replication Competent Adenovirus-Ad-IU-1

    DTIC Science & Technology

    2005-05-01

    PSMA postivie LNCaP and C4-2 cells. 6 Task 3. Investigate the capability of m6 to drive adenovirus replication in a prostate cancer-specific manner...of PSES to direct adenovirus replication , and HSV-TK gene, a pro-drug enzyme gene, under the control of another copy of PSES enhancer to restrict

  2. Avian influenza in ovo vaccination with replication defective recombinant adenovirus in chickens: Vaccine potency, antibody persistence, and maternal antibody transfer

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protective immunity against avian influenza (AI) can be elicited in chickens in a single-dose regimen by in ovo vaccination with a replication-competent adenovirus (RCA)-free human adenovirus serotype 5 (Ad)-vector encoding the AI virus (AIV) hemagglutinin (HA). We evaluated vaccine potency, antibo...

  3. RNAi suppressor P19 can be broadly exploited for enhanced adenovirus replication and microRNA knockdown experiments.

    PubMed

    Rauschhuber, Christina; Mueck-Haeusl, Martin; Zhang, Wenli; Nettelbeck, Dirk M; Ehrhardt, Anja

    2013-01-01

    RNA interference (RNAi) is a key regulator of various biological systems including viral infection. Within a virus life cycle gene products can be modulated by the RNA interference (RNAi) pathway which can crucially impact productive virus replication. Herein we explored the RNA interference suppressor protein P19 derived from a plant virus and we found that P19 enhanced adenovirus replication up to 100-fold. Critical factors responsible for this observation were overexpression of adenovirus encoded genes on mRNA and protein levels. To investigate the impact of this phenomenon on recombinant viruses, we exploited its feasibility for therapeutic and genomic applications. We found that P19 significantly increased recombinant adenovirus yields enabling up-scaling for preclinical and clinical studies. Moreover, adenoviruses possessed significantly higher oncolytic activity by expression of P19. Finally, we show that introducing a p19 expression cassette into high-capacity adenovirus provides a strategy to analyze RNAi knockdown in a tissue-specific manner.

  4. An adenovirus associated with intestinal impaction and mortality of male eiders (Somateria mollissima) in the Baltic Sea

    USGS Publications Warehouse

    Hollmén, Tuula E.; Franson, J Christian; Kilpi, Mikael; Docherty, Douglas E.; Myllys, V.

    2003-01-01

    We examined 10 common eider (Somateria mollissima) males found dead in 1998 during a die-off in the northern Baltic Sea off the southwestern coast of Finland. We diagnosed impaction of the posterior small intestine with mucosal necrosis as the cause of death in all 10 and isolated adenoviruses from cloacal samples of six birds. The adenovirus isolates were not neutralized by reference antisera to group I, II, or III avian adenoviruses. Cloacal swabs from 22 apparently healthy eider females nesting at the mortality area were negative for viruses. An adenovirus isolated from one of the eiders caused clinical signs of illness and gastrointestinal pathology in experimentally infected mallard (Anas platyrhynchos) ducklings. These findings suggest that the adenovirus contributed to the mortality of common eider males in the Finnish archipelago.

  5. Protection of non-human primates against rabies with an adenovirus recombinant vaccine

    SciTech Connect

    Xiang, Z.Q.; Greenberg, L.; Ertl, H.C.; Rupprecht, C.E.

    2014-02-15

    Rabies remains a major neglected global zoonosis. New vaccine strategies are needed for human rabies prophylaxis. A single intramuscular immunization with a moderate dose of an experimental chimpanzee adenovirus (Ad) vector serotype SAd-V24, also termed AdC68, expressing the rabies virus glycoprotein, resulted in sustained titers of rabies virus neutralizing antibodies and protection against a lethal rabies virus challenge infection in a non-human primate model. Taken together, these data demonstrate the safety, immunogenicity, and efficacy of the recombinant Ad-rabies vector for further consideration in human clinical trials. - Highlights: • Pre-exposure vaccination with vaccine based on a chimpanzee derived adenovirus protects against rabies. • Protection is sustained. • Protection is achieved with single low-dose of vaccine given intramuscularly. • Protection is not affected by pre-existing antibodies to common human serotypes of adenovirus.

  6. Attachment and Detachment Behaviour of Adenovirus and Surrogates in Fine Granular Limestone Aquifer Material

    NASA Astrophysics Data System (ADS)

    Stevenson, Margaret; Blaschke, Alfred Paul; Kirschner, Alexander; Farnleitner, Andreas; Sommer, Regina; Sidhu, Jatinder

    2015-04-01

    Comparison of transport of virus surrogates to the pathogenic virus is necessary to understand the differences between the virus and surrogate. Since experiments using pathogenic viruses cannot be done in the field, laboratory tests using flow through soil columns are used. Adenovirus, nanoparticles, PRD1 and MS2 bacteriophages were tested in fine granular limestone aquifer material taken from a borehole at a managed aquifer recharge site in Adelaide, Southern Australia. Results show that PRD1 is the most appropriate surrogate for adenovirus in an aquifer dominated by calcite material, although PRD1 did not mimic the detachment behaviour of adenovirus successfully under high pH conditions. It was also found that the charge of the colloid is not a dominant removal mechanism in this system. Implications from this study could influence how field tests using bacteriophages and nanoparticles are interpreted.

  7. An epizootic of adenovirus-induced hemorrhagic disease in captive black-tailed deer (Odocoileus hemionus).

    PubMed

    Boyce, W M; Woods, L W; Keel, M K; MacLachlan, N J; Porter, C O; Lehmkuhl, H D

    2000-09-01

    Ten fawns and four adult black-tailed deer (Odocoileus hemionus) in a captive herd died as a result of adenovirus-induced hemorrhagic disease. Acute, systemic infections were characterized by hemorrhagic enteropathy, pulmonary edema, and occasional ulceration of the upper alimentary tract. Localized infections were limited to the upper alimentary tract and included stomatitis, pharyngitis, mandibular osteomyelitis, and rumenitis. In deer with acute, systemic infections, a diagnosis was made by histopathology and immunohistochemistry. The serum neutralization test was useful for confirming a diagnosis in animals with prolonged, localized infections. Deer originating from herds with a history of adenovirus infection should not be transferred to other captive herds or released into free-ranging populations because they may serve as carriers of adenovirus.

  8. Adenoviruses as a model in the study of the effect of space flight factors

    NASA Astrophysics Data System (ADS)

    Nosach, L. M.; Povnitsa, O. Yu.; Zhovnovata, V. L.

    Simulated microgravity conditions, independently of multiplicity of infection, does not influence the reproduction of adenoviruses in cells which were clinorotated for 48 hours after adsorption of virus. The incubation of infected cells before clinorotation under static conditions at a temperature of 4 °C for three days (the conditions for keeping cells before the flight) does not change the number of infected cells relatively to control, but some changes of cell morphology are revealed, namely round off and aggregation of cells. The adenoviruses which were exposed in the medium keep infectivity under the conditions of clinorotation at 4 and 20-22 °C over prolonged periods (90 and 60 days, respectively). A model is elaborated for investigation of the influence of space flight factors on the interaction of the adenovirus and Epstein-Barr virus genomes at combined infection of limphoblastoid cells.

  9. Transcriptional and posttranscriptional regulation of class I major histocompatibility complex genes following transformation with human adenoviruses.

    PubMed Central

    Shemesh, J; Rotem-Yehudar, R; Ehrlich, R

    1991-01-01

    Transformation of rodent cells by human adenoviruses is a well-established model system for studying the expression, regulation, and function of class I antigens. In this report, we demonstrate that the highly oncogenic adenovirus type 12 operates at the transcriptional and posttranscriptional levels in regulating the activity of major histocompatibility complex class I genes and products in transformed cells. Adenovirus type 12 suppresses the cell surface expression of class I antigens in most cell lines. Nevertheless, in a number of cell lines suppression is the result of reduction in the amount of stable specific mRNA, while in another group of cell lines suppression involves interference with processing of a posttranscriptional product. The two mechanisms operate both for the endogenous H-2 genes and for a miniature swine class I transgene that is expressed in the cells. Images PMID:1895404

  10. Permissive growth of human adenovirus type 4 vaccine strain-based vector in porcine cell lines.

    PubMed

    Gao, Dong-sheng; Li, Xiao-jing; Wan, Wen-yan; Li, Hong-jie; Wang, Xiao-xue; Yang, Xia; Li, Yong-tao; Chang, Hong-tao; Chen, Lu; Wang, Chuan-qing; Zhao, Jun

    2016-02-01

    In recent years, there has been considerable interest in using adenoviruses as live vectors to develop recombinant vaccines. Previous studies have demonstrated the safety and effectiveness of HIV/SIV and influenza vaccine candidates based on human adenovirus type 4 (Ad4) replication-competent vectors in rhesus macaque and human model. To explore the possibility of human Ad4 vaccine strain used as a vector in developing porcine vaccines, the growth properties of replication-competent human Ad4 vaccine strain recombinant encoding EGFP in different porcine cell lines were investigated. All tested cell lines are permissive for Ad4 vaccine strain vector with varied replication efficiency. Thus, human Ad4 based vectors would be promising supplement to adenovirus vectors as a delivery vehicle for recombinant vaccines in swine industry.

  11. Chapter eight--Oncolytic adenoviruses for cancer immunotherapy: data from mice, hamsters, and humans.

    PubMed

    Cerullo, Vincenzo; Koski, Anniina; Vähä-Koskela, Markus; Hemminki, Akseli

    2012-01-01

    Adenovirus is one of the most commonly used vectors for gene therapy and two products have already been approved for treatment of cancer in China (Gendicine(R) and Oncorine(R)). An intriguing aspect of oncolytic adenoviruses is that by their very nature they potently stimulate multiple arms of the immune system. Thus, combined tumor killing via oncolysis and inherent immunostimulatory properties in fact make these viruses in situ tumor vaccines. When further engineered to express cytokines, chemokines, tumor-associated antigens, or other immunomodulatory elements, they have been shown in various preclinical models to induce antigen-specific effector and memory responses, resulting both in full therapeutic cures and even induction of life-long tumor immunity. Here, we review the state of the art of oncolytic adenovirus, in the context of their capability to stimulate innate and adaptive arms of the immune system and finally how we can modify these viruses to direct the immune response toward cancer.

  12. Dicer functions as an antiviral system against human adenoviruses via cleavage of adenovirus-encoded noncoding RNA

    PubMed Central

    Machitani, Mitsuhiro; Sakurai, Fuminori; Wakabayashi, Keisaku; Tomita, Kyoko; Tachibana, Masashi; Mizuguchi, Hiroyuki

    2016-01-01

    In various organisms, including nematodes and plants, RNA interference (RNAi) is a defense system against virus infection; however, it is unclear whether RNAi functions as an antivirus system in mammalian cells. Rather, a number of DNA viruses, including herpesviruses, utilize post-transcriptional silencing systems for their survival. Here we show that Dicer efficiently suppresses the replication of adenovirus (Ad) via cleavage of Ad-encoding small RNAs (VA-RNAs), which efficiently promote Ad replication via the inhibition of eIF2α phosphorylation, to viral microRNAs (mivaRNAs). The Dicer knockdown significantly increases the copy numbers of VA-RNAs, leading to the efficient inhibition of eIF2α phosphorylation and the subsequent promotion of Ad replication. Conversely, overexpression of Dicer significantly inhibits Ad replication. Transfection with mivaRNA does not affect eIF2α phosphorylation or Ad replication. These results indicate that Dicer-mediated processing of VA-RNAs leads to loss of activity of VA-RNAs for enhancement of Ad replication and that Dicer functions as a defence system against Ad in mammalian cells. PMID:27273616

  13. Intraductal delivery of adenoviruses targets pancreatic tumors in transgenic Ela-myc mice and orthotopic xenografts.

    PubMed

    José, Anabel; Sobrevals, Luciano; Miguel Camacho-Sánchez, Juan; Huch, Meritxell; Andreu, Núria; Ayuso, Eduard; Navarro, Pilar; Alemany, Ramon; Fillat, Cristina

    2013-01-01

    Gene-based anticancer therapies delivered by adenoviruses are limited by the poor viral distribution into the tumor. In the current work we have explored the feasibility of targeting pancreatic tumors through a loco-regional route. We have taken advantage of the ductal network in the pancreas to retrogradelly inject adenoviruses through the common bile duct in two different mouse models of pancreatic carcinogenesis: The transgenic Ela-myc mice that develop mixed neoplasms displaying both acinar-like and duct-like neoplastic cells affecting the whole pancreas; and mice bearing PANC-1 and BxPC-3 orthotopic xenografts that constitute a model of localized human neoplastic tumors. We studied tumor targeting and the anticancer effects of newly thymidine kinase-engineered adenoviruses both in vitro and in vivo, and conducted comparative studies between intraductal or intravenous administration. Our data indicate that the intraductal delivery of adenovirus efficiently targets pancreatic tumors in the two mouse models. The in vivo application of AduPARTKT plus ganciclovir (GCV) treatment induced tumor regression in Ela-myc mice. Moreover, the intraductal injection of ICOVIR15-TKT oncolytic adenoviruses significantly improved mean survival of mice bearing PANC-1 and BxPC-3 pancreatic xenografts from 30 to 52 days and from 20 to 68 days respectively (p less than 0.0001) when combined with GCV. Of notice, both AduPARTKT and ICOVIR15-TKT antitumoral responses were stronger by ductal viral application than intravenously, in line with the 38-fold increase in pancreas transduction observed upon ductal administration. In summary our data show that cytotoxic adenoviruses retrogradelly injected to the pancreas can be a feasible approach to treat localized pancreatic tumors.

  14. Adenovirus types 40 and 41 and rotaviruses associated with diarrhea in children from Guatemala.

    PubMed Central

    Cruz, J R; Cáceres, P; Cano, F; Flores, J; Bartlett, A; Torún, B

    1990-01-01

    From March 1987 to February 1988, fecal excretion of adenovirus types 40 and 41 and rotavirus serotypes in 194 children (age, 0 to 3 years) from a rural community of Guatemala was monitored. In total, 458 samples taken during 385 episodes of diarrhea and 191 specimens obtained during symptom-free periods were examined by enzyme-linked immunosorbent assay. Fifty-seven children hospitalized because of diarrhea were also studied. Among the rural children, 43 (22.2%) excreted adenovirus types 40 and 41 and 20 (10.3%) shed rotaviruses. Adenovirus types 40 and 41 were associated with 54 (14.0%) illnesses, and rotaviruses were associated with 18 (4.7%) illnesses. Asymptomatic infections with adenovirus types 40 and 41 were documented in nine children and with rotaviruses in two children. Fifteen typeable rotaviruses were identified as serotype 2. In the hospital population, 36 (63.2%) children had viral infections. Rotaviruses were identified in 29 (50.9%) and adenovirus types 40 and 41 were identified in 15 (31.2%) of 48 subjects tested. Dual infections by these viruses were found in eight children. Of 22 typeable strains of rotaviruses, 9 (34.6%) were serotype 1, 12 (46.1%) were serotype 2, and 1 (3.8%) was serotype 3. All the children infected with serotype 2 rotavirus were coinfected with other enteric pathogens, while only three (37.5%) of those infected with rotavirus serotype 1 excreted another pathogen. Adenovirus types 40 and 41 are an important cause of gastroenteritis in both ambulatory and hospitalized Guatemalan children. There seems to be a difference in the pathogenicity among rotavirus serotypes. PMID:2168438

  15. Intraductal Delivery of Adenoviruses Targets Pancreatic Tumors in Transgenic Ela-myc Mice and Orthotopic Xenografts

    PubMed Central

    José, Anabel; Sobrevals, Luciano; Camacho-Sánchez, Juan Miguel; Huch, Meritxell; Andreu, Núria; Ayuso, Eduard; Navarro, Pilar; Alemany, Ramon; Fillat, Cristina

    2013-01-01

    Gene-based anticancer therapies delivered by adenoviruses are limited by the poor viral distribution into the tumor. In the current work we have explored the feasibility of targeting pancreatic tumors through a loco-regional route. We have taken advantage of the ductal network in the pancreas to retrogradelly inject adenoviruses through the common bile duct in two different mouse models of pancreatic carcinogenesis: The transgenic Ela-myc mice that develop mixed neoplasms displaying both acinar-like and duct-like neoplastic cells affecting the whole pancreas; and mice bearing PANC-1 and BxPC-3 orthotopic xenografts that constitute a model of localized human neoplastic tumors. We studied tumor targeting and the anticancer effects of newly thymidine kinase-engineered adenoviruses both in vitro and in vivo, and conducted comparative studies between intraductal or intravenous administration. Our data indicate that the intraductal delivery of adenovirus efficiently targets pancreatic tumors in the two mouse models. The in vivo application of AduPARTKT plus ganciclovir (GCV) treatment induced tumor regression in Ela-myc mice. Moreover, the intraductal injection of ICOVIR15-TKT oncolytic adenoviruses significantly improved mean survival of mice bearing PANC-1 and BxPC-3 pancreatic xenografts from 30 to 52 days and from 20 to 68 days respectively (p<0.0001) when combined with GCV. Of notice, both AduPARTKT and ICOVIR15-TKT antitumoral responses were stronger by ductal viral application than intravenously, in line with the 38-fold increase in pancreas transduction observed upon ductal administration. In summary our data show that cytotoxic adenoviruses retrogradelly injected to the pancreas can be a feasible approach to treat localized pancreatic tumors. PMID:23328228

  16. Detection and analysis of six lizard adenoviruses by consensus primer PCR provides further evidence of a reptilian origin for the atadenoviruses.

    PubMed

    Wellehan, James F X; Johnson, April J; Harrach, Balázs; Benkö, Mária; Pessier, Allan P; Johnson, Calvin M; Garner, Michael M; Childress, April; Jacobson, Elliott R

    2004-12-01

    A consensus nested-PCR method was designed for investigation of the DNA polymerase gene of adenoviruses. Gene fragments were amplified and sequenced from six novel adenoviruses from seven lizard species, including four species from which adenoviruses had not previously been reported. Host species included Gila monster, leopard gecko, fat-tail gecko, blue-tongued skink, Tokay gecko, bearded dragon, and mountain chameleon. This is the first sequence information from lizard adenoviruses. Phylogenetic analysis indicated that these viruses belong to the genus Atadenovirus, supporting the reptilian origin of atadenoviruses. This PCR method may be useful for obtaining templates for initial sequencing of novel adenoviruses.

  17. Characterization of an upstream regulatory element of adenovirus L1 poly (A) site.

    PubMed

    Liu, Li

    2005-06-20

    The transition from early to late stage infection by adenovirus involves a change in mRNA expression from the adenovirus major late transcription unit (AdMLTU). This early to late switch centers around alternative selection of one of five poly (A) sites (L1-L5) that code for the major structural proteins of Adenovirus. During the early stage of infection, steady state mRNA is primarily derived from the L1 poly (A) site. During the late stage of infection, each of the MLTU poly (A) sites is represented in the steady state mRNA pool (Falck-Pedersen, E., Logan, J., 1989. Regulation of poly(A) site selection in adenovirus. J. Virol. 63 (2), 532-541.). Using transient transfection of a plasmid expressing Chloramphenicol Acetyl Transferase with a tandem poly (A) minigene system (L13) (DeZazzo, J.D., Falck-Pedersen, E., Imperiale, M.J., 1991. Sequences regulating temporal poly(A) site switching in the adenovirus major late transcription unit. Mol. Cell. Biol. 11 (12), 5977-5984; Prescott, J., Falck-Pedersen, E., 1994. Sequence elements upstream of the 3' cleavage site confer substrate strength to the adenovirus L1 and L3 polyadenylation sites. Mol. Cell. Biol. 14 (7), 4682-4693.), it has been demonstrated that the promoter-proximal L1 poly (A) site which is poorly recognized by the 3' end processing machinery, contains an upstream repressor element (URE) that influences steady state levels of mRNA (Prescott, J.C., Liu, L., Falck-Pedersen, E., 1997. Sequence-mediated regulation of adenovirus gene expression by repression of mRNA accumulation. Mol. Cell. Biol. 17 (4), 2207-2216.). In this study, we have further characterized the elements that mediate L1URE function. These studies indicate that the L1 upstream regulatory element (L1 URE) contains a complex RNA architecture that serves to repress gene expression through multiple sub-effectors. The L1URE functions when located upstream of a heterologous poly (A) site, and is able to strongly suppress steady state m

  18. Adenine arabinoside inhibition of adenovirus replication enhanced by an adenosine deaminase inhibitor.

    PubMed

    Wigand, R

    1979-01-01

    The inhibition of adenovirus multiplication by adenine arabinoside was determined by yield reduction in one-step multiplication cycle. Inhibition was greatly enhanced by an adenosine deaminase inhibitor (2-deoxycoformycin) in concentrations down to 10 ng/ml. Adenovirus types from four subgroups showed similar results. However, the enhancing effect of adenosine deaminase inhibitor was great in HeLa cells, moderate in human fibroblasts, and negligible in Vero cells. This difference could be explained by different concentrations of adenosine deaminase found in cell homogenates.

  19. Mouse polyoma virus and adenovirus replication in mouse cells temperature-sensitive in DNA synthesis.

    PubMed

    Sheinin, R; Fabbro, J; Dubsky, M

    1985-01-01

    Mouse adenovirus multiplies, apparently without impediment, in temperature-inactivated ts A1S9, tsC1 and ts2 mouse fibroblasts. Thus, the DNA of mouse adenovirus can replicate in the absence of functional DNA topoisomerase II, a DNA-chain-elongation factor, and a protein required for traverse of the G1/S interface, respectively, encoded in the ts A1S9, tsC1 and ts2 genetic loci. These results are compared with those obtained with polyoma virus.

  20. Molecular Epidemiology and Clinical Manifestations of Adenovirus Respiratory Infections in Taiwanese Children

    PubMed Central

    Wang, Ya-Fang; Shen, Fan-Ching; Wang, Shan-Li; Kuo, Pin-Hwa; Tsai, Huey-Pin; Liu, Ching-Chuan; Wang, Jen-Ren; Chi, Chia-Yu

    2016-01-01

    Abstract Human adenoviruses (HAdVs) are important causes of respiratory infections in children. They usually cause mild upper respiratory symptoms, but they can also produce severe pneumonia and other complications. The aims of this retrospective study were to better define the molecular epidemiology of respiratory adenoviruses circulating in Taiwanese children during 2002 and 2013, detect reinfections and co-infections, and characterize the clinical features and laboratory findings according to the causative genotypes. We collected a representative sample of 182 isolates of adenoviruses from 175 children during the 12-year study period. The most prevalent species was HAdV-B genotype 3 (HAdV-3) (92/182, 50.5%) followed by HAdV-C (HAdV-2) (38/182, 20.9%). A single outbreak of HAdV-E (6/182, 3.3%) was noted in 2007. The mean age of children with adenovirus infections was 3.7 ± 2.0 years, with a slight predominance of males (53.1%). Children with HAdV-B tended to be older, had more lower respiratory tract infections, gastrointestinal symptoms, and a higher rate of hospitalization than those with HAdV-C (P < 0.05). Adenovirus co-infections were noted in 25/175 (14.3%) of the children. The most frequent co-infections were with species B (HAdV-3) and C (HAdV-2) (14/25, 56.0%). Additional infections were noted in 23/175 (13.1%) of the children. Of these repeated infections, the initial isolates were always genotypes of HAdV-C. The second isolates were genotypes of HAdV-B or HAdV-E. The clinical features of the first HAdV-B infection and the reinfection of HAdV-B followed the HAdV-C were similar. In conclusion, HAdV-B, C, and E were the only adenovirus species that were isolated from children who were sufficiently ill with respiratory infections to require a visit to the hospital. Human adenovirus B (HAdV-3) accounted for half of these species. HAdV-B was more likely than other species to produce severe disease. The high incidence of adenovirus co-infection and

  1. A new role for the Kruppel-like transcription factor KLF6 as an inhibitor of c-Jun proto-oncoprotein function.

    PubMed

    Slavin, Daniela A; Koritschoner, Nicolás P; Prieto, Claudio C; López-Díaz, Fernando J; Chatton, Bruno; Bocco, José Luis

    2004-10-28

    Kruppel-like transcription factors (KLFs) represent one of the most diverse set of regulators in vertebrate organisms. KLF family members are involved in cell proliferation and differentiation control in normal as well as in pathological situations. Here, we demonstrate that KLF6 behaves as a functional antagonist of the c-Jun proto-oncoprotein. Thus, KLF6 overexpression downregulated c-Jun-dependent transcription and a physical interaction between c-Jun and KLF6 was detected. Moreover, cell proliferation induced by c-Jun was significantly decreased by KLF6. The inhibition of c-Jun functions correlates directly with c-Jun protein degradation induced by KLF6. We also show that all KLF6 effects on c-Jun were largely dependent on phorbol ester (TPA/ionomycin) extracellular stimulation, which enhanced KLF6 nuclear translocation and transcriptional activity and modified its phosphorylation status. Our data are consistent with a novel mechanism of KLF6's role as an inhibitor of cell proliferation by counteracting the function of the c-Jun proto-oncoprotein involving enhanced c-Jun degradation by the proteasome-dependent pathway, and further reinforces KLF6 as a potential tumor suppressor gene product.

  2. The oncoprotein HBXIP up-regulates FGF4 through activating transcriptional factor Sp1 to promote the migration of breast cancer cells.

    PubMed

    Shi, Hui; Li, Yinghui; Feng, Guoxing; Li, Leilei; Fang, Runping; Wang, Zhen; Qu, Jie; Ding, Peijian; Zhang, Xiaodong; Ye, Lihong

    2016-02-26

    We have reported that the oncoprotein hepatitis B X-interacting protein (HBXIP) is able to promote migration of breast cancer cells. Fibroblast growth factor 4 (FGF4) is a multipotent growth factor and is highly expressed in various human cancers. However, the regulatory mechanism of FGF4 in breast cancer remains poorly understood. In the present study, we report that HBXIP is able to up-regulate FGF4 to enhance the migration of breast cancer cells. Immunohistochemistry staining showed that HBXIP and FGF4 were highly expressed in clinical metastatic lymph nodes of breast tumor. The expression levels of HBXIP were positively related to those of FGF4 in clinical breast cancer tissues. Then, we validated that HBXIP up-regulated the expression of FGF4 at the levels of promoter, mRNA and protein by luciferase reporter gene assays, reverse transcription-polymerase chain reaction and Western blot analysis. Moreover, we found that HBXIP was able to activate FGF4 promoter through transcriptional factor Sp1 by luciferase reporter gene assays. Chromatin immunoprecipitation assays confirmed that HBXIP coactivated Sp1 to stimulate FGF4 promoter. In function, we showed that HBXIP promoted breast cancer cell migration through FGF4 by wound healing and transwell cell migration assays. Thus, we conclude that the oncoprotein HBXIP up-regulates FGF4 through activating transcriptional factor Sp1 to promote the migration of breast cancer cells. Therapeutically, HBXIP may serve as a novel target in breast cancer.

  3. The HPV16 E7 Oncoprotein Disrupts Dendritic Cell Function and Induces the Systemic Expansion of CD11b+Gr1+ Cells in a Transgenic Mouse Model

    PubMed Central

    Damian-Morales, Gabriela; Serafín-Higuera, Nicolás; Moreno-Eutimio, Mario Adán; Cortés-Malagón, Enoc M.; Bonilla-Delgado, José; Rodríguez-Uribe, Genaro; Ocadiz-Delgado, Rodolfo; Lambert, Paul F.

    2016-01-01

    Objective. The aim of this study was to analyze the effects of the HPV16 E7 oncoprotein on dendritic cells (DCs) and CD11b+Gr1+ cells using the K14E7 transgenic mouse model. Materials and Methods. The morphology of DCs was analyzed in male mouse skin on epidermal sheets using immunofluorescence and confocal microscopy. Flow cytometry was used to determine the percentages of DCs and CD11b+Gr1+ cells in different tissues and to evaluate the migration of DCs. Results. In the K14E7 mouse model, the morphology of Langerhans cells and the migratory activity of dendritic cells were abnormal. An increase in CD11b+Gr1+ cells was observed in the blood and skin of K14E7 mice, and molecules related to CD11b+Gr1+ chemoattraction (MCP1 and S100A9) were upregulated. Conclusions. These data suggest that the HPV16 E7 oncoprotein impairs the function and morphology of DCs and induces the systemic accumulation of CD11b+Gr1+ cells. PMID:27478837

  4. Overexpression of PP2A inhibitor SET oncoprotein is associated with tumor progression and poor prognosis in human non-small cell lung cancer.

    PubMed

    Liu, Hao; Gu, Yixue; Wang, Hongsheng; Yin, Jiang; Zheng, Guopei; Zhang, Zhijie; Lu, Minyin; Wang, Chenkun; He, Zhimin

    2015-06-20

    SET oncoprotein is an endogenous inhibitor of protein phosphatase 2A (PP2A), and SET-mediated PP2A inhibition is an important regulatory mechanism for promoting cancer initiation and progression of several types of human leukemia disease. However, its potential relevance in solid tumors as non-small cell lung cancer (NSCLC) remains mostly unknown. In this study, we showed that SET was evidently overexpressed in human NSCLC cell lines and NSCLC tissues. Clinicopathologic analysis showed that SET expression was significantly correlated with clinical stage (p < 0.001), and lymph node metastasis (p < 0.05). Kaplan-Meier analysis revealed that patients with high SET expression had poorer overall survival rates than those with low SET expression. Moreover, knockdown of SET in NSCLC cells resulted in attenuated proliferative and invasive abilities. The biological effect of SET on proliferation and invasion was mediated by the inhibition of the PP2A, which in turn, activation of AKT and ERK, increased the expression of cyclin D1 and MMP9, and decreased the expression of p27. Furthermore, we observed that restoration of PP2A using SET antagonist FTY720 impaired proliferative and invasive potential in vitro, as well as inhibited tumor growth in vivo of NSCLC cells. Taken together, SET oncoprotein plays an important role in NSCLC progression, which could serve as a potential prognosis marker and a novel therapeutic target for NSCLC patients.

  5. Bcl-2 anti-apoptotic oncoprotein suppresses angiogenesis in non-small cell lung cancer: implications in resistance to photodynamic treatment?

    NASA Astrophysics Data System (ADS)

    Koukourakis, M. I.; Giatromanolaki, A.; Skarlatos, J.; Kosma, L.; Apostolikas, N.; Beroukas, K.

    1998-07-01

    PDT cytotoxicity is likely to occur through photooxidative reactions. In that way mechanisms that define poor oxygenation should be involved in defining resistance to photo-dynamic treatment (PDT). On the other hand bcl-2 anti- apoptotic protein has been shown to delay cell death and protect cells from toxic oxidative products. We examined 134 specimens from T1,2-NO,1 staged patients treated with surgery alone. Specimens were immunohistochemically examined for vascular grade using the JC70 MoAb, and bcl-2 oncoprotein expression. Bcl-2 expression correlated with low vascular grade. Only 3/27 of bcl2+ case had high angiogenesis vs. 34/107 of cases without bcl-2 expression. In the present study we provide evidence that bcl-2 overexpression directly suppresses angiogenesis in non-small cell lung cancer, which obviously results in decreased blood supply and oxygenation. This finding implies that reduced intratumoral angiogenesis and immortalizing oncoprotein overexpression are linked to each other and may have a role in defining tumors resistant to PDT.

  6. Intracellular scFvs against the viral E6 oncoprotein provoke apoptosis in human papillomavirus-positive cancer cells

    SciTech Connect

    Lagrange, Magali; Boulade-Ladame, Charlotte; Mailly, Laurent; Weiss, Etienne; Orfanoudakis, Georges; Deryckere, Francois . E-mail: francois.deryckere@esbs.u-strasbg.fr

    2007-09-21

    The E6 protein of human papillomavirus type 16 (16E6) is involved in the tumorigenesis of human cervical cells by targeting numerous cellular proteins. We have designed a strategy for neutralizing 16E6 based on the intracellular expression of single-chain Fv antibodies (scFvs) specific to 16E6. Recombinant adenovirus vectors were constructed to allow expression of two 16E6-binding scFvs and one 16E6-non-binding scFv in HPV16-positive and -negative cells. Expression of the scFvs provoked two types of effects: (i) inhibition of proliferation of all cell lines tested, this aspecific toxicity being likely due to the aggregation of unfolded scFvs; and (ii) apoptosis observed only in HPV16-positive cervical cancer cell lines after expression of 16E6-binding scFvs, this specific effect being proportional to the intracellular solubility of the scFvs. These data demonstrate the feasibility of intracellular immunization with anti-16E6 scFvs and highlight the importance of the solubility of the intracellular antibodies.

  7. Human T-cell leukemia virus type 1 tax oncoprotein suppression of multilineage hematopoiesis of CD34+ cells in vitro.

    PubMed

    Tripp, Adam; Liu, Yingxian; Sieburg, Michelle; Montalbano, Joanne; Wrzesinski, Stephen; Feuer, Gerold

    2003-11-01

    Human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 are highly related viruses that differ in disease manifestation. HTLV-1 is the etiologic agent of adult T-cell leukemia and lymphoma, an aggressive clonal malignancy of human CD4-bearing T lymphocytes. Infection with HTLV-2 has not been conclusively linked to lymphoproliferative disorders. We previously showed that human hematopoietic progenitor (CD34(+)) cells can be infected by HTLV-1 and that proviral sequences were maintained after differentiation of infected CD34(+) cells in vitro and in vivo. To investigate the role of the Tax oncoprotein of HTLV on hematopoiesis, bicistronic lentiviral vectors were constructed encoding the HTLV-1 or HTLV-2 tax genes (Tax1 and Tax2, respectively) and the green fluorescent protein marker gene. Human hematopoietic progenitor (CD34(+)) cells were infected with lentivirus vectors, and transduced cells were cultured in a semisolid medium permissive for the development of erythroid, myeloid, and primitive progenitor colonies. Tax1-transduced CD34(+) cells displayed a two- to fivefold reduction in the total number of hematopoietic clonogenic colonies that arose in vitro, in contrast to Tax2-transduced cells, which showed no perturbation of hematopoiesis. The ratio of colony types that developed from Tax1-transduced CD34(+) cells remained unaffected, suggesting that Tax1 inhibited the maturation of relatively early, uncommitted hematopoietic stem cells. Since previous reports have linked Tax1 expression with initiation of apoptosis, lentiviral vector-mediated transduction of Tax1 or Tax2 was investigated in CEM and Jurkat T-cell lines. Ectopic expression of either Tax1 or Tax2 failed to induce apoptosis in T-cell lines. These data demonstrate that Tax1 expression perturbs development and maturation of pluripotent hematopoietic progenitor cells, an activity that is not displayed by Tax2, and that the suppression of hematopoiesis is not attributable to induction of apoptosis. Since

  8. Experimental study of Human Adenoviruses interactions with clays

    NASA Astrophysics Data System (ADS)

    Bellou, Maria; Syngouna, Vasiliki; Paparrodopoulos, Spyros; Vantarakis, Apostolos; Chrysikopoulos, Constantinos

    2014-05-01

    Clays are used to establish low permeability liners in landfills, sewage lagoons, water retention ponds, golf course ponds, and hazardous waste sites. Human adenoviruses (HAdVs) are waterborne viruses which have been used as viral indicators of fecal pollution. The objective of this study was to investigate the survival of HAdV in static and dynamic clay systems. The clays used as a model were crystalline aluminosilicates: kaolinite and bentonite. The adsorption and survival of HAdVs onto these clays were characterized at two different controlled temperatures (4 and 25o C) under static and dynamic batch conditions. Control tubes, in the absence of clay, were used to monitor virus inactivation due to factors other than adsorption to clays (e.g. inactivation or sorption onto the tubes walls). For both static and dynamic batch experiments, samples were collected for a maximum period of seven days. This seven day time - period was determined to be sufficient for the virus-clay systems to reach equilibrium. To infer the presence of infectious HAdV particles, all samples were treated with Dnase and the extraction of viral nucleid acid was performed using a commercial viral RNA kit. All samples were analyzed by Real - Time PCR which was used to quantify viral particles in clays. Samples were also tested for virus infectivity by A549 cell cultures. Exposure time intervals in the range of seven days (0.50-144 hours) resulted in a load reduction of 0.74 to 2.96 logs for kaolinite and a reduction of 0.89 to 2.92 for bentonite. Furthermore, virus survival was higher onto bentonite than kaolinite (p

  9. Adenovirus DNA synthesized in the presence of aphidicolin.

    PubMed Central

    Oguro, M; Yamashita, T; Ariga, H; Nagano, H

    1984-01-01

    Adenovirus types 2 and 5 DNA synthesized in vivo and in vitro in the presence of aphidicolin were studied. Inhibition of adenoviral DNA synthesis by aphidicolin was only 70% even at a concentration of 30 micrograms/ml of aphidicolin, at which the cellular DNA synthesis was completely inhibited. When initiation of the viral DNA synthesis was synchronized with hydroxyurea and labeled with [3H]thymidine for 60 min, the viral DNA synthesized in the presence of 30 micrograms/ml of aphidicolin was not of full length (35 kb) but small (approximately 12 kb) by analysis of alkaline sucrose density gradient centrifugation. When initiation of the viral DNA synthesis was not synchronized, the viral DNAs ranging from full size to 12 kb were synthesized in the presence of aphidicolin, indicating that the nascent DNAs longer than about 12 kb can continue to elongate in the presence of aphidicolin. This 12 kb DNA was not derived from the degradation products of newly synthesized full size adenoviral DNA. The viral DNA synthesis was restored and the full size of adenoviral DNA was attained within 15 min following removal of aphidicolin. About 20% of the entire viral genome length from the 5'-end was not inhibited by aphidicolin, while the synthesis of interior fragments of the adenoviral DNA was markedly inhibited by aphidicolin, judging from the electrophoretic pattern on neutral agarose gel after digestion of DNA with Hind III. These results indicate that aphidicolin inhibits adenoviral DNA replication at the internal region located approximately 20-30% from both terminals. Images PMID:6420772

  10. A tumor-stroma targeted oncolytic adenovirus replicated in human ovary cancer samples and inhibited growth of disseminated solid tumors in mice.

    PubMed

    Lopez, M Veronica; Rivera, Angel A; Viale, Diego L; Benedetti, Lorena; Cuneo, Nicasio; Kimball, Kristopher J; Wang, Minghui; Douglas, Joanne T; Zhu, Zeng B; Bravo, Alicia I; Gidekel, Manuel; Alvarez, Ronald D; Curiel, David T; Podhajcer, Osvaldo L

    2012-12-01

    Targeting the tumor stroma in addition to the malignant cell compartment is of paramount importance to achieve complete tumor regression. In this work, we modified a previously designed tumor stroma-targeted conditionally replicative adenovirus (CRAd) based on the SPARC promoter by introducing a mutated E1A unable to bind pRB and pseudotyped with a chimeric Ad5/3 fiber (Ad F512v1), and assessed its replication/lytic capacity in ovary cancer in vitro and in vivo. AdF512v1 was able to replicate in fresh samples obtained from patients: (i) with primary human ovary cancer; (ii) that underwent neoadjuvant treatment; (iii) with metastatic disease. In addition, we show that four intraperitoneal (i.p.) injections of 5 × 10(10) v.p. eliminated 50% of xenografted human ovary tumors disseminated in nude mice. Moreover, AdF512v1 replication in tumor models was enhanced 15-40-fold when the tumor contained a mix of malignant and SPARC-expressing stromal cells (fibroblasts and endothelial cells). Contrary to the wild-type virus, AdF512v1 was unable to replicate in normal human ovary samples while the wild-type virus can replicate. This study provides evidence on the lytic capacity of this CRAd and highlights the importance of targeting the stromal tissue in addition to the malignant cell compartment to achieve tumor regression.

  11. A Tumor-stroma Targeted Oncolytic Adenovirus Replicated in Human Ovary Cancer Samples and Inhibited Growth of Disseminated Solid Tumors in Mice

    PubMed Central

    Lopez, M Veronica; Rivera, Angel A; Viale, Diego L; Benedetti, Lorena; Cuneo, Nicasio; Kimball, Kristopher J; Wang, Minghui; Douglas, Joanne T; Zhu, Zeng B; Bravo, Alicia I; Gidekel, Manuel; Alvarez, Ronald D; Curiel, David T; Podhajcer, Osvaldo L

    2012-01-01

    Targeting the tumor stroma in addition to the malignant cell compartment is of paramount importance to achieve complete tumor regression. In this work, we modified a previously designed tumor stroma-targeted conditionally replicative adenovirus (CRAd) based on the SPARC promoter by introducing a mutated E1A unable to bind pRB and pseudotyped with a chimeric Ad5/3 fiber (Ad F512v1), and assessed its replication/lytic capacity in ovary cancer in vitro and in vivo. AdF512v1 was able to replicate in fresh samples obtained from patients: (i) with primary human ovary cancer; (ii) that underwent neoadjuvant treatment; (iii) with metastatic disease. In addition, we show that four intraperitoneal (i.p.) injections of 5 × 1010 v.p. eliminated 50% of xenografted human ovary tumors disseminated in nude mice. Moreover, AdF512v1 replication in tumor models was enhanced 15–40-fold when the tumor contained a mix of malignant and SPARC-expressing stromal cells (fibroblasts and endothelial cells). Contrary to the wild-type virus, AdF512v1 was unable to replicate in normal human ovary samples while the wild-type virus can replicate. This study provides evidence on the lytic capacity of this CRAd and highlights the importance of targeting the stromal tissue in addition to the malignant cell compartment to achieve tumor regression. PMID:22948673

  12. Pseudotyping Serotype 5 Adenovirus with the Fiber from Other Serotypes Uncovers a Key Role of the Fiber Protein in Adenovirus 5-Induced Thrombocytopenia.

    PubMed

    Raddi, Najat; Vigant, Frédéric; Wagner-Ballon, Oriane; Giraudier, Stéphane; Custers, Jerome; Hemmi, Silvio; Benihoud, Karim

    2016-02-01

    Adenovirus (Ad) infection in humans is associated with inflammatory responses and thrombocytopenia. Although several studies were conducted in mice models to understand molecular and cellular mechanisms of Ad-induced inflammatory responses, only few of them turned their interest toward the mechanisms of Ad-induced thrombocytopenia. Using different depletion methods, the present study ruled out any significant role of spleen, macrophages, and vitamin K-dependent factor in Ad-induced thrombocytopenia. Interestingly, mice displaying thrombocytopenia expressed high levels of cytokines/chemokines after Ad administration. Most importantly, pseudotyping adenovirus with the fiber protein from other serotypes was associated with reduction of both cytokine/chemokine production and thrombocytopenia. Altogether, our results suggest that capsid fiber protein (and more precisely its shaft) of Ad serotype 5 triggers the cytokine production that leads to Ad-induced thrombocytopenia.

  13. Cellular repressor of E1A-stimulated genes is a bona fide lysosomal protein which undergoes proteolytic maturation during its biosynthesis

    SciTech Connect

    Schaehs, Philipp; Weidinger, Petra; Probst, Olivia C.; Svoboda, Barbara; Stadlmann, Johannes; Beug, Hartmut; Waerner, Thomas; Mach, Lukas

    2008-10-01

    Cellular repressor of E1A-stimulated genes (CREG) has been reported to be a secretory glycoprotein implicated in cellular growth and differentiation. We now show that CREG is predominantly localized within intracellular compartments. Intracellular CREG was found to lack an N-terminal peptide present in the secreted form of the protein. In contrast to normal cells, CREG is largely secreted by fibroblasts missing both mannose 6-phosphate receptors. This is not observed in cells lacking only one of them. Mass spectrometric analysis of recombinant CREG revealed that the protein contains phosphorylated oligosaccharides at either of its two N-glycosylation sites. Cellular CREG was found to cosediment with lysosomal markers upon subcellular fractionation by density-gradient centrifugation. In fibroblasts expressing a CREG-GFP fusion construct, the heterologous protein was detected in compartments containing lysosomal proteins. Immunolocalization of endogenous CREG confirmed that intracellular CREG is localized in lysosomes. Proteolytic processing of intracellular CREG involves the action of lysosomal cysteine proteinases. These results establish that CREG is a lysosomal protein that undergoes proteolytic maturation in the course of its biosynthesis, carries the mannose 6-phosphate recognition marker and depends on the interaction with mannose 6-phosphate receptors for efficient delivery to lysosomes.

  14. HUMAN ADENOVIRUS TYPE 37 AND THE BALB/C MOUSE: PROGRESS TOWARD A RESTRICTED ADENOVIRUS KERATITIS MODEL (AN AMERICAN OPHTHALMOLOGICAL SOCIETY THESIS)

    PubMed Central

    Chodosh, James

    2006-01-01

    Purpose To establish a mouse model of adenovirus keratitis in order to study innate immune mechanisms in the adenovirus-infected cornea. Methods Balb/c 3T3 fibroblasts were inoculated with human adenovirus (HAdV) serotypes 8, 19, or 37 and observed for cytopathic effect. Viral growth titers were performed, and apoptosis was measured by TUNEL assay. Viral and host cytokine gene expression was assessed by RT-PCR in cultured Balb/c 3T3 fibroblasts and in the corneas of virus-injected Balb/c mice. Western blot analysis was performed to detect cell signaling in the virus-infected cornea. Results Only HAdV37 induced cytopathic effect in mouse cells. Viral gene expression was limited, and viral replication was not detected. Apoptotic cell death in HAdV37-infected Balb/c cells was evident 48 and 72 hours postinfection (P < .01). MCP-1, IL-6, KC, and IP-10 mRNA levels were increased maximally by 8.4, 9.6, 10.5, and 20.0-fold, respectively, at 30 to 90 minutes after HAdV37 infection. Similar cytokine elevations were observed in the corneas of Balb/c mice 4 hours after stromal injection of HAdV37, when viral gene expression for the viral capsid protein IIIa was not detected. Western blot showed increased phosphorylation of ERK1/2 at 4 and 24 hours after corneal infection. Conclusions Despite limited viral gene expression, HAdV37 infection of Balb/c 3T3 fibroblasts results in increased proinflammatory gene expression. A similar pattern of cytokine expression in the corneas of HAdV37-infected Balb/c mice suggests the mouse adenoviral keratitis model may be useful for the study of early innate immune responses in the adenovirus-infected corneal stroma. PMID:17471351

  15. STAT1 Interaction with E3-14.7K in Monocytes Affects the Efficacy of Oncolytic Adenovirus

    PubMed Central

    Spurrell, Emma; Gangeswaran, Rathi; Wang, Pengju; Cao, Fengyu; Gao, Dongling; Feng, Baisui; Wold, William; Tollefson, Ann

    2014-01-01

    Oncolytic viruses based on adenovirus type 5 (Ad5) have been developed as a new class of therapeutic agents for cancers that are resistant to conventional therapies. Clinical experience shows that these agents are safe, but virotherapy alone has not achieved long-term cure in cancer patients. The vast majority of oncolytic adenoviruses used in clinical trials to date have deletion of the E3B genes. It has been demonstrated that the antitumor potency of the E3B-deleted mutant (dl309) is inferior to adenovirus with