pTP Genes Research Articles

Bombyx mori nucleopolyhedrovirus ptp and egt genes are dispensable for triggering enhanced locomotory activity and climbing behavior in Bombyx mandarina larvae

Baculoviruses are classic pathogens that alter host behavior to enhance their dispersal and transmission. While viral protein tyrosine phosphatase (ptp) has been considered as a critical factor for inducing enhanced locomotory activity, preceding investigations have reported that viral ecdysteroid UDP-glucosyltransferase (egt) contributes to triggering climbing behavior in some virus and host species. Here we found that both egt and ptp were dispensable for these abnormal behaviors in Bombyx mandarina larvae induced by Bombyx mori nucleopolyhedrovirus, thus implying that there is an unknown core mechanism of baculovirus-induced alteration of host behaviors.

The sequence characteristics and functions on regulating trehalose metabolism of two PTP genes in Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

Protein tyrosine phosphatase is a type of enzyme commonly found in eukaryotes that specifically hydrolyzes tyrosine phosphate bonds on protein substrates to remove phosphate, thereby regulating protein functions or even cell functions. In this study, we used the model insect Tribolium castaneum to study the sequence characteristics and functions on trehalose metabolism of two PTP proteins in T. castaneum. We found that TcPTP1X1 and TcPTPIVA1 contain 190 and 177 amino acids, respectively. The results of phylogenetic analysis showed that PTP1 and PTPIVA1 are less well conserved across insect lineages. Examining the tissue and developmental expression patterns revealed that TcPTP1X1 and TcPTPIVA1 are expressed in various tissues and stages of T. castaneum; they are highly expressed in the tarsus during the pupal and adult stages. Compared with the negative control dsGFP group, almost all trehalase and 6-trehalose phosphate synthase genes were significantly down-regulated at 48 h after injection with dsPTP1X1 or dsPTPIVA1, except TRE1-3. At 72 h, many of the TRE and TPS genes appeared to return to normal levels or were even significantly upregulated compared with the control group. Silencing of TcPTP1X1 or TcPTPIVA1 altered the distribution of trehalose, glucose and glycogen in T. castaneum. Moreover, suppression of TcPTP1X1 or TcPTPIVA1 expression resulted in reduced adenosine triphosphate (ATP) content. We suggest that knockdown of TcPTP1X1 or TcPTPIVA1 impairs trehalose and energy metabolism in T. castaneum, and they may be new targets for control of storage pests.

CRISPR-Cas fluorescent cleavage assay coupled with recombinase polymerase amplification for sensitive and specific detection of Enterocytozoon hepatopenaei

Enterocytozoon hepatopenaei (EHP) is a parasite that infects pacific whiteleg shrimp, Penaeus vannamei, causing growth retardation and uneven size distributions that lead to severe losses in shrimp productivity. Routine monitoring is crucial to timely prevention and management of EHP, but field-deployable diagnostic kits for EHP are still scarce. Here, we proposed the use of recombinase polymerase amplification (RPA) and CRISPR-Cas12a fluorescence assay, henceforth RPA-Cas12a, for detection of EHP. Targeting ptp2 gene, RPA-Cas12a could detect as few as 50 copies of DNA and showed no reactivity with closely related microsporidia. The entire procedure could be performed at a temperature close to 37 °C within 1 h. Naked eye visualization was possible with UV/blue-light excitation or lateral flow detection. Thus, RPA-Cas12a is a rapid, sensitive and specific detection platform that requires no sophisticated equipment and shows promise for on-site surveillance of EHP.

Cotesia congregata Bracovirus Circles Encoding PTP and Ankyrin Genes Integrate into the DNA of Parasitized Manduca sexta Hemocytes.

Polydnaviruses (PDVs) are essential for the parasitism success of tens of thousands of species of parasitoid wasps. PDVs are present in wasp genomes as proviruses, which serve as the template for the production of double-stranded circular viral DNA carrying virulence genes that are injected into lepidopteran hosts. PDV circles do not contain genes coding for particle production, thereby impeding viral replication in caterpillar hosts during parasitism. Here, we investigated the fate of PDV circles of Cotesia congregata bracovirus during parasitism of the tobacco hornworm, Manduca sexta, by the wasp Cotesia congregata Sequences sharing similarities with host integration motifs (HIMs) of Microplitis demolitor bracovirus (MdBV) circles involved in integration into DNA could be identified in 12 CcBV circles, which encode PTP and VANK gene families involved in host immune disruption. A PCR approach performed on a subset of these circles indicated that they persisted in parasitized M. sexta hemocytes as linear forms, possibly integrated in host DNA. Furthermore, by using a primer extension capture method based on these HIMs and high-throughput sequencing, we could show that 8 out of 9 circles tested were integrated in M. sexta hemocyte genomic DNA and that integration had occurred specifically using the HIM, indicating that an HIM-mediated specific mechanism was involved in their integration. Investigation of BV circle insertion sites at the genome scale revealed that certain genomic regions appeared to be enriched in BV insertions, but no specific M. sexta target site could be identified.IMPORTANCE The identification of a specific and efficient integration mechanism shared by several bracovirus species opens the question of its role in braconid parasitoid wasp parasitism success. Indeed, results obtained here show massive integration of bracovirus DNA in somatic immune cells at each parasitism event of a caterpillar host. Given that bracoviruses do not replicate in infected cells, integration of viral sequences in host DNA might allow the production of PTP and VANK virulence proteins within newly dividing cells of caterpillar hosts that continue to develop during parasitism. Furthermore, this integration process could serve as a basis to understand how PDVs mediate the recently identified gene flux between parasitoid wasps and Lepidoptera and the frequency of these horizontal transfer events in nature.

Baculovirus PTP2 Functions as a Pro-Apoptotic Protein.

The family Baculoviridae encompasses a large number of invertebrate viruses, mainly infecting caterpillars of the order Lepidoptera. The baculovirus Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV) induces physiological and behavioral changes in its host Spodoptera exigua, as well as immunological responses, which may affect virus transmission. Here we show that the SeMNPV-encoded protein tyrosine phosphatase 2 (PTP2) induces mild apoptosis in Spodoptera frugiperda (Sf) 21 cells upon transient expression. Transient expression of a catalytic-site mutant of ptp2 did not lead to apoptosis, indicating that the phosphatase activity of PTP2 is needed to induce apoptosis. We also found that the caspase level (indicator of apoptosis) was higher in cells transfected with the ptp2 gene than in cells transfected with the catalytic mutant. Adding a caspase inhibitor reduced the level of ptp2-induced apoptosis. Moreover, deletion of the ptp2 gene from the viral genome prevented the induction of apoptosis in S. exigua hemocytes. The virus titer and virulence indices (the viral infectivity and the time to death) were not affected by deletion of the ptp2 gene. However, the viral occlusion body yield from S. exigua larvae infected with the mutant virus lacking the ptp2 gene was much lower than the yield from larvae infected with the wild-type (WT) virus. We hypothesize that the observed pro-apoptotic effects of PTP2 are the result of PTP2-mediated immune suppression in larvae, which consequently leads to higher viral occlusion body yields.

Anti-adenoviral Artificial MicroRNAs Expressed from AAV9 Vectors Inhibit Human Adenovirus Infection in Immunosuppressed Syrian Hamsters.

Infections of immunocompromised patients with human adenoviruses (hAd) can develop into life-threatening conditions, whereas drugs with anti-adenoviral efficiency are not clinically approved and have limited efficacy. Small double-stranded RNAs that induce RNAi represent a new class of promising anti-adenoviral therapeutics. However, as yet, their efficiency to treat hAd5 infections has only been investigated in vitro. In this study, we analyzed artificial microRNAs (amiRs) delivered by self-complementary adeno-associated virus (scAAV) vectors for treatment of hAd5 infections in immunosuppressed Syrian hamsters. In vitro evaluation of amiRs targeting the E1A, pTP, IVa2, and hexon genes of hAd5 revealed that two scAAV vectors containing three copies of amiR-pTP and three copies of amiR-E1A, or six copies of amiR-pTP, efficiently inhibited hAd5 replication and improved the viability of hAd5-infected cells. Prophylactic application of amiR-pTP/amiR-E1A- and amiR-pTP-expressing scAAV9 vectors, respectively, to immunosuppressed Syrian hamsters resulted in the reduction of hAd5 levels in the liver of up to two orders of magnitude and in reduction of liver damage. Concomitant application of the vectors also resulted in a decrease of hepatic hAd5 infection. No side effects were observed. These data demonstrate anti-adenoviral RNAi as a promising new approach to combat hAd5 infection.

Abstract B62: A novel combination HER2, brachyury, and MUC1 adenovirus based vaccines for a multitargeted immunotherapy approach to treat breast cancer

Abstract Many studies have shown that most breast cancers express a diverse array of tumor associated antigens (TAA), and the ability to simultaneously target multiple TAA as opposed to targeting a single TAA by immunotherapy could be of clinical benefit, especially in terms of breaking tolerance, impacting tumor growth and increasing overall survival. We report here the development and testing of two new recombinant TAA vaccines containing MUC1 and Brachyury that will be combined with our Ad5 [E1-, E2b-]-HER2/neu vaccine as a multi-target immunotherapeutic. MUC1 is a TAA that is over expressed in 70% of breast cancers and has been recognized as a priority TAA target for tumor immunotherapy. One of the major barriers to immunotherapy is the inability to target antigens that are involved in the progression of primary tumors to a metastatic state. Brachyury represents a new and attractive target as it plays an essential role in epithelial mesenchymal transition and targets cancer stem cells. A recently published study showed that Brachyury is over-expressed in breast cancer and is associated with poor prognosis. Our vaccines are based on an Ad5 [E1-, E2b-]- gene delivery platform that has an E1 gene deletion and additional early 2b (E2b) gene region deletions by removing the DNA polymerase (pol) and pre-terminal protein (pTP) genes. Potent T cell and antibody responses can be induced against vector-expressed antigens even in the presence of hyper Ad5 immunity. Using a truncated non-oncogenic HER2/neu gene in our delivery platform, we reported that this product candidate induces HER2/neu directed humoral and T cell directed immune responses. In addition, the Ad5 [E1-, E2b-]-HER2/neu significantly inhibits tumor growth of HER2/neu expressing tumors in a mouse model. This product candidate has been manufactured under cGMP and will initiate Phase I/2 clinical trials to evaluate immunotherapy treatment of patients with HER2+ breast cancer. To characterize our two new vaccines, Ad5 [E1-, E2b-]-MUC1 and Ad5 [E1-, E2b-]-Brachyury, immunogenicity studies were performed in mice. In collaboration with Dr. Jeffrey Schlom at the NCI we have generated an Ad5 [E1-, E2b-]-MUC1c vaccine that is modified with agonist peptides that results in increased immunogenic activity. In our studies, cell mediated immune (CMI) responses were determined by ELISpot assays and intracellular cytokine staining. Robust T cell responses and TNF-α/IFN-γ expressing polyfunctional CD4+ and CD8+ cells were detected in mice immunized with Ad5 [E1-, E2b-]-MUC1c but not control mice. In immunotherapy protocols, significant anti-tumor activity and increased survival was observed in mice bearing MUC1 expressing tumors and treated with Ad5 [E1-, E2b-]-MUC1c. These results indicate that our Ad5 [E1-, E2b-]-MUC1c can induce anti MUC1 directed anti-tumor responses. We have also generated an Ad5 [E1-, E2b-]-Brachyury modified to contain a HLA-A2 binding epitope that increases immunogenicity. After mice were immunized with this vaccine, splenocytes from immunized mice when stimulated with Brachyury peptide pools gave significant T cell responses compared to controls. As seen with our MUC1 vaccine, Ad5 [E1-, E2b-]-Brachyury also induced polyfunctional TNF-α/IFN-γ expressing CD4+ and CD8+ T cells. These results indicate that Ad5 [E1-, E2b-]-Brachyury can induce an immune response in vivo. This study lays the foundation to test whether a multi-targeted immunotherapy employing HER2/neu, MUC1 and Brachyury vaccines can result in greater anti-tumor activity against established tumors as well as prevent the development of metastatic disease in breast cancer patients. Citation Format: Yvette Latchman, Adrian Rice, Joseph Balint, Elizabeth Gabitzsch, Jeffrey Schlom, Frank Jones. A novel combination HER2, brachyury, and MUC1 adenovirus based vaccines for a multitargeted immunotherapy approach to treat breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B62.

Gene Content and Diversity of the Loci Encoding Biosynthesis of Capsular Polysaccharides of the 15 Serovar Reference Strains of Haemophilus parasuis

Haemophilus parasuis is the causative agent of Glässer's disease, a systemic disease of pigs, and is also associated with pneumonia. H. parasuis can be classified into 15 different serovars. Here we report, from the 15 serotyping reference strains, the DNA sequences of the loci containing genes for the biosynthesis of the group 1 capsular polysaccharides, which are potential virulence factors of this bacterium. We contend that these loci contain genes for polysaccharide capsule structures, and not a lipopolysaccharide O antigen, supported by the fact that they contain genes such as wza, wzb, and wzc, which are associated with the export of polysaccharide capsules in the current capsule classification system. A conserved region at the 3' end of the locus, containing the wza, ptp, wzs, and iscR genes, is consistent with the characteristic export region 1 of the model group 1 capsule locus. A potential serovar-specific region (region 2) has been found by comparing the predicted coding sequences (CDSs) in all 15 loci for synteny and homology. The region is unique to each reference strain with the exception of those in serovars 5 and 12, which are identical in terms of gene content. The identification and characterization of this locus among the 15 serovars is the first step in understanding the genetic, molecular, and structural bases of serovar specificity in this poorly studied but important pathogen and opens up the possibility of developing an improved molecular serotyping system, which would greatly assist diagnosis and control of Glässer's disease.

Expression of protein-tyrosine phosphatases in Acute Myeloid Leukemia cells: FLT3 ITD sustains high levels of DUSP6 expression

Protein-tyrosine phosphatases (PTPs) are important regulators of cellular signaling and changes in PTP activity can contribute to cell transformation. Little is known about the role of PTPs in Acute Myeloid Leukemia (AML). The aim of this study was therefore to establish a PTP expression profile in AML cells and to explore the possible role of FLT3 ITD (Fms-like tyrosine kinase 3 with internal tandem duplication), an important oncoprotein in AML for PTP gene expression. PTP mRNA expression was analyzed in AML cells from patients and in cell lines using a RT-qPCR platform for detection of transcripts of 92 PTP genes. PTP mRNA expression was also analyzed based on a public microarray data set for AML patients. Highly expressed PTPs in AML belong to all PTP subfamilies. Very abundantly expressed PTP genes include PTPRC, PTPN2, PTPN6, PTPN22, DUSP1, DUSP6, DUSP10, PTP4A1, PTP4A2, PTEN, and ACP1. PTP expression was further correlated with the presence of FLT3 ITD, focusing on a set of highly expressed dual-specificity phosphatases (DUSPs). Elevated expression of DUSP6 in patients harboring FLT3 ITD was detected in this analysis. The mechanism and functional role of FLT3 ITD-mediated upregulation of DUSP6 was then explored using pharmacological inhibitors of FLT3 ITD signal transduction and si/shRNA technology in human and murine cell lines. High DUSP6 expression was causally associated with the presence of FLT3 ITD and dependent on FLT3 ITD kinase activity and ERK signaling. DUSP6 depletion moderately increased ERK1/2 activity but attenuated FLT3 ITD-dependent cell proliferation of 32D cells. In conclusion, DUSP6 may play a contributing role to FLT3 ITD-mediated cell transformation.

Transcriptomic profiling of Microplitis demolitor bracovirus reveals host, tissue and stage-specific patterns of activity

The polydnaviruses (PDVs) are a family of DNA viruses that are symbiotically associated with parasitoid wasps. The transcription of particular genes or gene-family members have been reported for several PDVs, but no studies have characterized the spatio-temporal patterns of expression for the entire complement of predicted genes in the encapsidated genome of any PDV isolate. The braconid wasp Microplitis demolitor carries the PDV Microplitis demolitor bracovirus (MdBV) and parasitizes larval stage Pseudoplusia (Chrysodeixis) includens. The encapsidated genome consists of 15 genomic segments with 51 predicted ORFs encoding proteins ≥100 aa. A majority of these ORFs form four multimember gene families (ptp, ank, glc and egf) while the remaining ORFs consist of single copy (orph) genes. Here we used RT-PCR and quantitative real-time PCR methods to profile the encapsidated transcriptome of MdBV in P. includens and M. demolitor. Our results indicate that most predicted genes are expressed in P. includens. Spatial patterns of expression in P. includens differed among genes, but temporal patterns of expression were generally similar, with transcript abundance progressively declining between 24 and 120 h. A subset of ptp, ank and orph genes were also expressed in adult female but not male M. demolitor. Only one encapsidated gene (ank-H4) was expressed in all life stages of M. demolitor, albeit at much lower levels than in P. includens. However, another encapsidated gene (orph-B1) was expressed in adult M. demolitor at similar levels to those detected in P. includens.

Frameshift mutations in coding repeats of protein tyrosine phosphatase genes in colorectal tumors with microsatellite instability

BackgroundProtein tyrosine phosphatases (PTPs) like their antagonizing protein tyrosine kinases are key regulators of signal transduction thereby assuring normal control of cellular growth and differentiation. Increasing evidence suggests that mutations in PTP genes are associated with human malignancies. For example, mutational analysis of the tyrosine phosphatase (PTP) gene superfamily uncovered genetic alterations in about 26% of colorectal tumors. Since in these studies tumors have not been stratified according to genetic instability status we hypothesized that colorectal tumors characterized by high-level of microsatellite instability (MSI-H) might show an increased frequency of frameshift mutations in those PTP genes that harbor long mononucleotide repeats in their coding region (cMNR).ResultsUsing bioinformatic analysis we identified 16 PTP candidate genes with long cMNRs that were examined for genetic alterations in 19 MSI-H colon cell lines, 54 MSI-H colorectal cancers, and 17 MSI-H colorectal adenomas. Frameshift mutations were identified only in 6 PTP genes, of which PTPN21 show the highest mutation frequency at all in MSI-H tumors (17%).ConclusionAlthough about 32% of MSI-H tumors showed at least one affected PTP gene, and cMNR mutation rates in PTPN21, PTPRS, and PTPN5 are higher than the mean mutation frequency of MNRs of the same length, mutations within PTP genes do not seem to play a common role in MSI tumorigenesis, since no cMNR mutation frequency reached statistical significance and therefore, failed prediction as a Positive Selective Target Gene.

Relaxed template specificity in fowl adenovirus 1 DNA replication initiation

The fowl adenovirus 1 (FAdV-1) isolates PHELPS and OTE are highly similar, but have striking differences in the repeat region of the inverted terminal repeat (ITR). Whilst the repeat region in OTE conforms to the conventional human adenovirus repeat region (5'-CATCATC), that of PHELPS contains guanidine residues at positions 1, 4 and 7 (5'-GATGATG). This implies that the FAdV-1 isolates PHELPS and OTE have either distinct template specificity at replication initiation or, alternatively, a relaxed specificity for replication initiation. In this study, the distinct sequence variation at the origin of DNA replication in the ITRs of the FAdV-1 PHELPS and OTE isolates was confirmed. Sequence analyses of the pTP and Pol genes of both PHELPS and OTE did not reveal differences that could explain the distinct template specificity. Replication assays demonstrated that linear DNA fragments flanked by either 5'-CATCATC or 5'-GATGATG termini replicated in cells upon infection with FAdV-1 OTE and FAdV-1 PHELPS. This was evident from the appearance of DpnI-resistant fragments in a minireplicon assay. From these data, it is concluded that FAdV-1 has relaxed, rather than changed, its template specificity at replication initiation.

543. Ad DNA Polymerase and Preterminal Protein (pTP) Genes Each Mediate Inhibition of HIV-1 Replication in Human Alveolar Macrophages

Macrophages serve as a reservoir for HIV-1 even in the presence of highly active antiretroviral therapy. Adenovirus (Ad) gene transfer vectors (E1−E3−) inhibit HIV-1 replication in human alveolar macrophages (AM) at a step in the HIV-1 life cycle at, or prior to, HIV-1 long terminal repeat (LTR) transcription. The inhibition of HIV-1 replication by Ad is independent of the transgene, the Ad E4 region and the Ad capsid (Am J Respir Cell Mol Biol. 2002; 27: 214–219). We previously observed that both fully deleted Ad vectors or 1st generation vectors (E1−E3−) with an additional deletion of the Ad E2b region lose the ability to inhibit HIV-1 replication in AM, implicating E2b as the inhibitor of HIV-1 replication. The Ad E2b region includes the Ad DNA polymerase and the preterminal protein (pTP) genes that are needed for efficient initiation of Ad DNA replication and transcription of late proteins in wild type Ad. To determine their relative contributions, we used Ad vectors with an additional deletion in the Ad pol, but containing the pTP gene. One vector expressed the β-galactosidase gene (Adpol−lacZ); another expressed the human acid-alpha-glucosidase gene (Adpol−hGAA). Both Adpol−lacZ and AdE2b−lacZ mediated expression of the β-galactosidase gene in THP-1 cells at a dose of 2.5×104 particle units (pu)/cell. To quantify the relative contribution of pTP, AM (2×105/well), were infected with AdNull, AdE2b−lacZ, Adpol−lacZ, Adpol−hGAA, or empty Ad capsids 6×103 to 105 pu/cell or no Ad for 1 hr. After Ad infection, the AM were infected with HIV-1 strain JRFL, 103 TCID50/well and p24 in the media was quantified by ELISA. For the Ad dose of 2.5×104 pu/cell on day 25 post-infection, p24 levels were: AdNull 23±18, AdE2b-lacZ 5410±431, Adpol−lacZ 23±20, Adpol−hGAA 10±0, empty Ad capsids 9853±1170, no Ad 9942±217 pg/ml (p<0.05, ANOVA). This represents a greater than 2 log inhibition by AdNull, Adpol−lacZ and Adpol−hGAA, but little inhibition by empty Ad capsids or AdE2b−lacZ. To quantify the relative contribution of Ad pol, we used an Ad vector with an additional deletion in the Ad pTP, but containing the Ad DNA polymerase gene and expressing the green fluorescent protein gene (AdpTP−GFP). AZT 10 μg/ml was used as an additional control. For the Ad dose of 2.5×104 pu/cell on day 24 post-infection, p24 levels were: AdNull 931×338, AdpTP−GFP 940×88, AZT 932×68 no Ad 6368×1220 pg/ml (p<0.05, ANOVA). Taken together with our previous observations on the lack of inhibitory effect of E2b− and fully deleted Ad vectors, these data imply that the presence of either the Ad pTP or Ad DNA polymerase genes alone in the context of a 1st generation Ad vector are sufficient to inhibit HIV-1 replication in human AM.

Overexpression of the adenovirus type 12 (Ad12) pTP or E1A gene facilitates Ad12 DNA replication in nonpermissive BHK21 hamster cells.

In the adenovirus type 12 (Ad12) hamster cell system, abortive virus infection is one of the factors associated with the highly efficient oncogenesis in newborn Syrian hamsters. We have shown earlier that the replication and efficient late transcription of the Ad12 genome are blocked in Syrian hamster cells. Some of the early Ad12 functions are transcribed in these cells, although at a minimal rate. In the present study, we demonstrate that low expression levels of the E1A and precursor to terminal protein (pTP) genes of Ad12 seem to be responsible for the lack of Ad12 DNA replication in hamster cells. The Ad12 genes for the E1A functions or for pTP were tethered to the strong early promoter of the human cytomegalovirus and transfected into BHK21 cells. Subsequently, these cells were infected with Ad12 virions. In Ad12-infected BHK21 cells, which overexpressed pTP or E1A, full-length Ad12 DNA was de novo synthesized, as documented by metabolic labeling with [3H]thymidine and by zone velocity sedimentation in alkaline sucrose gradients followed by gel electrophoresis of the 3H-labeled DNA and Southern blot hybridization to 32P-labeled Ad12 DNA. Transfection of the cloned E1A region of Ad2 yielded similar results. The newly synthesized Ad12 DNA was covalently linked to pTP. The Ad12 DNA binding protein (DBP) and DNA polymerase (pol) genes were transcribed at levels similar to those in merely Ad12-infected cells. In pTP or E1A gene-transfected and Ad12-infected BHK21 cells, marginal levels of late Ad12 mRNA were detectable. Late Ad12 proteins were, however, not synthesized. Apparently, Ad12 DNA replication in hamster cells is rendered impossible due to insufficient threshold levels of the viral E1A and/or pTP.

Multiply deleted [E1, polymerase-, and pTP-] adenovirus vector persists despite deletion of the preterminal protein.

The inherent limitations of [E1-]Ad vectors as gene therapy vehicles suggest that further modifications may improve their overall performance profiles. However, Ad vector modifications can have untoward effects on their basic biology, e.g., some helper-virus dependent Ad vectors have been found to be unstable without the presence of preterminal protein (pTP) activities. Despite this concern, we generated a new class of helper-virus independent Ad vector that was multiply deleted for the E1, polymerase, and pTP genes, and investigated the ramifications of these deletions upon several vector performance parameters. The construction and propagation of an [E1-, polymerase-, pTP-]Ad vector was achieved with the use of trans-complementing cells co-expressing the Ad E1, polymerase and pTP genes. High titer production of the [E1-, polymerase-, pTP-]Ad vector was successfully accomplished via conventional Ad purification techniques. This unique class of Ad vector was capable of long-term gene transfer in vivo (despite lacking pTP functions) that was concomitant with a significantly decreased hepatic toxicity. Previous studies had suggested that Ad genome persistence in vivo may be dependent upon the presence of low level vector genome replication and/or pTP functions. Our results suggest that [E1-, polymerase-, pTP-]Ad vectors can overcome these barriers. The further benefits afforded by the use of this class of Ad vector (increased cloning capacity, high level growth, decreased propensity to generate replication competent Ad (RCA), decreased toxicity) suggests that they will be highly beneficial for use in several aspects of human gene therapy.

Regulation by protein-tyrosine phosphatase PTP2 is distinct from that by PTP1 during Dictyostelium growth and development.

We have cloned a gene encoding a second Dictyostelium discoideum protein-tyrosine phosphatase (PTP2) whose catalytic domain has approximately 30 to 39% amino acid identity with those of other PTPs and a 41% amino acid identity with D. discoideum PTP1. Like PTP1, PTP2 is a nonreceptor PTP with the catalytic domain located at the C terminus of the protein. PTP2 has a predicted molecular weight of 43,000 and possesses an acidic 58-amino-acid insertion 24 amino acids from the N terminus of the conserved catalytic domain. PTP2 transcripts are expressed at moderate levels in vegetative cells and are induced severalfold at the onset of development. Studies with a PTP2-lacZ reporter gene fusion indicate that PTP2, like PTP1, is preferentially expressed in prestalk and anterior-like cell types during the multicellular stages of development. PTP2 gene disruptants (ptp2 null cells) are not detectably altered in growth and show a temporal pattern of development similar to that of wild-type cells. ptp2 null slugs and fruiting bodies, however, are significantly larger than those of wild-type slugs, suggesting a role for PTP2 in regulating multicellular structures. D. discoideum strains overexpressing PTP2 from the PTP2 promoter exhibit growth rate and developmental abnormalities, the severity of which corresponds to the level of PTP2 overexpression. Strains with high overexpression of the PTP2 gene grow slowly on bacterial lawns and produce small cells in axenic medium. When development is initiated in these strains, cells are able to aggregate but then stop further morphogenesis for 6 to 8 h, after which time a variable fraction of these aggregates continue with normal timing, producing diminutive fruiting bodies. These disruption and overexpression phenotypes for PTP2 are distinct from the corresponding mutant PTP1 phenotypes. Immunoprobing PTP2 mutant strains during growth and development with antiphosphotyrosine antibodies reveals several changes in the tyrosine phosphorylation of proteins in PTP2 mutant strains compared with that in wild-type cells. These changes are different from those identified in the previously characterized corresponding PTP1 disruption and overexpression mutant strains. Thus, although PTP2 and PTP1 are nonreceptor PTPs with similar spatial patterns of expression, our findings suggest that they possess distinct regulatory functions in controlling D. discoideum growth and development.

Isolation and characterization of a second protein tyrosine phosphatase gene, PTP2, from Saccharomyces cerevisiae.

A putative protein tyrosine phosphatase (PTPase) gene, PTP2, was cloned from Saccharomyces cerevisiae. The complete yeast PTP2 gene encodes a 750-amino acid residue protein with a predicted mass of 86 kDa. The conserved PTPase domain was localized in the C-terminal half of the protein. Amino acid sequence alignment of the yeast PTPase domain with other phosphatases indicated approximately 20-25% sequence identity with the mammalian PTPase and a similar degree of identity with the PTPase encoded by the yeast PTP1 gene. The PTP2 gene is closely linked to the yeast RET1 and STE4 genes and is localized on the right arm of chromosome 15. Gene disruption experiments demonstrated that neither PTP2 alone nor PTP2 in combination with PTP1 was essential for growth under the conditions tested. The ability of PTP2 to complement the cdc25-22 mutant of Schizosaccharomyces pombe was also examined, and unlike the human T-cell PTPase, which was able to complement the cdc25-22 mutant, the S. cerevisiae PTP2 was unable to complement the cdc25-22 mutant of S. pombe.

Linker mutation scanning of the genes encoding the adenovirus type 5 terminal protein precursor and DNA polymerase

The replication of adenovirus DNA requires, in addition to several host factors, three virus-encoded proteins: a DNA binding protein, the precursor of the terminal protein (pTP), and a DNA polymerase (Ad poi). Ad poi and pTP form a tight complex that is necessary for the initiation step in DNA replication. To perform mutation scanning of the adenovirus type 5 pTP and Ad poi a series of in-frame linker insertions of a 12-mer oligonucleotide d(CCCATCGATGGG) were introduced into cloned viral DNA fragments containing coding sequences of these proteins. The insertions are located at recognition sites for several blunt end-cutting restriction endonucleases. Forty different sites were mutagenized and the mutated genes were transferred to a plasmid that contains the left 42% of the adenovirus genome. They were rebuilt into the viral genome by means of in vivo recombination between plasmid DNA and digested adenovirus DNA-TP complex. The resulting viral genomes were tested for viability and rescued virus was analyzed for the presence of the inserted linker oligonucleotide. This procedure resulted in recovery of a number of viable virus mutants with insertions in the pTP or Ad poi genes, all of which are phenotypically silent. The other mutations did not allow virus production. The positions of these apparent lethal codon insertion mutations were useful to identify regions of functional importance in both proteins. It can be concluded that the precursor-specific region of pTP plays an important role in virus multiplication.