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Biomedical subjects

J P Trempe

Publications and source records attributed to J P Trempe.

At least 19 recordsLinked to original sources
PubMed2001

Inhibition of adenovirus cytotoxicity, replication, and E2a gene expression by adeno-associated virus.

Adeno-associated virus (AAV) and the other parvoviruses have long been known to inhibit proliferation of nonpermissive cells. The mechanism of this inhibition is not thoroughly understood. To learn how AAV interacts with host cells, we have begun an investigation into AAV's relationship with adenovirus (Ad), AAV's most efficient helper virus. AAV, but not UV-inactivated AAV, delayed Ad-induced cytotoxicity and inhibited Ad E2a gene expression. AAV, but not UV-inactivated AAV or a recombinant AAV vector, inhibited Ad DNA replication. To determine whether AAV or its replication (Rep) proteins alter Ad early gene expression, we measured steady state E2a mRNA levels in AAV and Ad coinfected cultures and in a cell line (Neo6) that inducibly expresses the Rep proteins. AAV, but not UV-AAV, and Rep expression resulted in diminution of E2a protein and mRNA levels. To determine whether the AAV Rep proteins directly affect the individual Ad early promoters, we constructed luciferase reporter plasmids containing each of the five early promoters. Cotransfection of Ad-luciferase and an AAV rep gene-expressing plasmid in HeLa cells demonstrated that Rep78 repressed the E1a, E2a, and E4 promoters but trans-activated the E1b and E3 promoters. In the presence of a cotransfected E1a-expressing plasmid, Rep78 repressed expression from all five promoters. These results indicate that Rep may have different effects on the Ad early promoters dependent upon the presence of the E1a trans-activating protein.

Adenoviridae↗
PubMed2000

Luminometric quantitation of photinus pyralis firefly luciferase and Escherichia coli beta-galactosidase in blood-contaminated organ lysates.

Firefly luciferase and Escherichia coli beta-galactosidase chemiluminescent reporter gene assays are rapid and sensitive means of detecting reporter enzyme activities in cell lysates of both eukaryotic and prokaryotic systems. In these assays, expression vectors containing the luciferase or beta-galactosidase genes are transferred to cells in culture or animal tissues in vivo. Crude cell or organ lysates are then prepared and submitted to enzyme assays. The level of enzyme activity is proportional to the efficiency of gene delivery and expression. When used with modified substrates that emit light when cleaved by the appropriate enzyme, luciferase and beta-galactosidase activity can be detected luminometrically. Attempts to apply these assays to cell lysates contaminated with blood, as from any whole organ lysate, have had questionable results thus far because of light absorption by hemoglobin in the ranges of light emission by both of these assays. We have made several adjustments to standard chemiluminescent reporter gene assay protocols to minimize errors in quantitation contributed by hemoglobin. To this end, we have developed a method for quantitating the protein due to blood and due to the organ itself in a blood-contaminated organ lysate. We have also found that the use of a colorimetric protein assay that is unaffected by hemoglobin absorbance is preferred for protein quantitation. In conclusion, luciferase and beta-galactosidase assays can be applied to blood-contaminated organ lysates; however, the luciferase assay proved to be superior due to minimal endogenous activity and lower absorption by hemoglobin of light emitted by the enzyme product.

Animals↗
PubMed1999

A helper virus-free packaging system for recombinant adeno-associated virus vectors.

Adeno-associated virus (AAV) is a human parvovirus that is currently receiving widespread attention for its potential use as a gene therapy vector. Construction of the recombinant AAV vector (rAAV) involves replacing most of the viral genome with a transgene of interest and then packaging this recombinant genome into an infectious virion. Most current protocols for generating rAAV entail the co-transfection of a vector plasmid and a packaging plasmid that expresses the viral replication and structural genes onto adenovirus (Ad) infected cells growing in culture. Limitations of this procedure include (1) contamination of rAAV with the Ad helper virus, (2) low yields of rAAV and (3) production of replication-competent AAV. In this report we describe new helper plasmids (pSH3 and pSH5) that eliminate the Ad co-infection requirement. The helper plasmids express the AAV rep and cap genes and the Ad E2A, VAI and E4 genes. When the helper plasmids are co-transfected onto human 293 cells with a vector plasmid in the absence of Ad infection, the rAAV vector yield is up to 80-fold greater than those obtained with the pAAV/Ad packaging plasmid. Moreover, replication competent AAV in the rAAV preparations is less than 0.00125%. The major advantages of this system are (1) the absence of infectious adenovirus and (2) the use of only two plasmids, which enhances transfection efficiencies and hence vector production. We believe that this two-plasmid transfection system will allow for more widespread use of the AAV vector system because of its simplicity and high yields. This system will be especially useful for preclinical analyses of multiple rAAV vectors.

Cell Line↗
PubMed1999

Induction of apoptosis by cadmium and the adeno-associated virus Rep proteins.

The parvoviruses exert antiproliferative effects on transformed cells in culture. The development of cell lines that inducibly express the parvovirus nonstructural proteins have implicated these proteins in the limitation of cell growth. To study the host cell interactions of the nonstructural proteins we have developed a human 293 cell line that expresses the adeno-associated virus (AAV) rep gene upon induction with heavy metal salts. When induced with both Zn(2+) and Cd(2+), Rep protein expression correlates with a cell cycle block in S phase (Yang, Q., Chen, F., and Trempe, J. P. (1994). J. Virology 68,7169-7177). However when induced with Cd(2+) alone, the Rep proteins are expressed and the cells are killed. Production of a nucleosomal DNA repeat pattern and degradation of poly-ADP ribose polymerase (PARP) suggest that killing occurs by apoptosis. These results demonstrate that AAV Rep protein expression in chemically stressed cells is cytotoxic due to induction of apoptotic pathways.

Apoptosis↗
PubMed1999

Spectral and physical characterization of the inverted terminal repeat DNA structure from adenoassociated virus 2.

An oligodeoxynucleotide (ODN) that includes elements found in secondary structures at the 5'- and 3'- ends of adenoassociated virus 2 virion DNA was synthesized by ligation of three overlapping ODNs. The most stable secondary structure was calculated to be branched, with a 61 bp duplex stem, terminating in a three-way junction with 9 bp arms. The electrophoretic mobility of the ODN is slower than expected for normal duplex DNA of the same size, suggesting a bent or branched conformation. CD spectra indicate that the ITR structure is largely B form DNA, although there is a slight blue shift compared to the spectra of the isolated stem and loop elements. Thermal melting experiments indicate that the hairpin is significantly more stable than the isolated stem and loop elements. Singular value decomposition of UV spectra obtained as a function of temperature indicates that four species contribute to changes in the spectra upon denaturation, indicating that the melting is not a simple two-state process. Characterization of the branched ODN by differential scanning calorimetry permits estimation of the enthalpy of melting by a model-free analysis, yielding DeltaHcal= 614 kcal mol-1. This value agrees with the enthalpy computed for the most stable secondary structure.

Base Sequence↗
PubMed1999

Enhancement of UV-induced cytotoxicity by the adeno-associated virus replication proteins.

Adeno-associated virus (AAV) normally requires co-infection of a helper virus to complete its life cycle. However, under conditions of cellular stress, such as treatment with carcinogens or ultraviolet (UV) light, a permissive intracellular environment is established and AAV completes its replicative cycle producing low levels of progeny virus. AAV DNA replication is dependent upon viral replication proteins, Rep78 and Rep68. The detailed mechanism by which these proteins interact with host cell factors is unknown. We have used a cell line (Neo6) that inducibly expresses the AAV Rep proteins to study their effects on cells that have undergone UV-induced DNA damage. Induction of Rep protein expression immediately after a sub-lethal dose of UV irradiation resulted in rapid cell killing. Those cells that die had chromatin condensation while cellular membranes remained intact, suggesting that concurrent Rep expression and UV damage induces an apoptosis-like response. However, we did not observe any DNA degradation. Thus we believe that the combination of Rep expression and UV irradiation induces cell death that shares some of the characteristics of apoptosis. UV irradiation and Rep expression induced an increase in the level of the CDK inhibitor, p21Cip, and the appearance of modified forms of both p21Cip and Bcl-2. Alteration of normal expression of these cytostatic/apoptotic proteins provides insight into the intracellular targets of the AAV replication proteins.

Apoptosis↗
PubMed1998

Optimization of packaging of adeno-associated virus gene therapy vectors using plasmid transfections.

Adeno-associated Virus (AAV) is attracting wide attention as a potential human gene therapy vector. The advantages of this vector system are that it is naturally defective, it readily integrates into the target cell's genome and is considered to be nonpathogenic. AAV infects a wide variety of cell and tissue types. The major disadvantages of the vector are its small size and the labor-intensive procedures required to prepare large amounts of the vector for clinical studies. In this manuscript we have systematically tested a number of variables in the packaging procedure to determine the optimal conditions for successful vector preparation. Using an AAV vector that expresses the green fluorescent protein and the most commonly used packaging plasmid, pAAV/Ad, we determined the optimal conditions for; lysis of the transfected cultures, ratio of packaging to vector plasmids, day of harvest after transfection, storage conditions, multiplicity of infection of helper adenovirus, and the time of adenovirus infection. These results have important relevance for investigators that are using AAV vectors for heterologous gene transfer studies.

Cytopathogenic Effect, Viral↗
PubMed1997

Phosphorylation of the adeno-associated virus replication proteins.

The adeno-associated virus (AAV) replication proteins Rep78 and Rep68 regulate viral gene expression and DNA amplification. Their effects on both processes suggest that they play roles in all phases of the virus life cycle. We have investigated Rep protein phosphorylation to determine if this modification might alter Rep function. All four Rep proteins were found to be phosphorylated in AAV and adenovirus co-infected cell cultures, and Rep proteins contained phospho-serine whereas no phospho-threonine or -tyrosine was detected. We also observed that when viral DNA synthesis was inhibited, there was a significant decrease in the level of Rep78/68 phosphorylation. Our results suggest a plausible mechanism whereby AAV Rep 78/68 function may be regulated by phosphorylation.

Cell Line, Transformed↗
PubMed1997

Characterization of the Rep78/adeno-associated virus complex.

Adeno-associated virus (AAV) replication proteins Rep78 and Rep68 play major roles in the life cycle of AAV. We have recently provided in vivo evidence for the existence of a covalent association between Rep78 and virion single-stranded (ss) AAV DNA (K. M. R. Prasad and J.P. Trempe(1995) Virology 214, 360-370). In this work we have further characterized the Rep78 protein-AAV DNA covalent linkage. Exonuclease and primer extension analyses revealed that in the majority of isolated ssDNA, Rep78 protein is covalently linked to one of the 5' terminal thymidines. Pulse-chase experiments with radiolabeled methionine suggest that Rep protein remains associated with the virus particle for up to 8 hr after labeling in infected cells. Quantitative immunoprecipitation indicated that approximately 30% of the ssDNA remains associated with the Rep protein after cell fractionation and partial purification. When cells are infected with Rep-associated AAV particles, a significant proportion of viral DNA remains attached to Rep after entry into the nucleus. However, the linkage does not persist after nuclear entry. These observations suggest that covalently linked Rep78 protein may play a key role during wild-type AAV infections and AAV vector transductions.

Base Sequence↗
PubMed1995

The adeno-associated virus Rep78 protein is covalently linked to viral DNA in a preformed virion.

Resolution of the covalently closed terminus of adeno-associated Virus (AAV) DNA is mediated by viral replication protein Rep78 or Rep68. In vitro studies with purified Rep proteins indicate that concurrent with this resolution is a covalent attachment of one of the proteins to the 5' end of the viral genome. The in vivo existence and fate of the covalently associated Rep protein during the virus life cycle has not yet been elucidated. In this report, we use immunoprecipitation analyses to demonstrate that the Rep78 protein is covalently attached to viral DNA in a preformed virion. The attached Rep78 is susceptible to antibody binding and protease digestion, and the DNA linkage is susceptible to nuclease digestion, therefore Rep78 is probably located on the outside of the particle. Rep proteins are also attached to double-stranded replicative-form monomer (RFM) DNA in extracts from AAV and adenovirus coinfected cells. Rep protein attachment to RFM and encapsidated AAV DNA suggest that the covalent complex is an intermediate in virus assembly. These observations are similar to those noted by others for the autonomous parvoviruses and provide additional insights into parvovirus assembly.

Animals↗
PubMed1995

Inhibition of cellular and SV40 DNA replication by the adeno-associated virus Rep proteins.

In order to define the mechanism used by the adeno-associated virus replication (rep) gene to mediate inhibition of cell proliferation, we have studied its effects on SV40 and cellular DNA replication. SV40 DNA replication was inhibited by the presence of the rep gene in human 293 cells, and the inhibition was not linked to suppression of SV40 early gene expression. Using double-immunofluorescence assays that measured both rep gene expression and bromodeoxyuridine incorporation, we found that the presence of the Rep78 and Rep68 proteins correlated with inhibition of cellular DNA synthesis in NIH3T3 cells. This links the rep gene's anti-proliferative effects to either: (i) a direct inhibition of DNA synthesis or (ii) a possible cell cycle block.

3T3 Cells↗
PubMed1994

Characterization of cell lines that inducibly express the adeno-associated virus Rep proteins.

The replication (rep) gene of adeno-associated virus (AAV) is involved in AAV DNA replication, gene regulation, and inhibition of cellular transformation induced by various oncogenes. To study the rep gene's antiproliferative effects, we have developed cell lines which express the replication proteins under the control of an inducible mouse metallothionein transcription promoter. The Rep78 protein produced in these cell lines binds to the AAV terminal repeat sequences in vitro and supports AAV DNA replication and trans activation of the AAV p40 transcription promoter in vivo. These cell lines are capable of assembling infectious viruses containing a mutant rep gene or a vector bearing a heterologous gene. Growth rate and colony formation efficiency assays indicated that rep gene expression substantially altered cellular proliferation. Long-term induction of the cell lines followed by removal of the inducing agent suggested that constitutive expression of the Rep proteins does not necessarily result in cell death and that the cells can recover from the cytostatic effects. Flow cytometry analysis indicated that the presence of the Rep proteins increased the population of cells in the S phase of the cell cycle. Thus the rep gene's antiproliferative effects may be realized by interference with cellular DNA replication.

Cell Division↗
PubMed1993

Analysis of the terminal repeat binding abilities of mutant adeno-associated virus replication proteins.

The adeno-associated virus (AAV) Rep78 and Rep68 proteins play essential roles in viral DNA replication, trans activation of viral gene expression, and suppression of oncogene-mediated cellular transformation. By using an extensive set of linker insertion and deletion mutations in the replication gene, we mapped the regions of the Rep78 protein that mediate binding to the AAV origin of replication in vitro. Deletions that removed amino acid codons 25 to 62, 88 to 113, 125 to 256, and 346 to 400 abolished binding. Alterations in several other regions of the protein affected the binding affinity of the mutant proteins. All of the mutant proteins that support AAV DNA replication or p40 trans activation bound to the terminal repeat sequence, thus verifying the importance of binding for these functions. Several mutant rep genes that failed to suppress oncogene-mediated cellular transformation produced proteins that were capable of binding to the AAV terminal repeat sequences.

DNA Mutational Analysis↗
PubMed1992

Mutational analysis of the adeno-associated virus rep gene.

The replication (rep) gene of the human parvovirus adeno-associated virus (AAV) is a pleiotropic effector of numerous viral functions and experts profound effects on cellular transformation. Of the four Rep proteins, the primarily nuclear Rep78 and Rep68 direct AAV DNA replication, trans activation of the capsid (cap) gene promoter, and inhibition of cellular proliferation mediated by various oncogenes. In an initial attempt to define functional domains in Rep78, we have constructed a comprehensive set of XhoI linker insertion and deletion mutations in the rep gene. Each of the mutant genes has been expressed in cell culture and assayed for the following functions: (i) nuclear localization, (ii) AAV DNA replication, (iii) trans activation of the AAV capsid gene transcription promoter, and (iv) suppression of cellular transformation mediated by the adenovirus E1a and an activated ras oncogene pair. Modest disruptions in the normal conformation of Rep78 inactivated its AAV DNA replication function and trans activation of the cap gene promoter. Linker insertion mutations in the amino-terminal one-third of the protein inactivated Rep78's ability to suppress oncogene-mediated cellular transformation. The transformation suppression domains are not limited to the amino-terminal regions, however, since deletions throughout the protein altered its suppression capabilities. A putative nuclear localization signal that is essential for each of the above functions was found in the Rep proteins. These results provide a preliminary screening of the functional domains in the AAV Rep proteins and pave the way for more subtle mutational analysis.

3T3 Cells↗
PubMed1991

Adeno-associated virus rep proteins produced in insect and mammalian expression systems: wild-type and dominant-negative mutant proteins bind to the viral replication origin.

The adeno-associated virus (AAV) rep gene proteins, Rep78 and Rep68, are required for replication of AAV DNA and bind to the AAV replication origin. An AAV genome having a Lys340 to His (K340H) mutation in the consensus purine nucleotide binding site of the rep gene protein exhibited a dominant-negative phenotype for DNA replication. We synthesized both wild-type and the K340H mutant Rep78 protein in a baculovirus expression system. Nuclear extracts of Sf9 cells containing these proteins were examined in gel mobility-shift assays with radiolabeled AAV terminal repeat DNA. Each protein bound specifically to the hairpin configuration of the AAV terminal repeat DNA to yield three shifted components. However the mobility of these components observed with the mutant Rep protein was slightly decreased compared to that with the wild-type Rep78. The addition of an antibody made against an oligopeptide from the carboxyl terminal region of the Rep78 protein generated novel shifted bands in the presence of either extract. Similar results were observed when the wild-type and mutant Rep proteins were expressed from an inducible expression system employing the human immunodeficiency virus type 1 transcription promoter in human 293 cells. These results suggest that the dominant-negative phenotype of the K340H mutation may be mediated by binding of the mutant protein to the AAV replication origin.

Amino Acid Sequence↗
PubMed1991

Inhibition of cellular transformation by the adeno-associated virus rep gene.

The replication, or rep, gene of the human parvovirus, adeno-associated virus (AAV), is a pleiotropic effector of numerous viral functions. The rep gene trans-regulates viral DNA replication, mRNA transcription, and assembly of the infectious virion. In addition to its roles in the virus life cycle the rep gene also represses gene expression from viral or cellular transcription promoters in both transient and long-term assays. In this report we have investigated the ability of the rep gene to inhibit cellular transformation mediated by SV40 DNA or the adenovirus E1a and human ras oncogene pair. In DNA transfection assays, the complete AAV rep gene inhibited SV40 DNA and E1a/ras gene-mediated transformation of mouse fibroblasts. AAV DNA plasmids that expressed the Rep68/40 or Rep52/40 proteins alone did not suppress transformation. AAV DNA replication was not required for suppression. Due to the antiproliferative effect of the AAV rep gene, we propose that it acts a viral analogue of cellular anti-oncogenes and is a useful model system for studying the regulation of cellular proliferation.

Adenovirus Early Proteins↗
PubMed1991

Adeno-associated virus Rep protein inhibits human immunodeficiency virus type 1 production in human cells.

The adeno-associated virus (AAV) rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40) required for AAV DNA replication and AAV gene regulation. In addition, the Rep proteins may have pleiotropic regulatory effects in heterologous systems, and in particular Rep78 may mediate a negative regulatory effect. We analyzed the effects of the AAV rep gene on human immunodeficiency virus type 1 (HIV-1) gene expression. The rep gene proteins of AAV type 2 (AAV2) inhibited the trans-activating ability of HIV-1. Constructs containing the AAV2 rep gene (pHIVrep) or a CAT gene (pBennCAT) expressed from the 5' HIV-1 long terminal repeat were inducible for Rep78 and Rep68 or CAT expression, respectively, when cotransfected with a plasmid containing the HIV-1 tat gene (pARtat). When equivalent amounts of pHIVrep and pBennCAT were cotransfected with increasing amounts of pARtat, expression of CAT activity was decreased. The pHIVrep construct was more inhibitory than plasmids expressing rep from the wild-type AAV2 p5 transcription promoter. rep expression from pHIVrep almost completely inhibited the replication of an HIV-1 proviral clone as measured by reverse transcriptase activity and p24 protein levels. Inhibition of HIV-1 production by Rep protein was also seen at the transcriptional level in that all HIV-1 transcripts were decreased when pHIVrep was present. The inhibitory effects of pHIVrep appear to be mediated primarily by Rep78 and perhaps Rep68. These results suggest that a trans-acting protein from a heterologous virus might be used to inhibit HIV-1 growth.

Cell Line↗