Biomedical subjects

D Derse

Publications and source records attributed to D Derse.

At least 55 records · Page 3Linked to original sources

PubMed1990

Construction of a recombinant bovine leukemia virus vector for analysis of virus infectivity.

A recombinant bovine leukemia virus (BLV) was constructed in which the X region was replaced with the bacterial neomycin resistance gene controlled by the simian virus 40 early promoter. This virus, termed BLV-SVNEO, is a self-packaging, activator-dependent retroviral vector. Introduction of the plasmid pBLV-SVNEO into mammalian cells resulted in constitutive expression of the neo gene, whereas the BLV structural genes, gag, pol, and env, were expressed only in the presence of the two regulatory proteins, Tax and Rex. The production and release of recombinant virus by cells transfected with pBLV-SVNEO were proportional to the number of G418-resistant colonies that developed after susceptible cells were exposed to the filtered culture medium. BLV-SVNEO was able to infect cell lines of human, bovine, canine, feline, and murine origin. BLV-producing cell lines were resistant to superinfection with BLV-SVNEO. This cell-virus system should facilitate molecular genetic studies of BLV and will provide a rapid, quantitative measure of BLV infectivity in a variety of cell types. These studies also demonstrate the feasibility of using activator-dependent retroviral vectors such as BLV-SVNEO to deliver foreign genes into cells and eventually animals.

Animals↗

PubMed1990

Equine infectious anemia virus tat: insights into the structure, function, and evolution of lentivirus trans-activator proteins.

Equine infectious anemia virus (EIAV) contains a tat gene which is closely related to the trans-activator genes of the human and simian immunodeficiency viruses. Nucleotide sequence analysis of EIAV cDNA clones revealed that the tat mRNA is composed of three exons; the first two encode Tat and the third may encode a Rev protein. Interestingly, EIAV Tat translation is initiated at a non-AUG codon in exon 1 of the mRNA, perhaps allowing an additional level of gene regulation. The deduced amino acid sequence of EIAV tat, combined with functional analyses of tat cDNAs in transfected cells, has provided some unique insights into the domain structure of Tat. EIAV Tat has a C-terminal basic domain and a highly conserved 16-amino-acid core domain, but not the cysteine-rich region, that are present in the primate immunodeficiency virus Tat proteins. Thus, EIAV encodes a relatively simple version of this kind of trans activator.

Amino Acid Sequence↗

PubMed1990

Cloning and characterization of cDNAs encoding equine infectious anemia virus tat and putative Rev proteins.

We isolated and characterized six cDNA clones from an equine infectious anemia virus-infected cell line that displays a Rev-defective phenotype. With the exception of one splice site in one of the clones, all six cDNAs exhibited the same splicing pattern and consisted of four exons. Exon 1 contained the 5' end of the genome; exon 2 contained the tat gene from mid-genome; exon 3 consisted of a small section of env, near the 5' end of the env gene; and exon 4 contained the putative rev open reading frame from the 3' end of the genome. The structures of the cDNAs predict a bicistronic message in which Tat is encoded by exons 1 and 2 and the presumptive Rev protein is encoded by exons 3 and 4. tat translation appears to be initiated at a non-AUG codon within the first 15 codons of exon 1. Equine infectious anemia virus-specific tat activity was expressed in transient transfections with cDNA expression plasmids. The predicted wild-type Rev protein contains 30 env-derived amino acids and 135 rev open reading frame residues. All of the cDNAs had a frameshift in exon 4, leading to a truncated protein and thus providing a plausible explanation for the Rev-defective phenotype of the original cells. We used peptide antisera to detect the faulty protein, thus confirming the cDNA sequence, and to detect the normal protein in productively infected cells.

Amino Acid Sequence↗

PubMed1990

Structure and expression of the equine infectious anemia virus transcriptional trans-activator (tat).

Equine infectious anemia virus (EIAV) encodes a tat gene which is closely related to the trans-activators encoded by the human and simian immunodeficiency viruses. Nucleotide sequence analysis of EIAV cDNA clones revealed that the tat message is composed of three exons; the first two encode tat and the third may encode rev.. Interestingly, EIAV tat translation is initiated at a non-AUG codon in the first exon of the message, perhaps allowing an additional level of gene regulation. The deduced amino acid sequence of EIAV tat, combined with functional analyses of tat cDNAs in transfected cells, have provided some unique insights into the domain structure of this protein. EIAV Tat has a C-terminal basic domain, a highly conserved 16 amino acid core domain, but not the cysteine-rich region, that is present in the primate immunodeficiency virus Tat proteins. Thus EIAV encodes a relatively simple version of this kind of trans-activator.

Amino Acid Sequence↗

PubMed1989

Cross-activation of the Rex proteins of HTLV-I and BLV and of the Rev protein of HIV-1 and nonreciprocal interactions with their RNA responsive elements.

The Rex regulatory proteins of human T-cell leukemia virus type I (HTLV-I) and bovine leukemia virus (BLV), and the Rev protein of human immunodeficiency virus type 1 (HIV-1), promote the cytoplasmic accumulation and translation of viral messenger mRNAs encoding structural proteins. Rev and Rex act through cis-acting elements on the viral RNA; these elements are named Rev- and Rex-responsive elements, or RRE and RXRE, respectively. We show that the Rex proteins of HTLV-I and BLV are interchangeable, but only the Rex protein of HTLV-I can substitute for Rev of HIV-1. Rex of HTLV-I and Rev of HIV-1 appear to act on RRE by similar mechanisms. Rev of HIV-1 does not act on the RXRE of HTLV-I or BLV. The nonreciprocal action of Rev and Rex suggests that these factors interact directly with the cis-acting RNA elements of the two viruses.

Animals↗

PubMed1988

trans-acting regulation of bovine leukemia virus mRNA processing.

Bovine leukemia virus (BLV) and the human T-cell leukemia virus types I and II comprise a unique retrovirus subfamily which has evolved complex strategies for the regulation of gene expression. A transcriptional control circuit has been characterized in both human and bovine systems in which cis-acting promoter control elements are responsive to trans-acting factors encoded in the pX region of the virus. The BLV pX mRNA encoding the transcriptional trans-acting factor is translated in an alternate reading frame to produce an 18-kilodalton nuclear phosphoprotein, p18. A function for this protein was revealed in cotransfection experiments using mutated BLV proviruses in combination with pX expression plasmids. These experiments indicated that p18 was required for the accumulation of viral mRNAs representing full-length (genomic) and single-spliced (env) transcripts. In contrast, synthesis of the double-spliced pX mRNA was not influenced by p18 expression. Large regional deletions and substitutions of provirus sequences localized elements essential for p18 regulation to the 3' long terminal repeat. Furthermore, sequences within a 250-nucleotide region between the AATAAA signal and poly(A) site were found to be essential for efficient virus mRNA 3'-end processing and response to p18 regulation.

Animals↗

PubMed1988

cis- and trans-acting regulation of gene expression of equine infectious anemia virus.

Deletion analysis of the equine infectious anemia virus long terminal repeat revealed that sequences responsive to virus-specific transactivation are located within the region spanning the transcriptional start site (-31 to +22). In addition, an active exon of a trans-acting factor (tat) was identified downstream of pol and overlapping env (nucleotides 5264 to 5461). Activation by tat is accompanied by an increase in the steady-state levels of mRNA directed by the equine infectious anemia virus long terminal repeat.

Animals↗

PubMed1987

Characterization of equine infectious anemia virus long terminal repeat.

The long terminal repeats (LTRs) of equine infectious anemia virus (EIAV) were examined with respect to their ability to function as transcriptional promoters in various cellular environments. Nucleotide sequence analyses of the LTRs derived from two unique proviral clones revealed the requisite consensus transcription and processing signals. One of the proviruses possessed a duplication of a 16-base-pair sequence in the CCAAT box region of the LTR which was absent in the other provirus. To assess its functional activity, each LTR was coupled to the bacterial chloramphenicol acetyltransferase gene and transfected onto various cell lines, including matched cultures of EIAV-infected and uninfected cells. The levels of chloramphenicol acetyltransferase activity directed by the EIAV LTRs were between 250 and 900 times greater in EIAV-infected cells compared with their uninfected counterparts. Thus, EIAV expression appears to be activated by a virus-induced trans-activation phenomenon analogous to that recently shown to amplify expression of certain other lentiviruses.

Base Sequence↗

PubMed1987

Bovine leukemia virus transcription is controlled by a virus-encoded trans-acting factor and by cis-acting response elements.

Bovine leukemia virus (BLV) gene expression is exquisitely regulated at multiple levels, including a transcriptional control effected by virus-encoded trans-acting factors and cis-acting target sequences. Like the human T-cell leukemia viruses type I and type II, but unlike other RNA tumor viruses, BLV contains several open reading frames at the 3' end of its genome. A subgenomic mRNA which encodes two overlapping reading frames from this region could produce proteins of 38 and 18 kilodaltons (kDa). A series of cis-trans experiments using transfected virus gene constructs in different combinations revealed that expression of the 38-kDa protein was both necessary and sufficient to activate, in trans, the BLV promoter. This activation was specific for the BLV long terminal repeat, as a variety of related retroviral promoters were not responsive to the expression of the 38-kDa protein p38(XBL). Deletion analysis and construction of chimeric promoters identified a 75-base-pair long terminal repeat region which functions like a p38(XBL)-dependent enhancer element.

Cloning, Molecular↗

PubMed1986

Two elements in the bovine leukemia virus long terminal repeat that regulate gene expression.

The bovine leukemia virus, like the human T-cell leukemia viruses (HTLV-I and HTLV-II), are unusual biologically in that viral transcripts are not detected in tumors or infected tissues. The bovine leukemia virus long terminal repeat (BLV LTR) functions as a transcriptional promoter only in cell lines productively infected with BLV. Deletion mapping indicated that at least two regions of the LTR, on the 5' and 3' sides of the RNA start site, influenced gene expression. An analysis has now been made of the effects of coupling sequences from these LTR regions to a heterologous core promoter derived from the SV40 early promoter unit. Through the use of the transient expression of the bacterial chloramphenicol acetyltransferase (CAT) gene to monitor transcriptional activity in vivo, two independent, regulatory elements were identified in the BLV LTR. One was present in a fragment of 75 base pairs derived from the U3 region of the LTR and behaved much like other enhancer elements. It may be a major determinant of BLV expression in productively infected cell lines, since it enhanced transcription controlled by the heterologous core promoter only in these cells. The second element was contained in a 250-bp fragment derived from LTR sequences in the R region, located downstream from the RNA start site. Its activation of CAT expression was not dependent on BLV infection and was evident only when the fragment was located immediately downstream from the RNA start site. BLV expression thus appears to be regulated in part by a cell-specific enhancer element upstream from the core promoter and a novel sequence downstream from the RNA initiation site in the viral LTR.

Animals↗

PubMed1986

Transactivation induced by human T-lymphotropic virus type III (HTLV III) maps to a viral sequence encoding 58 amino acids and lacks tissue specificity.

The acquired immune deficiency syndrome (AIDS) retrovirus, HTLV-III/LAV, encodes a transacting factor which directly or indirectly stimulates the expression of genes linked to its LTR. To further dissect this phenomenon, we have cotransfected a biologically active molecular clone of HTLV-III and a recombinant plasmid containing an indicator gene, the bacterial gene for chloramphenicol acetyltransferase (CAT), under the control of the HTLV-III LTR. Amplified CAT activity was detected in both lymphoid cells and fibroblasts from a number of species in the presence of the proviral DNA. Deletion experiments confirm the previous assignment of the gene required for transactivation to a region immediately 5' to the envelope gene, and further narrow down the critical functional domain to a coding sequence of 58 codons.

Acetyltransferases↗

PubMed1985

Bovine leukemia virus long terminal repeat: a cell type-specific promoter.

The functional activity of the promoter unit contained within the long terminal repeat (LTR) of bovine leukemia virus (BLV) was examined by monitoring transient expression of a heterologous gene placed under its control. Various cell lines were transfected with recombinant plasmids carrying the bacterial chloramphenicol acetyltransferase (CAT) gene coupled to the BLV LTR (pBL-cat). Transient expression of CAT activity directed by the BLV LTR was observed only in the established BLV-producer cell lines derived from fetal lamb kidney (FLK) cells and bat lung cells. The amount of CAT activity transiently expressed in FLK-BLV cells was decreased approximately tenfold by deletion of LTR sequences located within a region 100 to 170 nucleotides upstream of the RNA start site. Surprisingly, removal of the region 50 base pairs downstream of the RNA initiation site to the 3'-end of the LTR reduced the expression of CAT activity by 87 percent. The BLV LTR thus appears to be an unusual promoter unit, functioning in a cell type-specific manner and possessing sequences on both the 5' and 3' sides of the RNA start site that influence gene expression.

Animals↗

PubMed1985

Nucleotide sequence and structure of integrated bovine leukemia virus long terminal repeats.

Bovine leukemia virus (BLV) proviruses, harbored by the productively infected fetal lamb kidney (FLK-BLV) cell line, were cloned in bacteriophage lambda L47. The nucleotide sequence of the proviral long terminal repeats (LTR) with flanking cell and virus DNA have been determined. The BLV LTR is 531 bp in length and is bounded by the dinucleotides 5'-TG...CA-3', which are part of a 3-bp inverted repeat. The integrated provirus is flanked by 6-bp direct repeats of cellular DNA. A tRNApro primer binding site is present starting 2 bp downstream of the 5' LTR. In addition to sequencing integrated proviral DNA clones, the nucleotide sequence of a cDNA clone, representing the 3' end of genomic viral RNA, was determined; thus revealing the RNA polyadenylation site and R:U5 boundary within the LTR. Unlike most other retroviruses, a consensus polyadenylation signal, "AATAAA," is not located proximal to the BLV polyadenylation site. The RNA initiation site, defining the U3:R boundary, was located in the BLV LTR by S1 nuclease mapping. This site is approximately 25 bp downstream of an A + T-rich region which probably encompasses a Goldberg-Hogness ("TATAA") box and about 90 bp downstream of a potential "CCAAT" box. The BLV LTR possesses a U3 region of 204 bp, an unusually long R region of 241 bp, and a U5 region of 86 bp.

Base Sequence↗

PubMed1983

Properties of purified enzymes induced by pathogenic drug-resistant mutants of herpes simplex virus. Evidence for virus variants expressing normal DNA polymerase and altered thymidine kinase.

The DNA polymerases and thymidine kinases induced by three drug-resistant mutants of herpes simplex virus type 1 (S1, Tr7, and B3) and their common parent strain, SC16, have been purified and their properties compared. No significant differences were seen in the affinities of the polymerases for TTP and dGTP, or for the triphosphates of 9-(2-hydroxyethyloxymethyl)guanine (acyclovir) or (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVdU) (drugs used in their isolation). In contrast all three mutants induced abnormal thymidine kinases. Those induced by the acyclovir-resistant mutants, S1 and Tr7, showed reduced affinities for thymidine, acyclovir, and also BVdU. Thymidine kinase induced by the BVdU-resistant mutant B3 showed reduced affinity for BVdU, but its affinities for thymidine and acyclovir were similar to those of the wild type enzyme. Thus, it appears that these variants of herpes simplex virus express altered thymidine kinases with impaired ability to phosphorylate particular nucleoside analogue drugs and these characteristics probably account for the drug resistance of the viruses. This strategy for resistance is important as it may result in variants with undiminished pathogenicity.

Acyclovir↗

PubMed1982

Characterization of the DNA polymerases induced by a group of herpes simplex virus type I variants selected for growth in the presence of phosphonoformic acid.

Five independently derived variants of a herpes simplex virus type I (HSV-1) strain were plaque purified from a virus population passaged in 1 mM phosphonoformic acid (PFA). The DNA polymerase induced by the parent and PFA-resistant viruses were purified and characterized. No differences were observed among the enzymes with respect to their chromatographic properties, specific activities, or polypeptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The variant enzymes exhibited levels of PFA resistance which ranged from 15- to 25-fold. Resistance to PFA was always associated with a similar degree of resistance to its congener phosphonoacetic acid, but cross-resistance to beta-phenylphosphonoacetic acid was only seen with two of the five variant enzymes. PFA and pyrophosphate were mutually competitive in PPi exchange reactions, but in DNA synthetic reactions the levels of resistance to PFA and PPi were not equal. The apparent affinities of the enzymes for Mg2+ did parallel their affinities for PFA. Km values of dNTPs were about 2-fold higher than the parent virus enzyme for all of the variant enzymes except one which was 4-fold higher. The processivity of polymerization was apparently unaffected by the enzyme changes related to PFA resistance although one variant enzyme had a lower value. Resistance among the variant enzymes to the triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 2',3'-dideoxyguanosine was directly related to the level of resistance to PFA. The data presented here indicated that (i) PFA resistance may result from several types of active site alterations, since the PFA-resistant enzymes were of three kinetically distinct types. Also, additional enzyme alterations, probably unrelated to PFA resistance, were detected in one enzyme. (ii) PFA and PPi possess some different binding determinants within the active center of herpes simplex virus type I DNA polymerase. (iii) PFA and the triphosphates of 9-(2-hydroxyethoxymethyl)guanine and 2',3'-dideoxyguanosine may have a common ultimate inhibitory mechanism.

Antiviral Agents↗

PubMed1982

In vitro biological activity of 9-beta-D-arabinofuranosyl-2-fluoroadenine and the biochemical actions of its triphosphate on DNA polymerases and ribonucleotide reductase from HeLa cells.

9-beta-D-Arabinofuranosyl-2-fluoroadenine (2-F-araA) inhibited the growth in vitro of HeLa cells by 50% at a concentration of 0.25 microM and depressed the replication of herpes simplex virus Types 1 and 2 by 99% at 25 microM. The analogue served as a substrate for cytoplasmic but not mitochondrial deoxycytidine (dCyd) kinase partially purified from human peripheral chronic lymphocytic leukemic blast cells. The Km values of dCyd and 2-F-araA for the cytoplasmic enzyme were 5 microM and 213 microM, respectively. However, at concentrations of 0.4 mM, the analogue was phosphorylated 2.9 times faster than dCyd. The 5'-triphosphate of 2-F-araA was examined for its biochemical effects on partially purified ribonucleotide reductase and highly purified DNA alpha- and beta-polymerases from HeLa cells. 2-F-araATP was a potent inhibitor of ribonucleotide reductase; the concentration required for 50% inhibition of ADP reduction (0.3 mM ADP; 5 mM GTP or dGTP) was 1 microM and for CDP reduction (0.15 mM CPD; 5 mM ATP) was 8.5 microM. Furthermore, 2-F-araATP was a competitive inhibition (Ki = 1.2 microM) with respect to dATP (Km = 3.8 microM) of DNA alpha-polymerase, whereas DNA beta-polymerase was relatively insensitive to the drug. The results suggest that the cytotoxic actions of 2-F-araA may be due, in part, to a "self-potentiating" inhibition of DNA synthesis. This is, by inhibiting the formation of competing dATP, 2-F-araATP may potentiate its inhibition of DNA synthesis.

Antiviral Agents↗

PubMed1981

Inhibition of purified human and herpes simplex virus-induced DNA polymerases by 9-(2-hydroxyethoxymethyl)guanine triphosphate. Effects on primer-template function.

The inhibition of highly purified herpes simplex virus (HSV)-induced and host cell DNA polymerases by the triphosphate form of 9-(2-hydroxyethoxymethyl)guanine (acyclovir; acycloguanosine) was examined. Acyclovir triphosphate (acyclo-GTP) competitively inhibited the incorporation of dGMP into DNA, catalyzed by HSV DNA polymerase; apparent Km and Ki values of dGTP and acyclo-GTP were 0.15 microM and 0.003 microM, respectively. HeLa DNA polymerase alpha was also competitively inhibited; Km and Ki values of dGTP and acyclo-GTP were 1.2 microM and 0.18 microM, respectively. In contrast, HeLa DNA polymerase beta was insensitive to the analogue. The "limited" DNA synthesis observed when dGTP was omitted from HSV or alpha DNA polymerase reactions was inhibited by acyclo-GTP in a concentration-dependent manner. Prior incubation of activated DNA, acyclo-GTP, and DNA polymerase (alpha or HSV resulted in a marked decrease in the utilization of the primer-template in subsequent DNA polymerase reactions. This decreased ability of preincubated primer-templates to support DNA synthesis was dependent on acyclo-GTP, enzyme concentration, and the time of prior incubation. Acyclo-GMP-terminated DNA was found to inhibit HSV DNA polymerase-catalyzed DNA synthesis. Kinetic experiments with variable concentrations of activated DNA and fixed concentrations of acyclo-GMP-terminated DNA revealed a noncompetitive inhibition of HSV-1 DNA polymerase. The apparent Km of 3'-hydroxyl termini was 1.1 X 10(-7) M, the Kii and Kis of acyclo-GMP termini in activated DNA were 8.8 X 10(-8) M and 2.1 X 10(-9) M, respectively. Finally, 14C-labeled acyclo-GMP residues incorporated into activated DNA by HSV-1 DNA polymerase could not be excised by the polymerase-associated 3',5'-exonuclease activity.

Acyclovir↗

PubMed1981

Herpes simplex virus type I DNA polymerase. Kinetic properties of the associated 3'-5' exonuclease activity and its role in araAMP incorporation.

An exonuclease activity copurified with herpes simplex virus type I (HSV-1) DNA polymerase through DNA-cellulose column chromatography and comigrated with DNA polymerase activity on nondenaturing gel electrophoresis at varied polyacrylamide concentrations. A gapped duplex DNA was the preferred substrate for this exonuclease activity since the hydrolytic activity on this type of DNA was much greater than the hydrolysis of either native or heat-denatured DNA. Using 3'-terminally labeled activated calf thymus DNA as substrate, the exonuclease activity was found to be activated by salt and spermidine in a manner identical with HSV-1 DNA polymerase. This activation was accompanied by increases in apparent Km and Vmax values of the activated DNA substrate. Phosphonoformic acid inhibited both DNA polymerase and exonuclease activities uncompetitively with respect to activated DNA and had a Ki of 2.4 microM at an ionic strength of 0.25 mu. Of the nucleoside 5'-monophosphates tested only the purine ribonucleotides inhibited the exonuclease activity. The inhibition was noncompetitive with respect to DNA, and GMP was about twice as potent as AMP or IMP. 9-beta-D-arabinosyladenine 5'-monophosphate (araAMP) could be incorporated into DNA by HSV-1 DNA polymerase; however, 9-beta-D-arabinosyladenine 5'-triphosphate would not replace dATP in supporting in vitro HSV-1 DNA synthesis. AraAMP incorporated into primer termini caused a significant decrease in the rate of subsequent primer elongation. These 3'-terminal araAMP residues could be removed by the HSV-1 DNA polymerase-associated exonuclease activity in a manner dependent on GMP concentration.

Arabinonucleotides↗