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Biomedical subjects

B Fleckenstein

Publications and source records attributed to B Fleckenstein.

At least 127 records · Page 7Linked to original sources
PubMed1991

Discordant expression of the immediate-early 1 and 2 gene regions of human cytomegalovirus at early times after infection involves posttranscriptional processing events.

Expression of the immediate-early 1 and 2 (IE-1 and IE-2) gene region of human cytomegalovirus (HCMV) was studied during initial phases of the replicative cycle. Accumulation of RNA from IE-1 and -2 was found to be differential. Transcripts from IE-2 reached peak levels very early in infection between 3 and 5 h, whereas IE-1 RNA peak levels were detected later, between 6 and 8 h. A strong decrease in steady-state levels of a 2.2-kb IE-2 RNA was observed at a time when IE-1 transcripts showed a further increase in abundance. Northern (RNA) blot experiments revealed that expression of both the IE-1 RNA and the 2.2-kb IE-2 transcript is controlled by the IE-1 enhancer-promoter. Nuclear run-on experiments demonstrated equal rates of primary transcription for IE-1 and -2 at a time when different steady-state levels of RNA were observed. Concomitant with down-regulation of IE-2 RNAs, a decrease in the size of the IE-1 transcript was detected. At 2 to 5 h after infection the IE-1 transcript migrated at 1.95 kb, whereas later in the replicative cycle the RNA was found at 1.8 kb. RNase H blot analysis revealed that this size discrepancy is due to a shorter poly(A) tail of the IE-1 RNA at early times after infection. These experiments suggest that in addition to transcriptional regulation, specific posttranscriptional mechanisms are involved in controlling expression from the IE-1 and -2 gene region of HCMV.

Antigens, Viral↗
PubMed1991

In vitro binding of human T-cell leukemia virus rex proteins to the rex-response element of viral transcripts.

Human T-cell leukemia virus (HTLV-I, HTLV-II) rex protein function is required for the cytoplasmic expression of incompletely spliced viral transcripts encoding structural proteins. The effect is mediated by a cis-acting rex-response element (RRX) which is located near the 3' end of all viral mRNAs. We show that rex polypeptides of HTLV-I and HTLV-II expressed in Escherichia coli are capable of specifically binding RRX-containing transcripts of both viruses in cell-free assays. Binding analyses with deletion variants of rex proteins revealed a domain with RNA-binding activity in the first 77 N-terminal amino acids. Removal of a basic peptide of 19 amino acids from the N terminus abrogated RNA binding, whereas a beta-galactosidase fusion protein containing this peptide bound to the RRX. These results suggest that direct binding of rex protein to the RRX is important for rex-mediated regulation of viral gene expression and that a short stretch of positively charged amino acids contributes to the specific binding of rex to its target RNA.

Carrier Proteins↗
PubMed1990

A rev/beta-galactosidase fusion protein binds in vitro transcripts spanning the rev-responsive element of human immunodeficiency virus type 1 (HIV-1).

The rev protein of human immunodeficiency virus type 1 (HIV-1), a phosphoprotein of 20 kDa apparent molecular mass, is essential to target the mRNA for virion polypeptides into the cytoplasm. This effect is mediated by a specific RNA stretch (rev-responsive element = RRE) localized within a 3'-terminal segment of the mRNA encoding virion proteins. We present evidence that rev expressed as a beta-galactosidase fusion protein in E. coli forms a complex with in vitro transcripts containing the RRE; it can be precipitated by monoclonal antibodies with rev or beta-galactosidase specificity. In addition, specific binding of rev protein to RNA could be demonstrated by Northwestern blotting.

Animals↗
PubMed1990

The divergence between two oncogenic Herpesvirus saimiri strains in a genomic region related to the transforming phenotype.

Herpesvirus saimiri strains can be divided into at least three subgroups (A, B, C) based on sequence divergence at the left end of viral unique sequence DNA. Strains of subgroups A and C are highly oncogenic and readily transform simian T-lymphocytes in vitro to interleukin-2 independent growth, while subgroup B strains do not. A left terminal reading frame of a H. saimiri subgroup A strain was shown previously to correlate with the oncogenic phenotype and in vitro transforming potential; the deduced polypeptide was termed STP-A. Furthermore, this same region contains an open reading frame (ORF) for dihydrofolate reductase (DHFR) and genes for five virus-specific U RNAs (HSURs). We now show by sequence analysis of the corresponding region in a subgroup C strain that DHFR and HSUR genes are present in both virus subgroups; however, no sequence homologous to the STP-A reading frame was found in this subgroup C virus. At a position and orientation similar to STP-A, two ORFs were found for peptides sharing a putative transmembrane domain. One of them encodes a peptide with collagen-like repetitions. In addition to the lack of similarity to STP-A, these two reading frames also did not show any similarity to known oncogenes. The organization of sequences at the left junction of unique L- and repetitive H-DNA of H. saimiri suggests frequent recombinational events, possibly accelerating the uptake of foreign genes by the virus.

Amino Acid Sequence↗
PubMed1990

Structural organization of the conserved gene block of Herpesvirus saimiri coding for DNA polymerase, glycoprotein B, and major DNA binding protein.

Lymphotropic herpesviruses such as Epstein-Barr virus and Herpesvirus saimiri are commonly grouped as gamma-herpesviruses, although overall genome organization and numerous biological properties are quite different in the viruses. To define the relationship more precisely, we sequenced the Kpnl fragments F (6.5 kb) and C (9.8 kb) of the H.saimiri strain No. 11 genome; these DNA fragments were found to contain the genes coding for equivalents of the major DNA binding protein, a putative glycoprotein transport polypeptide, the glycoprotein B, and the DNA polymerase of herpes simplex virus. This DNA segment represents the longest block of contiguous genes with pronounced sequence homologies between herpesviruses of known DNA primary structure. Comparisons confirmed that the two gamma-herpesviruses are related; the group is, however, even more diverse than the alpha-herpesviruses represented by their prototypes, herpes simplex virus and varicella-zoster virus. H. saimiri DNA is strongly depleted in the dinucleotide CpG, possibly the consequence of de novo methylation of persisting viral DNA in lymphoid cells.

Amino Acid Sequence↗
PubMed1990

Monoclonal antibodies directed against the rev protein of human immunodeficiency virus type 1.

The rev (art/trs) protein of human immunodeficiency virus type 1 (HIV-1), a phosphoprotein of 20 K apparent molecular weight, is essential to target the mRNA for virion polypeptides into the cytoplasm. The rev protein was expressed in Escherichia coli as a beta-galactosidase fusion protein with a cleavage site for proteinase factor Xa. The rev-specific fragment was isolated to immunize mice. Five stable hybridoma cell lines were obtained producing monoclonal antibodies that reacted with rev protein in Western blot and ELISA. Using the monoclonal antibodies in indirect immunofluorescence, the rev protein could be localized in the nucleus, mostly in the nucleoli, of Hela cells that were transfected with a eukaryotic rev expression plasmid.

Animals↗
PubMed1990

Cell type-specific induction of the major immediate early enhancer of human cytomegalovirus by cyclic AMP.

The major immediate early enhancer of human cytomegalovirus (HCMV) is known to exert a strong constitutive transcription stimulation in a broad spectrum of cells. This basal activity can be augmented considerably by elevated levels of intracellular cAMP in a cell type-specific manner. Cyclic AMP induction was observed in several lymphoid cell lines and in HeLa cells. One of the functionally important enhancer sequence modules, the 19 bp repeat element, mediates this effect as a cAMP-responsive element (CRE). It acts more efficiently than the corresponding sequence from the human chorionic gonadotropin gene. It is suggested that protein kinase C is involved in the pathway which leads to the activation of CRE-containing genes in lymphoid cells. Gel retardation assays indicated that similar, but not identical complexes are formed between nuclear protein extracts and the CREs of HCMV and the gonadotropin gene.

Alkaloids↗
PubMed1990

trans activation of the thymidylate synthase promoter of herpesvirus saimiri.

Herpesvirus saimiri has been shown to possess a thymidylate synthase (TS) gene that is unusual in its transcriptional regulation. Although TS is believed to be required for viral DNA synthesis, the TS-specific 2.5-kb mRNA was found most abundantly during the late phases of asynchronous virus replication in permissive cultures. To study the kinetics of gene activation, the TS promoter and regulatory sequences were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene. No CAT expression or transcripts were found after transfection of fusion genes into permissive owl monkey kidney (OMK) cells. However, the promoter was strongly activated when CAT plasmids were cotransfected with intact herpesvirus saimiri virion DNA or were transferred to OMK cells that were lytically infected with herpesvirus saimiri or a related herpesvirus, herpesvirus ateles. CAT was expressed at reduced levels in cultures when viral DNA replication was inhibited by phosphonoacetic acid; this indicates that the gene is activated during the delayed-early phase. However, the highest amounts of mRNA were present in the late period of replication. Deletion analyses localized essential response elements for trans activation in the promoter upstream region between nucleotides -311 and -56; they consisted of related tandem repeats and perfect palindromes. A sequence with two overlapping palindromes of 16 and 18 bp was found to be a major target for activation of the herpesvirus saimiri TS promoter. These palindromes did not have any significant homologies with known sequences of herpesviruses or cellular DNA; the 18-bp palindrome had, however, a certain structural similarity with a conserved sequence of the E2-responsive cis sequence that is required for transcription regulation of early papillomavirus genes.

Animals↗
PubMed1989

CMER, an RNA encoded by human cytomegalovirus is most likely transcribed by RNA polymerase III.

Through computer analysis of a human cytomegalovirus (HCMV) genomic region, previously identified to be homologous to human genomic DNA, an element showing significant similarity to the 3'-internal control region (3'-ICR or B-block) of a eukaryotic RNA polymerase III promoter could be detected. This region-located on the EcoRI b fragment within the UL segment of the viral genome of HCMV strain AD 169-cannot be transcribed in vitro in an RNA polymerase III specific transcription system. However, this part of the viral genome is able to compete for components of the RNA polymerase III transcription complex as shown in template exclusion experiments and by gel retardation assays. Two different synthetic oligonucleotides complementary to the 3'-ICR and to nucleotides located immediately downstream of this promoter element can anneal specifically to a HCMV-encoded ribonucleic acid (termed CMER) synthesized in human foreskin fibroblasts (HFF) late in virus replication. As a consequence of identifying the transcription initiation point by primer extension analyses the position of the 5'-internal control region (5'-ICR or A-block) of the CMER gene could be uncovered. Both identified control regions (the A-block as well as the B-block) of the transcription unit exhibit significant similarities to corresponding regulatory elements of other class III genes, including virus encoded class III genes. Initiation of in vivo transcription occurs 15 nucleotides upstream of the 5'-border of the 5'-ICR and the two non-contiguous gene internal promoter elements are separated by 79 nucleotides.

Base Sequence↗
PubMed1989

Transformation to continuous growth of primary human T lymphocytes by human T-cell leukemia virus type I X-region genes transduced by a Herpesvirus saimiri vector.

The role of the X region of the genome of the human T-cell leukemia virus type I (HTLV-I) in the immortalization of lymphocytes has been difficult to distinguish from its role in viral replication as this region encodes at least two genes, tax and rex, required for replication and the expression of viral proteins. To determine whether the X region does encode immortalizing functions, a fragment of the HTLV-I provirus capable of expressing known X-region proteins was inserted into the genome of a transformation-defective, replication-competent Herpesvirus saimiri. Infection of fresh mitogen-activated human cord blood and thymocytes yielded immortal T-cell lines that had the same phenotype (CD4+, CD5+, HLA class II+, interleukin 2 receptor alpha-chain +) as lymphocytes transformed by cocultivation with HTLV-I. These experiments demonstrate that the X region encodes the functions of HTLV-I that immortalize a distinct subpopulation of human T cells. The experiments also demonstrate the utility of the H. saimiri vector for the transduction of heterologous genes into human T cells.

Cell Transformation, Neoplastic↗
PubMed1989

The role of a repetitive palindromic sequence element in the human cytomegalovirus major immediate early enhancer.

The major enhancer, extending from nucleotides -530 to -120 upstream of the transcription initiation site of immediate early (IE) genes 1 and 2 in human cytomegalovirus (HCMV), contains four groups of repeated sequence motifs that consist of 17, 18, 19 or 21 bp, respectively. One of these elements, the 19 bp repeat, is a symmetrical palindrome that is also part of IE regulatory sequences of other cytomegalovirus-type herpesviruses, but not of unrelated members of the herpesvirus group. Synthetic oligonucleotides representing the 19 bp repeat unit strongly reduced the activity of the IE1/2 enhancer/promoter in cotransfection assays after transient expression. The HCMV enhancer can substitute for the 72 bp repeats of simian virus 40 (SV40). Replication-competent deletion mutants of SV40/HCMV enhancer recombinants were constructed that contained a single palindromic 19 bp repeat with a central cleavage site for AhaII. If deletions were introduced into the single remaining 19 bp repeat most of the mutant viruses were still replication-competent in CV-1 monkey kidney cells. Insertion of two nucleotides into the single AhaII site did not significantly alter transient SV40 T antigen expression. Deletion of four nucleotides or more from the single 19 bp palindrome reduced the stimulation of T antigen synthesis by the HCMV enhancer/SV40 promoter unit down to about 50%. More extended deletions (28 to 80 bp) did not further reduce T antigen expression. All mutants without an intact 19 bp repeat contained the 18 bp and/or the 21 bp sequence motif. DNase I footprinting and gel retardation assays indicated sequence-specific protein binding by the 19 bp palindrome. Altered palindromes, correlating with reduced enhancer activity, lost most of their protein-binding properties. Thus, the 19 bp repeat element is one of several protein-binding sites that contribute to enhancer strength. However, the 19 bp sequence motif can be deleted entirely to leave reduced activity. The HCMV IE1/2 upstream sequence appears to be the perfect model of an enhancer as a complex of multiple binding sites for trans-activating proteins in a modular fashion.

Animals↗
PubMed1989

Selectable recombinant herpesvirus saimiri is capable of persisting in a human T-cell line.

Strategies were developed to insert the neo resistance marker into the junction between L DNA and the right terminal repetitive H DNA of herpesvirus saimiri. Recombinant viruses were selectable in permissive epithelioid cultures. The human T-cell line Jurkat could be infected persistently with the Neor virus; the cells contained episomal viral DNA in high copy number. This selectable vector should be generally applicable for gene expression in human T cells.

Cell Line↗
PubMed1988

Delayed seroconversion to HIV in a hemophiliac.

In a 14-year-old boy with severe hemophilia B, HIV seroconversion was observed about 2 years after substitution therapy had been changed to heat-treated PPSB concentrates. This shows that HIV-antibodies may appear in hemophiliacs after time periods longer than generally assumed.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Human cytomegalovirus DNA in the temporal cortex of a schizophrenic patient.

Using highly sensitive nucleic acids hybridization techniques, which allow the detection of 0.1-0.5 single copy gene equivalents per cell, DNA from the temporal cortex of seven definite schizophrenics, five persons with schizophrenia-like psychoses, three patients with Huntington's chorea and nine mentally normal individuals were probed with human cytomegalovirus (HCMV) DNA. A clear hybridization signal was obtained with DNA from the temporal lobe of a young schizophrenic patient, whereas DNA from the temporal cortex of controls did not hybridize to the HCMV probe. This finding is in agreement with the cytomegalovirus hypothesis of schizophrenia and hints at the possibility that viral infection of the temporal cortex may in some sporadic cases be a contributing factor to the development of schizophrenic psychoses. There is no indication, however, that infection of the central nervous system with HCMV is an aetiological factor in the great majority of schizophrenic disorders. Clearly further studies, preferably in situ hybridizations of whole brains, are needed to prove or disprove the cytomegalovirus hypothesis of schizophrenia.

Adult↗
PubMed1988

Detection of human cytomegalovirus DNA and viral antigens in tissues of different manifestations of CMV infection.

Biopsy and autopsy specimens from 22 patients with cytomegalovirus (CMV) infections were investigated by means of in situ hybridization (ISH) to detect viral DNA and by immunohistochemistry (IHC) to visualize viral proteins. Both methods proved to be valuable tools for histopathology. ISH sometimes recognized cells that did not show typical CMV inclusions. An antiserum against the full spectrum of viral proteins (non-infectious enveloped particles) detected most cytomegalic cells in disseminated and organ-limited infections. An antiserum against a recombinant polypeptide (XP1) was particularly useful in connatal CMV infections and organ-limited infections. We have demonstrated that IHC and ISH studies in parallel are the best approach to the detection of CMV infections in pathological specimens.

Adolescent↗
PubMed1988

Reactivation of the methylation-inhibited late E2A promoter of adenovirus type 2 by a strong enhancer of human cytomegalovirus.

Promoter inactivation by sequence-specific methylation was demonstrated by using a construct which contained the late E2A promoter of adenovirus type 2 (Ad2) DNA and the prokaryotic gene for chloramphenicol acetyltransferase (CAT) as indicator. After the in vitro methylation of 5'-CCGG-3' sequences at positions -215, +6, and +24 relative to the cap site of the promoter, the construct was inactive upon transfection into mammalian cells. The same pAd2E2AL-CAT construct was active in the unmethylated form. Promoter inactivation could be overcome when the strong immediate early enhancer of human cytomegalovirus DNA, which lacked 5'-CCGG-3' sites, was inserted into the construct either in a position immediately antecedent to the promoter or in a location several thousand nucleotides remote from it. Reactivation of the 5'-CCGG-3' methylated pAd2E2AL-CAT construct entailed initiation of transcription at the authentic cap site of the late E2A promoter and maintenance of methylation at least during the duration of the transient expression experiment. Reactivation of the methylated late E2A promoter had also been demonstrated by the trans-activating 289 amino acid protein which was encoded in the E1A region of adenoviruses (B. Weisshaar et al., 1988, J. Mol. Biol. 202, 255-270). Thus there are several ways in which a methylated and silenced promoter can be reactivated in mammalian cells.

Adenovirus Early Proteins↗