Biomedical subjects

B A Spalholz

Publications and source records attributed to B A Spalholz.

13 recordsLinked to original sources

PubMed1996

Sp1 is critical for basal and E2-transactivated transcription from the bovine papillomavirus type 1 P89 promoter.

The bovine papillomavirus type 1 (BPV-1) long control region (LCR) contains at least three consensus binding sites for the transcription factor Sp1 at nucleotides (nt) 7800, 7833 and 7854. A high basal-level P89 expression vector consisting of an origin-deleted LCR fused to the chloramphenicol acetyltransferase (CAT) gene was utilized to determine the role of these Sp1 sites in the regulation of transcription from the BPV-1 P89 promoter. The three Sp1 sites were capable of binding Sp1 in vitro. Mutation of these sites in the background of the origin-deleted LCR-CAT or a wild-type LCR-CAT construct resulted in decreased basal expression from P89. In addition, mutation of the Sp1 sites in the wild-type background caused a reduction in E2-transactivation potential. These data illustrate the importance of these Sp1 sites in regulating both basal and E2-transactivated P89 expression.

Animals↗

PubMed1993

Binding of bovine papillomavirus E1 to the origin is not sufficient for DNA replication.

Replication of the bovine papillomavirus (BPV-1) DNA requires both the viral E1 and E2 gene products. The minimal origin of replication, which resides in a 60-basepair fragment centered on the unique HpaI site of the BPV-1 genome, can be bound by the E1 protein and is flanked by E2 binding sites. The integrity of the region surrounding the unique HpaI restriction enzyme site is important for E1 binding and DNA replication, but the E2 binding sites are not required. The ability of E1 to complex with E2 and the requirement for both factors in DNA replication have led to the hypothesis that the E1/E2 complex may have a direct role in replication. Therefore we have studied mutations in the BPV-1 origin region for E1, E2, and E1/E2 complex binding and for their effect on transformation and replication in the context of the complete viral genome. Transformation and DNA replication were observed with some mutated viral genomes dramatically reduced for E1 binding. These mutated origins could bind significant amounts of E1 in the presence of E2, suggesting that binding of the E1/E2 complex could compensate for the loss of E1 binding in DNA replication. One mutation, with an eight-nucleotide insertion at the HpaI site, was still bound by E1 and the E1/E2 complex, yet did not replicate or transform efficiently. Thus although the binding of E1 or the E1/E2 complex to the origin appears to be necessary for replication, it is not sufficient.

Amino Acid Sequence↗

PubMed1993

Importance of the bovine papillomavirus P2443 promoter in the regulation of E2 and E5 expression.

The full-length bovine papillomavirus E2 gene product (E2TA), which has a direct role in DNA replication and functions as a transcriptional activator, can be expressed from an unspliced mRNA transcribed from the P2443 promoter or from spliced mRNAs transcribed from other upstream promoters. The regulation of E2 expression from these promoters is still in question. In the background of wild-type protein coding sequences, this study identified the P2443 promoter as the major source of E2TA as well as E5 expression in C127 cells.

Animals↗

PubMed1993

Repression of bovine papillomavirus type 1 transcription by the E1 replication protein.

Bovine papillomavirus type 1 (BPV-1) is the prototype virus for the study of papillomavirus gene regulation. The functions of the BPV-1 E2 proteins in transcriptional regulation have been well characterized. The BPV-1 E1 protein is required for viral DNA replication and can bind to the origin of replication alone or in a complex with the E2 transactivator protein. In this study, we demonstrated that the BPV-1 E1 protein is also involved in transcriptional regulation. The E1 protein significantly repressed E2-transactivated transcription from the major early promoter P89. This activity is consistent with the elevated level of P89 transcription observed in BPV-1 E1 open reading frame mutants. Transcriptional repression by E1 correlated with the ability of an E1-E2 protein complex to bind the replication origin but was not dependent on viral DNA replication. These studies identify a new mechanism involved in the regulation of papillomavirus transcription which has implications regarding expression of the viral transforming functions.

Animals↗

PubMed1991

Characterization of the cis elements involved in basal and E2-transactivated expression of the bovine papillomavirus P2443 promoter.

Transcriptional transactivation and repression by the viral E2 proteins are important regulatory mechanisms for the papillomaviruses. In the bovine papillomavirus type 1 (BPV-1), several viral promoters can be transactivated by E2 through E2-dependent enhancer elements located in the viral long control region (LCR), including promoters involved in E2 expression itself. This report demonstrates that the BPV-1 P2443 promoter is transactivated by E2-responsive elements in the LCR and that this promoter is responsible for a major part of the expression of the E2 and E5 gene products. Characterization of the cis elements involved in P2443 regulation indicated that the single E2-binding site directly upstream of P2443 is not required for either the E2 transactivation or for any E2 repression of the basal or transactivated activity of this promoter. Therefore, cooperative interactions between E2 bound at the LCR and E2 bound near P2443 do not have any role in the regulation of this promoter. Further definition of the cis regulatory elements of this promoter indicated that a binding site for the transcriptional factor Sp1 exists directly upstream of the P2443 TATA box and is critical for the basal level of transcription from this promoter. Disruption of this Sp1 site eliminated P2443 promoter activity in transient expression assays for E2 and E5 and resulted in a loss of transforming activity when introduced into the full viral genome.

Animals↗

PubMed1988

The bovine papillomavirus P2443 promoter is E2 trans-responsive: evidence for E2 autoregulation.

The bovine papillomavirus type 1 (BPV-1) P2443 promoter is located just upstream of the E2, E3, E4 and E5 open reading frames (ORFs) and is active in both transformed rodent cells and in productively infected warts. Analysis of viral RNA structures suggests that transcripts from this promoter encode the E2 transactivator as well as the E5 oncoprotein. To study expression of P2443, the chloramphenicol acetyltransferase (CAT) reporter gene was placed downstream of this promoter, deleting the E2 and E5 ORFs, in a plasmid which contained all BPV-1 upstream sequences including the long control region (LCR). By itself, this plasmid demonstrated a low level of activity in transient assays and could be transactivated to a high level by the full-length E2 product. Transactivation of P2443 expression by E2 required the LCR in cis in an orientation- and position-independent manner, suggesting that this transactivation was mediated through the E2 responsive enhancer elements (E2RE) located within the LCR. Primer extension analysis of the 5' ends of the viral transcripts from pooled cells expressing these P2443/CAT plasmids confirmed that E2 transactivation results in an increase in the steady-state levels of RNA initiated from the P2443 promoter. Furthermore, E2 transactivation of the P2443 promoter could be inhibited by the trans-repressor encoded by the 3' portion of the E2 ORF. Thus, expression of the E2 transactivator and the E5 oncoprotein is directly regulated by transcriptional factors encoded by the E2 ORF.

Animals↗

PubMed1988

Evidence for cooperativity between E2 binding sites in E2 trans-regulation of bovine papillomavirus type 1.

The long control region of bovine papillomavirus type 1 (BPV-1) can function in an orientation- and position-independent manner as an E2-dependent enhancer. Dissection of the long control region has revealed two E2-responsive elements, E2RE1 and E2RE2, which map, respectively, between nucleotides 7611 and 7806 and between nucleotides 7200 and 7386 of the BPV-1 genome. In this study, we have carried out a detailed analysis of E2RE1, which has previously been shown to be involved in the regulation of the BPV-1 promoters P89 and P7940. One characteristic of E2RE1 is the presence of a pair of ACCN6GGT motifs (E2 binding sites) at each end of the element. To determine the contribution of these sites, as well as other sequences within E2RE1, to enhancer function, specific mutations and deletions were generated by oligonucleotide reconstruction. The functional analysis of these mutations confirmed that a pair of E2 binding sites was essential for E2-dependent enhancer activity but also indicated that cooperativity between the motifs at each end of E2RE1 creates a highly responsive element. Isolated ACCN6GGT motif pairs could also act as E2-dependent enhancers but at a significantly reduced level in comparison to the intact element. The sequences between the E2 binding sites in E2RE1 were not required for enhancer function and could actually block the enhancer activity of an isolated pair of E2 binding sites when positioned between the binding sites and the enhancer-deleted simian virus 40 early promoter.

Animals↗

PubMed1987

A transcriptional repressor encoded by BPV-1 shares a common carboxy-terminal domain with the E2 transactivator.

A negative-acting transcriptional regulatory factor encoded by bovine papillomavirus type 1 (BPV-1) was identified. This factor inhibits BPV-1-mediated transformation of mouse C127 cells; inhibition is BPV-1-specific and occurs only when the BPV-1 transforming genes are regulated by authentic transcriptional control elements. Plasmids expressing the inhibition function also repress E2 transactivation of the BPV-1 E2-dependent enhancer, and this repression is mediated by the same cis-acting element required for E2 transactivation. Inhibition of transformation may result from down-regulation of E2-dependent viral gene expression. Analysis of cDNA expressing the inhibition/repression activities mapped the function to the 3' domain of the E2 open reading frame. The E2 open reading frame thus encodes both positive and negative transcriptional regulatory factors, and these factors share a carboxy-terminal domain.

Bovine papillomavirus 1↗

PubMed1987

Bovine papillomavirus transcriptional regulation: localization of the E2-responsive elements of the long control region.

The long control region (LCR) of the bovine papillomavirus type 1 genome can function as a conditional transcriptional enhancer which can be specifically trans-activated by the viral E2 gene product. To precisely map the target(s) of this trans-activation, BAL 31 exonuclease was used to generate two overlapping series of deleted DNA segments through the LCR. These fragments were assayed for their ability to activate transcription from the enhancer-deleted simian virus 40 early promoter of pA10CAT in the presence or absence of the viral E2 gene product. Two different E2-responsive elements were localized within the LCR. The major target for E2 trans-activation (E2-responsive element 1) was mapped to a 196-base-pair fragment between nucleotides 7611 and 7806, just upstream from promoters P7940 and P89. Further deletions which destroyed or impaired enhancer function revealed that the ACCN6GGT sequence motifs at each end of E2-responsive element 1 are critical components of this element. Primer extension analysis of RNA extracted from acute transfections with plasmids containing the bovine papillomavirus type 1 LCR driving the CAT gene revealed that each of the P7940 and P89 promoters is responsive to E2 trans-activation.

Base Sequence↗

PubMed1986

Papillomavirus transforming functions.

The bovine papillomavirus type 1 (BPV-1) has served as a model for unravelling the molecular genetics of the papillomaviruses. BPV-1 transformation of rodent cells in tissue culture has provided a means to study the viral functions involved in latent infection of cells and in the induction of cellular proliferation functions. BPV-1 has been shown to encode two independent transforming genes, each of which can induce cellular transformation in susceptible rodent cells. These two genes apparently act synergistically in transforming mouse C127 cells. Deletion mutagenesis studies have shown that the expression of one of these genes (E6) is required for efficient tumorigenesis and anchorage independence. BPV-1 also encodes functions which may act indirectly to affect transformation. BPV-1 contains transcriptional enhancers which can act in a position-independent and orientation-independent manner to increase the transcriptional activity of a heterologous gene. One of these elements, which is located in a non-coding region of the genome, can be trans-activated by a specific viral gene product encoded by the E2 open reading frame. Mutations which eliminate this trans-activation function also have a dramatic effect on transformation and on stable plasmid maintenance.

Animals↗

PubMed1985

Transactivation of a bovine papilloma virus transcriptional regulatory element by the E2 gene product.

We have mapped a transcriptional regulatory sequence within the 1.0 kb noncoding region of the bovine papilloma virus (BPV-1) genome, using an enhancer dependent expression vector for chloramphenicol acetyltransferase. This transcriptional regulatory element works independently of position and orientation, and its function is significantly augmented in BPV-1 transformed C127 cells and in monkey CV-1 cells acutely transfected with plasmids expressing BPV-1 early gene products. Using defined deletion mutants of the BPV-1 DNA and full-length viral cDNAs expressed from an SV40 early promoter, we demonstrate that the expression of this trans-activating factor maps to the 3' open reading frames of the viral transforming region. A premature termination codon engineered into the E2 ORF eliminates expression of this diffusible transactivation function establishing the E2 gene product as the diffusible trans-activating factor.

Acetyltransferases↗

PubMed1983

Interaction of minute virus of mice with differentiated cells: strain-dependent target cell specificity is mediated by intracellular factors.

The prototype strain of minute virus of mice and the immunosuppressive strain are unable to grow lytically in each other's murine host cell type. To characterize these strain-dependent virus-host cell interactions further, we have compared the early events of both productive and restrictive infections. Each virus binds to specific receptors on the surface of both productive and restrictive cell types. Competition experiments show that both viruses recognize the same receptor on each cell type. Penetration and uncoating are presumed to be similar in both productive and restrictive infections, since incoming viral genomes are converted to parental replicative form DNA independent of the final outcome of the virus-host cell interaction. In contrast to the majority of other systems studied to date, these differences in minute virus of mice target cell specificity are not mediated at the cell surface, but by the interaction of a strain-specific viral determinant with intracellular host factors that are expressed in particular cell types as a function of differentiation. These cellular factors catalyze a step in viral replication which occurs after the initiation of viral DNA synthesis, but before the detectable expression of the viral capsid polypeptide genes.

Animals↗

PubMedDate not supplied

Dissociation of transforming and trans-activation functions for bovine papillomavirus type 1.

It has been shown that genetic information encoded by the 3' open reading frames (ORFs), E2, E3, E4 and E5, of bovine papillomavirus type 1 (BPV-1), is sufficient to induce cellular transformation of certain mouse cells. The product of the E2 ORF has further been shown to be responsible for the trans-activation of a transcriptional regulatory element located in the noncoding region (NCR) of the BPV-1 genome. To examine whether or not the E2 trans-activation function is encoded by the same gene that encodes the 3' ORF viral transformation function, we have now analysed the expression of the trans-activation function in series of mouse C127 cells transformed by BPV-1 deletion mutants. In addition, using mutated complementary DNA clones generated by the insertion of a premature translational termination linker into different sites of a BPV-1 cDNA clone containing the 3' ORFs intact, we demonstrate that transformation and transcriptional trans-activation functions can be dissociated and that they map respectively to the E5 and E2 ORFs.

Acetyltransferases↗