Characterization of a cell type-specific enhancer found in the human papilloma virus type 18 genome.

We have previously isolated long-range acting enhancer elements from the HeLa genome by functional selection. In this report, the structural and functional characteristics of one (GA1) of the enhancers are reported. By cloning various restriction fragments and by deletion mutagenesis, the activity of GA1 was located in a 230-bp region. The nucleotide sequence of GA1 and genomic Southern blot analysis indicated that GA1 is derived from human papilloma virus (HPV) 18 DNA that had been integrated into the HeLa genome. The enhancer is located in the non-coding region of the HPV 18 genome. The HPV 18 enhancer consists of two functional domains, both of which have full enhancer activity in HeLa cells. The enhancer does not contain enhancer core sequences but contains several blocks of potential Z-DNA sequence. Like SV40 and polyoma virus enhancers, the activity of the HPV 18 enhancer was repressed by adenovirus E1a products. The HPV 18 enhancer shows a narrow cell type specificity: it is active in some cervical carcinoma cell lines, but inactive in all non-cervical cells except for one neuroblastoma cell line. These results suggest that the HPV 18 enhancer plays an important role in regulation of the viral genes.     

Interspersed Homologous DNA of Autographa californica Nuclear Polyhedrosis Virus Enhances Delayed-Early Gene Expression

The five regions of homologous DNA which are interspersed in the genome of the baculovirus Autographa californica nuclear polyhedrosis virus increased the expression of a delayed-early gene of this virus. Although this activity was first observed as a 10-fold trans effect, the homologous region 5 (hr5) enhanced the expression of linked genes 1,000-fold. The hr5 enhancer also exhibited the other characteristics associated with viral enhancer elements, including orientation independence and the abilities to function at a distance from the linked promoter, to regulate heterologous promoters, and to increase the number of RNA polymerase molecules transcribing the linked genes. The expression of the immediate-early regulatory gene was not enhanced by cis-linked hr5, although the enhancer function may require the immediate-early regulatory gene product. The hr5 enhancer was relatively insensitive to competition by an excess of enhancer molecules. The nucleotide sequence of hr5 revealed two different conserved repeats separated by nonhomologous DNA. Deletion analysis of the hr5 enhancer indicated that a 30-base-pair inverted repeat was essential for enhancer function.     

Structure and role of the herpes simplex virus DNA termini in inversion, circularization and generation of virion DNA

The herpes simplex virus genome consists of two components, L and S, which invert relative to each other during viral replication. The a sequence is present at the genomic termini in direct orientation and at the L-S junction in inverted orientation. Previously, we showed that insertion of a fragment spanning the L-S junction into the viral genome causes additional inversions. In this study, we determine the nucleotide sequence of the genomic termini and show that insertion of either the free S terminus or the L terminus causes inversions in the viral genome. We conclude that the a sequence is the inversion-specific sequence, that linear unit-length molecules packaged in virions are generated by cleavage between adjacent copies of the a sequence, that cleavage produces 3' single-base extensions on the genomic termini and that the signal for cleavage is contained within the a sequence.     

Amplification of a short nucleotide sequence in the repeat units of defective herpes simplex virus type 1 Angelotti DNA

It has been shown earlier that the reiterated regions TRS and IRS bracketing the Us segment of herpes simplex virus type 1 Angelotti DNA are heterogeneous in size by stepwise insertion of one to six copies of a 550-base-pair nucleotide sequence. Considerably higher amplification of this sequence was observed in defective viral DNA: up to 14 copies were detected to be inserted in the repeat units of a major class of defective herpes simplex virus type 1 Angelotti DNA, dDNA1, which originated from noncontiguous sites located in UL and the inverted repeats of the S component of the parental genome. Physical maps were established for the cleavage sites of KpnI, PstI, XhoI, and BamHI restriction endonucleases on the repeats of dDNA1. The map position of the insertion sequence was determined. It was demonstrated that the amplified inserts were not distributed at random among or within the repeats. A given total population of dDNA1 molecules consisted of different homopolymers, each of which contained a constant number of inserts in all of its repeats. Assuming that a rolling-circle mechanism is involved in the generation of full-length defective herpes simplex virus type 1 Angelotti DNA from single repeat units, these data suggest that the 550-base-pair sequence is amplified in the repeats before the replication process.     

Simian virus 40 T antigen activates the late promoter by modulating the activity of negative regulatory elements.

Late promoter activity measured before viral DNA replication results from a complex involvement of negative and positive cis-acting elements located both in the enhancer and in the 21-bp repeats. GC motifs located within the 21-bp repeats act in cooperation with sequences overlapping the early TATA box to down-regulate the late promoter activity. Analysis of insertion mutants indicates that the late promoter might be negatively regulated at least partially by the early promoter machinery. The GTI motif located within the enhancer as well as the GC motifs lose the ability to down-regulate the late promoter in the presence of T antigen. Results obtained with tsA58 protein indicate that two different domains of T antigen are involved in the negative autoregulation of the early promoter activity and in the release of the down-regulation of the late promoter by the GC motifs.     

Integration of the adeno-associated virus genome into cellular DNA in latently infected human Detroit 6 cells.

A clone of human cells (Detroit 6) latently infected by adeno-associated virus (AAV) has been characterized with regard to the status of the viral DNA. In both early (9 to 10) and late (118) passages of the clone, AAV-DNA was recombined with host DNA, at least in some cases as a head-to-tail tandem repeat, via the terminal sequences of the viral genome. However, it was not possible to distinguish between integration into chromosomal DNA and very large plasmids (< 20 x 10(6) molecular weight) which contain both viral and cellular DNA sequences. Although evidence for some modifications of the viral sequence was obtained, most of the integrated sequences appeared to be intact. In some cases sequences of undetermined origin separated adjacent copies of the viral genome. Free copies of the AAV genome were detectable in late passage cells, but not in early passage cells. The orientation of nucleotide sequences present in the free AAV DNA from late passage cells was indistinguishable from that of virion DNA. With the notable exception, the organization of the integrated AAV sequences as determined by restriction enzyme digestion remained constant with continued passage. Digestion with SmaI, which cleaves within the palindromic region of the terminal repetition in AAV DNA, produced reproducibly different patterns when early and late passage DNAs were compared. Several models for rescue of free copies of the genome from the integrated DNA are possible, all of which involve the terminal repetition.     

A calcium ionophore-inducible cellular promoter is highly active and has enhancerlike properties.

We examined the regulatory/promoter sequence of a calcium ionophore-inducible gene isolated from the rat genome. Whereas the promoter of this ubiquitously expressed gene is active under noninduced conditions, after induction by calcium ionophore A23187 this promoter is 10- to 25-fold more active than the simian virus 40 early promoter, as measured by chloramphenicol acetyltransferase activities. Within this regulatory/promoter region, we have identified a DNA fragment with enhancer-like properties immediately 5' to the TATA sequence. This 291-nucleotide fragment acts in cis to enhance expression of the neomycin phosphotransferase (neo) gene driven by the herpes simplex virus thymidine kinase promoter in an orientation-independent manner. In addition, this fragment can confer A23187 inducibility to the neo gene and effectively compete for positive regulatory factors involved in A23187 induction. Sequence analysis of this promoter reveals homology with viral core enhancer sequences, and the apparent organization of direct repeat domains is similar to those observed in viral enhancers.     

Expression of an enhancer-binding protein in insect cells transfected with the Autographa californica nuclear polyhedrosis virus IE1 gene.

The baculovirus Autographa californica nuclear polyhedrosis virus contains an element known as homologous region 5 (hr5) which is an enhancer of delayed-early viral gene expression. To begin to identify proteins that interact with hr5, DNA-protein interactions were analyzed by using extracts from Spodoptera frugiperda cells and a fragment of DNA containing the left half of the hr5 enhancer. This 252-bp DNA fragment contains two copies of a 30-bp direct repeat (DR30) and two copies of a 24-bp imperfect palindrome contained within a 60-bp direct repeat (DR60). Extracts prepared from normal S. frugiperda cells and cells transfected with pUC8 lacked enhancer-binding proteins. However, when gel shift assays were performed with extracts from cells transfected with a plasmid containing the viral trans-activator IE1 gene, two DNA-protein complexes were formed. Both DNA-protein complexes were specifically inhibited by competition with a 60-bp oligonucleotide corresponding to DR60 but not by competition with a different oligonucleotide corresponding to DR30. Formation of the two complexes did not appear to involve cooperative interactions between binding proteins. When DR60 was used as a probe, a single complex was formed. To measure the enhancer activity of DR60, a reporter plasmid was constructed that contained DR60 cloned upstream of the reporter chloramphenicol acetyltransferase gene under the control of the delayed-early 39K promoter. Transient expression analysis indicated that the oligonucleotide increased expression of this gene 300-fold over the level obtained in the absence of any enhancer sequences.     

Free and integrated forms of hepatitis B virus DNA in human hepatocellular carcinoma cells (PLC/342) propagated in nude mice.

Primary hepatocellular carcinoma cells (PLC/342) propagated in nude mice produce hepatitis B surface antigen of subtype adr, as well as core particles containing viral DNA and DNA polymerase. Free and integrated forms of hepatitis B virus (HBV) DNA in the tumor were isolated by molecular cloning, and their nucleotide sequences were determined. Both of the two representative clones of free HBV DNA had the same genomic length (3,158 base pairs) and had two stop codons as well as two deletions in the envelope gene. None of the seven distinct clones of integrated HBV DNA possessed the entire viral genome. The integrated clone sequences had deletions and rearrangements, and only two clones possessed the envelope gene including the promoter and enhancer sequences. The C gene, which codes for core protein, was preserved in the two free clones and one of the integrated clones. The P gene, which codes for DNA polymerase, had deletions at two positions of 21 and 36 base pairs in both free clones, but was carried in toto by one of the integrated clones. The nucleotide sequences of the S genes of two free and four integrated clones, as well as their two inverted repeats, were compared. All of the eight sequences of the S gene possessed two nucleotide substitutions in common that were not displayed by any of the reported HBV genomes. The sequences differed from one another by only 1.2%. They differed, however, from 11 reported HBV genomes of subtype adr by 2.4%, from an ayr genome by 1.9%, from 2 adw genomes by 6.9%, and from 2 ayw genomes by 5.9%. These results indicate that all free and integrated HBV DNA species in the PLC/342 tumor cell evolved from a common progenitor. The free HBV DNA underwent nucleotide substitutions during several integration events, resulting in integrated HBV DNA copies that were similar in sequence but distinct from the reported HBV genomes.     

Identification of the 12-O-tetradecanoylphorbol-13-acetate-responsive enhancer of the MS gene of the Epstein-Barr virus.

We previously located two 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive enhancers, MSTRE-I and MSTRE-II, in the upstream sequence of the MS gene of Epstein-Barr virus (Liu, Q., and Summers, W.C. (1989) J. Virol. 63, 5062-5068). The core sequence of the MSTRE-I enhancer is now determined to be between -718 and -708 of the upstream sequence of the MS gene. The activity of the enhancer is also sensitive to its immediate surrounding sequence on either side. A single copy of a 30-base pair (bp) fragment containing the MSTRE-I sequence was able to confer TPA responsiveness upon the MS promoter even in the absence of an AP-1 binding site. Multiple tandem copies of this 30-bp fragment, regardless of their relative orientations to each other, could function synergistically to enhance the MS promoter activity. At least two copies of the 30-bp fragment were required to bestow TPA induction upon the thymidine kinase gene promoter of herpes simplex virus type 1. The MSTRE-I sequence could also be bound by a Fos-GCN4 chimeric protein but with an affinity much lower than that between the chimeric protein and the AP-1 binding site. This MSTRE-I region has strong homology to one of the TPA-responsive elements (the ZII domain) in the upstream sequence of the EBV BZLF1 gene. In addition, a putative negative regulatory region or silencer was found immediately downstream of the MSTRE-I enhancer. This potential silencer region contains a 14-bp sequence that is homologous to the silencer consensus sequence of the BZLF1 gene. Therefore, the regulation of the MS gene may share the same pathway with the immediate early gene BZLF1.     

Deletion mapping of Sindbis virus DI RNAs derived from cDNAs defines the sequences essential for replication and packaging

Defective-interfering (DI) genomes of a virus contain sequence information essential for their replication and packaging. They need not contain any coding information and therefore are a valuable tool for identifying cis-acting, regulatory sequences in a viral genome. To identify these sequences in a DI genome of Sindbis virus, we cloned a cDNA copy of a complete DI genome directly downstream of the promoter for the SP6 bacteriophage DNA dependent RNA polymerase. The cDNA was transcribed into RNA, which was transfected into chicken embryo fibroblasts in the presence of helper Sindbis virus. After one to two passages the DI RNA became the major viral RNA species in infected cells. Data from a series of deletions covering the entire DI genome show that only sequences in the 162 nucleotide region at the 5' terminus and in the 19 nucleotide region at the 3' terminus are specifically required for replication and packaging of these genomes.     

Negative transcriptional regulatory element that functions in embryonal carcinoma cells.

We have cloned the polyomavirus mutant fPyF9, which persists in an episomal state in F9 embryonal carcinoma cells (K. Ariizumi and H. Ariga, Mol. Cell. Biol. 6:3920-3927, 1986). fPyF9 carries three copies of exogenous sequences, the prototype of which is a 21-base-pair repeat (box DNA), in the region of the enhancer B domain of wild-type polyomavirus DNA. The consensus sequence, GCATTCCATTGTT, is 13 base pairs long. The box DNA inserted into fPyF9 appeared to come from a cellular sequence and was present in many kinds of DNAs, including F9 chromosomal DNA. The biological function of box DNA was analyzed by chloramphenicol acetyltransferase expression assays, using chimeric plasmids containing box DNA conjugated with simian virus 40 promoter elements. The results showed that box DNA repressed the activities both of the simian virus 40 promoter and enhancer only in transfected undifferentiated F9 cells and not in differentiated LTK- cells. Box DNA functioned independently of orientation and position with respect to the promoter in an enhancerlike manner, although the effect of box DNA was opposite that of the enhancer. The XhoI linker insertion into the consensus sequences of box DNA abolished the repression activity, and the protein(s) recognizing the consensus sequences was identified only in F9 cells, not in L cells. These analyses suggest that box DNA may be a negative regulatory element that functions in undifferentiated cells.     

Human cellular retinol-binding protein gene organization and chromosomal location

The gene encoding the human cellular retinol-binding protein (CRBP) has been isolated from genomic libraries and its structure determined. Only one copy of the gene is present in the human genome. We have located the CRBP gene to segment 3p11-3qter on human chromosome 3 using hybridizations to mouse-human, rat-human and hamster-human cell hybrids. The gene harbors four exons encoding 24, 59, 33, and 16 amino acid residues respectively. The second intervening sequence alone occupies 19 kb of the 21 kb of the CRBP gene. The nucleotide sequence of the gene has been determined with the exception of the second intron. The positions of the introns agree with those in the rat CRBPII, the rat liver fatty-acid-binding protein and the mouse adipose P2 protein genes encoding molecules belonging to the same protein family as CRBP. In contrast to the other sequenced members of this family the promoter of the CRBP gene resembles those found in the 'housekeeping' genes in that it is (G + C)-rich, contains multiple copies of the CCGCCC sequence and lacks TATA box. A 9-bp homology containing the core sequence of the simian virus 40 enhancer repeat was found in the 5' upstream region. A genomic Southern blot probed with CRBP cDNA revealed hybridizing bands in restricted chicken and frog DNA.     

Isolation and characterization of polyoma virus mutants which grow in murine embryonal carcinoma and trophoblast cells.

Mouse trophoblast cell lines established from cultured midterm placenta and a cell line obtained from cultured blastocyst resemble trophectoderm cells. These cells are resistant to infection by wild-type polyoma virus. We have isolated six polyoma virus mutants capable of growing in trophoblast cell lines. Restriction enzyme analyses and marker rescue experiments revealed that the genetic changes necessary for the growth of these mutants ( PyTr mutants) in trophoblast cells were located in a regulatory region of the genome between the origin of viral DNA replication and the region encoding the viral structural proteins. PyTr mutants are, therefore, similar to PyEC mutants, described by others, which are able to grow in embryonal carcinoma cell lines such as F9 or PCC4. The nucleotide sequence of two independently obtained PyTr mutants has an identical 26-bp deletion from nucleotide 5131 to 5156. This deleted region is replaced by either the sequence GGGA or by viral DNA sequences that flank this deletion. PyECF9 mutants grow well in trophoblast and trophectoderm cells, but PyTr mutants do not grow in F9 or PCC4 cells.