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PubMed1989

Nuclear factor I represses the reverse-oriented transcription from the adenovirus type 5 DNA terminus.

The promoter activity of a cloned inverted terminal repeat (ITR) of human adenovirus (Ad) type 5 was found to be oriented in the opposite direction with respect to that of DNA replication in a cell-free transcription system using HeLa nuclear extracts. The major transcript was initiated outside the Ad sequence about 30 nucleotides downstream from the putative TATA-box located between nucleotide positions 9-18 of Ad 5 left terminus. Competitive transcription experiments using double-stranded oligonucleotides and isolated nuclear factor I revealed that nuclear factor I and its cognate binding site located upstream of the putative TATA-box are involved in negative regulation of the transcription from ITR promoter.

Adenoviruses, Human↗
PubMed1986

A novel member of the serpin superfamily is encoded on a circular plasmid-like DNA species isolated from rabbit cells.

A novel member of the serpin family of serine protease inhibitors is presented. A plasmid-like DNA was isolated from rabbit cells by its homology to the genome of Shope fibroma virus (SFV), a tumorigenic poxvirus of rabbits, and was shown elsewhere to encode a serpin-like protein [(1986) Mol. Cell. Biol. 6, 265-276]. Although significant DNA homology exists between the rabbit plasmid serpin open reading frame and the SFV terminal inverted repeat DNA there is no intact serpin counterpart encoded by this region of the SFV genome. The alignment of the novel plasmid-borne polypeptide with the serpin family of proteins confirms its status within this group.

Animals↗
PubMed1986

Functional promoters created by the insertion of transposable element IS1.

We have isolated several insertions of the transposable element IS1 into the proximal promoter (P3) of the beta-lactamase gene of plasmid pBR322, which do not abolish resistance to ampicillin. Using a transcription termination module (omega), we have shown that the gene can be expressed from hybrid promoters, created by the insertion of IS1. The terminal inverted repeats of IS1 carry sequences partially homologous to the "-35" consensus region. Splicing either of these sequences to the existing "-10" region of the beta-lactamase promoter by transposition of IS1 at the proper distance results in the formation of an active hybrid promoter. This interpretation was confirmed by transcription studies in vitro. Gene expression from the hybrid promoters was found to be less efficient than from P3. However, the orientation of IS1 that contributes a "-35" with the greater homology to the known "-35" consensus sequence is significantly more efficient than the other. In addition, we were able to assign a strong determinant of IS1 polarity to a 254 base-pair internal segment of IS1. An examination of the ends of many insertion sequences leads us to expect that the phenomenon described here may occur with several of these transposable elements, and may have an unexpected evolutionary significance.

Base Sequence↗
PubMed1988

DNA sequences required for the initiation of adenovirus type 4 DNA replication in vitro.

In-vivo studies have demonstrated that adenovirus type 2 and adenovirus type 4 have different DNA sequence requirements for the initiation of DNA replication. To investigate the basis of these differences an in-vitro system has been developed which will faithfully initiate adenovirus type 4 DNA replication. A plasmid containing 140 base-pairs of the right terminus of adenovirus type 4 supported initiation of DNA replication in vitro, provided that the plasmid was linearized in such a way as to locate the viral terminal sequences at the molecular ends of the DNA. Initiation by adenovirus type 4-infected cell extracts was also supported by a plasmid containing the complete adenovirus type 2 inverted terminal repeat (ITR). Deletion analysis of both adenovirus types 2 and 4 ITRs revealed that only the terminal 18 base-pairs of the genomes (perfectly conserved between the 2 viruses) were required for initiation in vitro. Thus, initiation was not enhanced by the presence of either the NFI site, the NFIII site or both sites together. Fractionation of a HeLa cell nuclear extract, by ion-exchange chromatography, identified a nuclear factor that stimulated the initiation reaction four- to fivefold. The stimulatory factor did not correspond to either of the cellular proteins NFI or NFIII which stimulate adenovirus type 2 DNA replication in vitro. Initiation in vitro was also supported by single-stranded DNA templates, albeit at a lower efficiency. Studies with synthetic oligonucleotides indicated a surprising specificity for initiation: whereas the strand used as template during initiation in vivo was active as a template for initiation in vitro, the complementary strand was inactive.

Adenoviridae↗
PubMed1989

Regulation of IS1 transposition by the insA gene product.

The IS1 element contains two adjacent genes called insA and insB, both required for IS1 transposition and IS1-mediated plasmid cointegration. These two genes are transcribed polycistronically from the promoter in the left terminal inverted repeat of IS1 (insL). We constructed overexpression systems of these genes with the tac promoter, which are regulated by an exogenous inducer, isopropyl-beta-D-thiogalactopyranoside (IPTG). Then we have examined, under various conditions of induction with IPTG, how overexpression of these genes affects IS1 transposition, using an assay based on plasmid cointegration. When the insA and insB genes were organized identically to the wild-type IS1 genes and simultaneously expressed using low concentrations of IPTG, activity of a mutant IS1 in cis was restored, but not in trans. Higher IPTG concentrations resulted in lower transposition activity. Expression in trans of insA and insB results in a 50 to 100-fold reduction of the frequency of cointegration mediated by wild-type IS1. Such a reduction is also observed when only the insA gene is overexpressed in trans. Overexpression of either mutant insA or insB does not affect the cointegration event. Tests with the insA-lacZ fusion gene showed that the InsA product inhibits the expression of IS1 genes directed by its own promoter in insL. These results suggest that the InsA product regulates IS1 transposition by inhibiting expression of IS1 transposition genes in addition to acting as part of a transposase complex.

Bacterial Proteins↗
PubMed1989

Interactions between the termini of adeno-associated virus DNA.

The adeno-associated virus (AAV) genome is a linear, single polynucleotide chain with inverted terminal repeats of 145 bases. In order to test whether the terminal repeats at opposite ends of the genome have to be able to completely base-pair during DNA replication, we have created chimeric genomes in which an 11 base symmetrical sequence has been deleted from the terminal repeat at one end of the genome and replaced by a different 12 base symmetrical sequence. We have used these chimeric constructs either as a duplex insert in pBR322 or as purified duplex virion DNA to transfect adenovirus-infected HeLa cells. When chimeric duplex virion DNA was used, all of the progeny virions obtained after two cell passages contained DNA with wild-type sequences in both terminal repeats. When plasmid clones were used, the structure of virion DNA depended on the original orientation. If the mutant terminal repeat was originally at the right end of the genome (terminus of genetic map), all progeny terminal repeat sequences were again wild-type. However, if the original construct contained the mutant sequence in the left terminal repeat, the majority of progeny molecules were parental in type (i.e. mutant left and wild-type right terminal repeat). We conclude (1) although the terminal repeats at opposite ends of the genome may interact during DNA replication, it is not necessary that they be perfectly complementary. (2) In direct competition, the wild-type sequence displays an advantage over the mutant allele. (3) In a plasmid clone, the terminal repeat on the left end of the genome is at an advantage in a competitive situation. We note that the left terminal repeat is adjacent to a transcriptional promoter.

Base Sequence↗
PubMed1991

Sequence requirements for protein-primed DNA replication of bacteriophage PRD1.

In vitro studies have demonstrated that linear duplex, protein-free DNA molecules containing an inverted terminal repeat (ITR) sequence of the PRD1 genome at one end can undergo replication by a protein-primed mechanism. No DNA replication was observed when the ITR sequence was deleted or was not exposed at the terminus of the template DNA. We have determined the minimal origin of replication by analyzing the template activity of various deletion derivatives. Our results showed that the terminal 20 base-pairs of ITR are required for efficient in vitro DNA replication. We have found that, within the minimal replication origin region, there are complementary sequences. A site-specific mutagenesis analysis showed that most of the point mutations in the complementary sequences markedly reduced the template activity. The analyses of the results obtained with synthetic oligonucleotides have revealed that the specificity of the replication origin is strand specific and even on a single-stranded template a particular DNA sequence including a 3'-terminal C residue is required for the initiation of PRD1 DNA replication in vitro.

Bacteriophages↗
PubMed1983

Structure and reversion of an adenovirus type 2 mutant containing a duplication of the left end of the genome at the right end.

A mutant (dp 201) of human adenovirus type 2 has been isolated that has the left 3% (1110 bp) of the genome duplicated at the right terminus. The structure of the mutant genome was studied by restriction endonuclease analysis and by direct DNA sequence analysis. The duplication has resulted in a deletion of 10 bp of the right inverted terminal repeat (ITR) at the junction of right and left end sequences. This internal ITR is not active in initiation or termination of DNA synthesis. Two subclones of dp 201 that bear a deletion between map positions 2.1 and 6.8 encompassing early gene blocks E1a and E1b were also isolated. One of these isolates (dl 201.1) retains the left end duplication and the other isolate (dl 201.2) lacks the left end duplication and is an apparent revertant of dl 201.1. The original mutant dp 201 also reverts to a wild type sequence at a low rate. Since 10 base pairs of the right ITR are deleted in the mutant, reversion must involve a repair mechanism using one of the two complete left end ITRs in the mutant genome. The mechanisms by which this reversion may occur are discussed.

Adenoviruses, Human↗
PubMed1984

Nucleotide sequence at the termini of the DNA of Streptococcus pneumoniae phage Cp-1.

The 5' ends of Cp-1 DNA, which have a covalently linked terminal protein, can be partially unblocked by treatment with 1 M NaOH (E. Garcia, A. Gomez, C. Ronda, C. Escarmis, and R. Lopez (1983) Virology 128, 92-104) and labeled with polynucleotide kinase and [gamma-32P]ATP. The sequence of the first 444 and 520 nucleotides at the termini of Cp-1 DNA has been determined. A 236-nucleotide-long inverted terminal repeat was found and, in addition, the 116 nucleotides following the repeat show 93% homology. The first 352 nucleotides at both ends have an adenine plus thymine content of 75%. More than 50% of the nucleotides of the sequenced regions are involved in repeats of a minimum of 8 nucleotides. Three promoter-like sequences were also found at each end of Cp-1 DNA.

Bacteriophages↗
PubMed1985

Transposon-mediated mutagenesis of a baculovirus.

Spontaneous mutants of insect nuclear polyhedrosis viruses of Autographa californica (AcMNPV) and Galleria mellonella (GmMNPV) were analyzed. These mutants produce few polyhedra in infected cells and have insertions of host cell DNA. All the insertions mapped to two adjacent sites in the genome. The junctions between two host insertions and the viral DNA were sequenced. One of the insertions contained a perfect 7-bp inverted terminal repeat, and had caused a direct duplication of 4 bp of viral DNA at the point of insertion. Therefore, this insertion sequence has properties of a transposon of the host cell Trichoplusia ni.

Animals↗
PubMed1985

Characterization, molecular cloning, and physical mapping of the Shope fibroma virus genome.

Five strains of Shope fibroma virus (SFV), a strain of rabbit myxoma virus, and a strain of vaccinia virus were compared by restriction endonuclease digestion of their viral DNAs. Restriction digest patterns revealed that SFV and rabbit myxoma, both members of the Leporipoxvirus genus, were distinct from vaccinia, an Orthopoxvirus. All strains of SFV examined had a high degree of nucleotide sequence homology as shown by conservation of restriction sites within their genomes. However, restriction patterns of SFV and myxoma were quite different from one another suggesting that the genomes from these two viruses of the Leporipoxvirus genus do not share a large, highly conserved region of homology as do the viruses belonging to the Orthopoxvirus genus. Restriction mapping identified inverted terminal repeats of approximately 12 kb in length. Restriction fragments representing all but 400 bp of the termini were cloned in plasmid vectors.

Animals↗
PubMed1985

Tumorigenic poxviruses: genomic organization of malignant rabbit virus, a recombinant between Shope fibroma virus and myxoma virus.

The genome of malignant rabbit virus (MRV), a newly discovered tumorigenic poxvirus of rabbits, has been analyzed using cloned DNA probes from Shope fibroma virus (SFV) and myxoma virus. Under high stringency conditions for Southern blotting such that SFV probes do not cross-hybridize with myxoma virus DNA, it is demonstrated that greater than 90% of the MRV genome has been derived from myxoma virus, and that approximately 10 kb of SFV-derived sequences have substituted for a similar amount of myxoma sequences. Mapping of the MRV genome indicates that the SFV sequences are present in two regions of the genome, one in each copy of the MRV terminal inverted repeat sequence. Furthermore, fine mapping studies of the integration sites for SFV into the myxoma background show that these SFV sequences are not symmetrical with respect to the left and right genomic termini. At the left end, 4 kb of SFV-derived DNA maps between 6 and 10 kb from the terminus, while at the right end about 5.5 kb of SFV sequences are found to extend at least 1 kb further toward the unique internal sequences. Based on this asymmetrical bipartite distribution of SFV sequences in MRV, a two-stage model to rationalize the origin of MRV is proposed. This model postulates an initial recombination event similar to gene conversion between myxoma and SFV at the right terminus of myxoma, followed by an incomplete transposition of only part of these SFV sequences to the left terminus.

Animals↗
PubMed1988

Physical organization of the enteric adenovirus type 41 early region 1A.

Enteric adenovirus types 40 and 41 (Ad40 and Ad41), representing subgenus F, differ from all other human adenoviruses by being so fastidious that productive replication does not occur in conventional established cell lines. They are dependent of the Ad5 early regions E1A and E1B since they can not grow in HEK cells, only in 293 HEK cells transformed by Ad5 E1. The overall genetic organization of Ad41 E1A is similar to the E1A region of other characterized human adenoviruses but it is slightly shorter, comprising 1350 bp. The inverted terminal repeat (ITR) at the 5' end of both Ad40 and AD41 consists of 163 nucleotides, being similar to the ITR of Ad12 (subgenus A) and longer than the ITRs of adenoviruses of subgenera B, C, and E. The early mRNA products (12 and 13 S) can be translated into a 222-amino acid (aa) and a 251-aa tentative protein, respectively. In a comparison of the Ad41 251-aa protein with corresponding peptides of Ad12, Ad7, Ad5, and Ad4, three conserved amino acid sequences CS1-CS3 can be found. In the second conserved domain CS2, which is particularly acidic, the homology is very high within all five serotypes compared. Only one among eight conserved amino acids differs in the Ad41 251-aa protein. Within CS1 and CS3 which exhibit a hydrophilic and a hydrophobic character, respectively, the amino acid composition of the Ad41 protein is less conserved than the corresponding regions in all other analyzed adenovirus types. Ten of 16 conserved amino acids in CS1 are shared by Ad41 and 18 of 23 conserved amino acids in CS3 are shared by Ad41.

Adenoviridae Infections↗
PubMed1989

Transposon mutagenesis of baculoviruses: analysis of Trichoplusia ni transposon IFP2 insertions within the FP-locus of nuclear polyhedrosis viruses.

The transposable IFP2 element of Trichoplusia ni was originally isolated as a host DNA insertion in spontaneous FP mutants of Galleria mellonella or Autographa californica nuclear polyhedrosis viruses (NPVs). The termini of IFP2 insertions from five independently isolated FP mutants were sequenced. In all cases IFP2 is flanked by 13-bp terminal inverted repeats and has additional inverted repeats of 19 bp in length located asymmetrically with respect to the ends of the element. Insertion of IFP2 into the viral genome always generated a duplication of the tetranucleotide target site, TTAA. There was an apparent preference for insertion within a 12-bp A + T-rich imperfect palindromic sequence surrounding the target site. Sequence analysis of three independent IFP2 elements revealed an internal domain of 2.475 kb containing an RNA polymerase II promoter region and two large open reading frames. Primer extension analysis of IFP2-specific mRNA positioned the 5' terminus of the transcript. The element is present in DNA isolated from T. ni cell lines TN-368 and TN-5B1, but is not apparent in DNAs isolated from the TN-R2 cell line or our laboratory colony of T. ni larvae, suggesting IFP2 was recently introduced into the T. ni genome.

Base Sequence↗
PubMed1989

Tumorigenic poxviruses: characterization of an early promoter from Shope fibroma virus.

A strong early promoter from the T1 open reading frame (ORF) within the terminal inverted repeat (TIR) of Shope fibroma virus (SFV) has been isolated and characterized. Promoter activity was determined by a transient gene expression assay in poxvirus-infected cells using the bacterial chloramphenicol acetyltransferase as a reporter gene. The sequences which constitute the boundaries of the promoter element were determined by 5' and 3' deletion analysis. The functional SFV T1 promoter domain comprises about 28 bp and includes, in addition to the transcriptional initiation site, a stretch of eight continuous A residues from position -18 to -11 which is critical for promoter function. Both the SFV T1 promoter and the vaccinia 7.5-kDa early/late promoter are active in the transient expression assay when the cells are infected with either the leporipoxvirus SFV or the orthopoxvirus vaccinia. To look more closely at the conservation of promoter function between poxvirus genera, a recombinant vaccinia virus containing the CAT gene driven by the SFV T1 promoter and a recombinant SFV containing the CAT gene driven by the vaccinia 7.5-kDa early/late promoters was constructed. The SFV T1 promoter behaves as an early promoter in the vaccinia genome, and both the T1 and the 7.5-kDa early/late promoters use transcriptional initiation sites in their heterologous genomic environment that are identical to the ones used in the native viral genome. The results from this work indicate that despite the relative lack of absolute sequence conservation, the transcriptional machinery, at least with respect to temporal regulation of early promoters and the position of transcript initiation, is conserved between these two poxvirus genera.

DNA Mutational Analysis↗
PubMed1989

A capripoxvirus pseudogene whose only intact homologs are in other poxvirus genomes.

Equivalent regions from within the inverted terminal repeats (ITRs) of the genomes of two capripoxviruses, KS-1 and InS-1, were sequenced. The sequence from KS-1 DNA covers the major part of three contiguous open reading frames (ORFs), which match three contiguous ORFs from within the genomic ITRs of the leporipoxvirus Shope Fibroma Virus (SFV). The sequenced region of InS-1 DNA contains only two of the three ORFs. The region homologous to the third ORF has no coding potential due to the presence of several stop codons, resulting from small frameshifting deletions and insertions. The significance of a degenerate poxvirus gene, intact homologs of which are only found in other poxvirus genomes, is discussed.

Amino Acid Sequence↗
PubMed1990

Myxoma virus and malignant rabbit fibroma virus encode a serpin-like protein important for virus virulence.

The leporipoxviruses Shope fibroma virus (SFV), the myxoma virus (MYX), and the SFV/MYX recombinant malignant rabbit fibroma virus (MRV) are closely related yet induce profoundly different diseases in the European rabbit. SFV, which produces a benign tumor at the site of inoculation, is cleared by the immune system after approximately 2 weeks whereas MYX and MRV induce a rapidly lethal systemic infection characterized by generalized suppression of host immune functions. DNA sequencing studies reveal that MRV and MYX possess homologous gene members of the T6/T8/T9 family originally described in the terminal inverted repeat (TIR) of SFV. We also describe a gene present in both MYX and MRV genomes, but which has apparently evolved in the SFV genome into a fragmented pseudogene that appears to contribute to the aggressive nature of MYX and MRV infections. Translation of this open reading frame, designated MYXOMA SERPIN 1 (SERP1), reveals a protein sequence with highly significant homology to the super-family of serine protease inhibitors (serpins) which also includes a number of other poxviral proteins. In the MYX genome the SERP1 gene lies entirely within the TIR sequences and is thus present as two copies, while in the MRV genome SERP1 is present in the unique sequences adjacent to the TIR boundary and hence is a single copy. The amino acid homology between the putative active site of SERP1 and those of other serpins predicts that the target enzyme will be different from the known catalog of serine antiprotease substrates. Deletion of this gene from MRV significantly attenuates the disease spectrum induced by the normally lethal virus. Although the MRV-S1 deletion construct (MRV with SERP1 gene deleted) grows in all tissue culture cells tested in a fashion identical to the MRV parent, the majority of rabbits infected with MRV-S1 are able to mount an effective immune response and totally recover from the virus infection to become resistant to subsequent challenge by MRV or MYX.

Amino Acid Sequence↗
PubMed1990

Cloning of the genome of a densovirus and rescue of infectious virions from recombinant plasmid in the insect host Spodoptera littoralis.

We have cloned an infectious genome of the Junonia coenia densonucleosis virus (JcDNV) into the bacterial plasmid pBR322. The viral genome could be rescued from the recombinant plasmid pBRJ by transfection of pBRJ DNA to sensitive Spodoptera littoralis larvae. pBRJ DNA produced a typical viral infection and a comparable percentage of larvae became infected following inoculation of equivalent amounts of purified virion DNA or cloned viral DNA. Virions extracted from transfected larvae were indistinguishable from wild-type (wt) virions with regard to their biophysical and biological properties. In particular, rescued virions were as infectious as wt virions and showed identical restriction profiles of their genome. In contrast, subcloning of JcDNV DNA deleted at both extremities of a sequence of ca 250 or ca 100 bp resulted in the inability of the recombinant plasmids to initiate a viral infection. These data suggest that, as for vertebrate parvoviruses, the inverted terminal repeats display essential functions in the rescue process and replicative cycle of densoviruses. This is the first report of the molecular cloning of the infectious genome from an insect parvovirus, and more generally from an invertebrate virus. pBRJ should provide an efficient tool to further define the organization of the JcDNV genome and compare it to other parvoviruses.

Animals↗