Termini Of Adenovirus Research Articles

Long-term transgene expression in proliferating cells mediated by episomally maintained high-capacity adenovirus vectors.

High-capacity "gutless" adenovirus vectors (HC-AdV) mediate long-term transgene expression in resting cells in vitro and in vivo because of low toxicity and immunogenicity. However, in proliferating cells, expression is transient since HC-AdV genomes do not possess elements that allow for replication and segregation of the replicated genomes to daughter cells. We developed a binary HC-AdV system that, under certain conditions, allows for significantly prolonged episomal maintenance of HC-AdV genomes in proliferating tissue culture cells, resulting in sustained transgene expression. After transduction of target cells the linear HC-AdV genomes were circularized by the DNA recombinase FLPe, which was expressed from the second HC-AdV. The oriP/EBNA-1 replication system derived from Epstein-Barr virus, as well as the human replication origin from the lamin B2 locus, were used as cis elements to test for replication of the 28-kb circular vector genomes with or without selective pressure. Depending on the system, up to 98% of the circularized genomes were replicated and segregated to daughter cells, as demonstrated by Southern assays and as confirmed by monitoring EGFP transgene expression. Surprisingly, in the absence of FLPe recombinase, a small but significant number of HC-AdV genomes spontaneously circularized after transduction of target cells. These circles, found to contain end-to-end joined adenovirus termini, replicated with increased efficiency compared to vectors circularized by FLPe. After further improvements, this HC-AdV system might be suitable for gene therapy applications requiring long-term transgene expression.

Spliced segments at the 5? terminus of adenovirus 2 late mRNA.Susan M. Berget, Claire Moore and Phillip A. Sharp; An amazing sequence arrangement at the 5? ends of adenovirus 2 messenger RNA.Louise T. Chow, Richard E. Gelinas, Thomas R. Broker and Richard T. Roberts

Spliced segments at the 5? terminus of adenovirus 2 late mRNA.Susan M. Berget, Claire Moore and Phillip A. Sharp; An amazing sequence arrangement at the 5? ends of adenovirus 2 messenger RNA.Louise T. Chow, Richard E. Gelinas, Thomas R. Broker and Richard T. Roberts

Adenovirus subviral particles and cores can support limited DNA replication.

Adenovirus type 2 cores can function effectively as templates in an in vitro replication system. Viral DNA replication assays using cores as templates do not differ in their requirements to the well characterized assays using DNA-complex templates, i.e. there is a dependence on terminal protein precursor (pTP), DNA polymerase and DNA binding protein and the assay is greatly stimulated by certain host transcription factors. The products of initiation and limited elongation are easily distinguishable and, in the system described, there is specific proteolysis of the pTP adducts as a function of the adenovirus-coded protease, present in the nuclear extracts from infected cells, or the core templates. Substitution of Mn2+ ions for Mg2+ ions in the replication assay has a dramatic effect on the nature of the replication events, in most cases resulting in the stimulation of initiation without elongation. Similar results can be achieved by utilizing subviral particles as templates, obtained by dialysis of purified adenovirus in a hypotonic buffer at pH 6.4. Restriction enzyme analysis of the replicated products confirmed that DNA synthesis proceeds from the adenovirus termini using both the core and subviral templates. By adding an ATP-regenerating system elongation can be further stimulated, particularly in the case of the subviral templates. Quantification of nucleotide incorporation into the appropriate restriction fragments indicates that for the subviral templates replication can proceed for at least 2000 to 3000 bases from either terminus. These results suggest that the adenovirus genome is packaged in the virion in a conformation readily available for at least the initial replication events. Such a conformation might also be appropriate for early transcription.

Helper-free stocks of recombinant adeno-associated viruses: normal integration does not require viral gene expression

A method is described for the production of recombinant adeno-associated virus (AAV) stocks that contain no detectable wild-type helper AAV. The recombinant viruses contained only the terminal 191 nucleotides of the AAV chromosome bracketing a nonviral marker gene. trans-Acting AAV functions were provided by a helper DNA in which the terminal 191 nucleotides of the AAV chromosome were substituted with adenovirus terminal sequences. Although the helper DNA did not appear to replicate, it expressed AAV functions at a substantially higher level than did DNA molecules that contained neither AAV nor adenovirus termini. Since the recombinant viruses with AAV termini contained no sequence homology to the helper DNA, no wild-type AAV was generated by homologous recombination within infected cells. Since the terminal region of the AAV chromosome is required for replication and encapsidation, only recombinant DNAs were amplified and packaged into AAV virions. When human cells were infected at a high multiplicity with a recombinant virus carrying a drug resistance marker gene, approximately 70% of the infected cells gave rise to colonies stably expressing the marker. The recombinant virus gene was then used to generate drug-resistant human cell lines subsequent to infection. These cells contained stably integrated copies of the recombinant viral DNA which could be excised, replicated, and encapsidated by infection with wild-type AAV plus adenovirus. Thus, AAV gene expression is not required for normal integration of an infecting DNA containing AAV termini.

Viable viruses with deletions in the left inverted terminal repeat define the adenovirus origin of DNA replication.

A series of human adenovirus type 2 genomes with deletions in the left inverted terminal repeat (ITR) have been constructed. Viral genomes that contained a minimum of 45 base pairs (bp) from the terminus of the genome were fully infectious and gave rise to progeny virus which maintained the deletion. In contrast, genomes containing 36 bp or less from the termini of the genome were not infectious. The boundary of a cis-acting element required for viral replication is therefore between 36 and 45 bp from the adenovirus termini and corresponds to the previously identified viral origin of replication, defined using a transfection assay to measure ori activity in vivo. The growth parameters of viruses with deletions in the left ITR were examined. These deletions had no measurable effect on plaque formation or morphology, viral DNA synthesis or early viral mRNA synthesis. Thus, it appears that DNA sequences in the left ITR, outside the replication origin, are completely dispensable for lytic viral growth in tissue culture cells.

The origin of adenovirus DNA replication: minimal DNA sequence requirement in vivo.

Adenovirus mini-chromosomes which contain two cloned, inverted adenovirus termini replicate in vivo when supplied with non-defective adenovirus as a helper. This system has been used to define the minimum cis acting DNA sequences required for adenovirus DNA replication in vivo. Deletions into each end of the adenovirus inverted terminal repeat (ITR) were generated with Bal31 exonuclease and the resulting molecules constructed into plasmids which contained two inverted copies of the deleted ITR separated by the bacterial neomycin phosphotransferase gene. To determine the effect of the deletion in vivo plasmids cleaved to expose the adenovirus termini were co-transfected with adenovirus type 2 DNA into tissue culture cells. The replicative ability of the molecules bearing adenovirus termini was assayed by Southern blotting of extracted DNA which had been treated with DpnI, a restriction enzyme which cleaves only methylated and therefore unreplicated, input DNA. Molecules containing the terminal 45 bp of the viral genome were fully active whereas molecules containing only 36 bp were in-active in this assay. Therefore sequences required for DNA replication are contained entirely within the terminal 45 bp of the viral genome. Thus, both the previously described highly conserved region (nucleotides 9-18) and the binding site for the cellular nuclear factor I (nucleotides 19-48) are essential for adenovirus DNA replication in vivo.

Replication of adenovirus mini-chromosomes

We have isolated adenovirus origins of DNA replication from both the right and left ends of the genome, which are functional on linear autonomously replicating mini-chromosomes. The mini-chromosomes contain two cloned inverted adenovirus termini and require non-defective adenovirus as a helper. Replicated molecules are covalently attached to protein, and DNA synthesis is initiated at the correct nucleotide even when the origins are not located at molecular ends. The activity of embedded origins leads to the generation of linear minichromosomes from circular or linear molecules. These observations therefore suggest that sequences within the adenovirus origin of replication position the protein priming event at the adenovirus terminus. Experiments investigating the regeneration of deleted viral inverted terminal repeat sequences show a sequence-independent requirement for inverted sequences in this process. This result strongly suggests that repair results from the formation of a panhandle structure by a displaced single strand. On the basis of these observations we propose a model for the generation of adenovirus mini-chromosomes from larger molecules.

A rapid and simple method for preparation of adenovirus DNA from infected cells.

Microbiology and ImmunologyVolume 27, Issue 9 p. 817-822 NoteFree Access A Rapid and Simple Method for Preparation of Adenovirus DNA from Infected Cells Morikazu Shinagawa, Morikazu Shinagawa Department of Veterinary Public Health, School of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080Search for more papers by this authorAkihisa Matsuda, Akihisa Matsuda Department of Veterinary Public Health, School of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080Search for more papers by this authorToshiro Ishiyama, Toshiro Ishiyama Department of Veterinary Public Health, School of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080Search for more papers by this authorHitoshi Goto, Hitoshi Goto Department of Veterinary Microbiology, School of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080Search for more papers by this authorGihei Sato, Gihei Sato Department of Veterinary Public Health, School of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080Search for more papers by this author Morikazu Shinagawa, Morikazu Shinagawa Department of Veterinary Public Health, School of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080Search for more papers by this authorAkihisa Matsuda, Akihisa Matsuda Department of Veterinary Public Health, School of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080Search for more papers by this authorToshiro Ishiyama, Toshiro Ishiyama Department of Veterinary Public Health, School of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080Search for more papers by this authorHitoshi Goto, Hitoshi Goto Department of Veterinary Microbiology, School of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080Search for more papers by this authorGihei Sato, Gihei Sato Department of Veterinary Public Health, School of Veterinary Medicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido, 080Search for more papers by this author First published: September 1983 https://doi.org/10.1111/j.1348-0421.1983.tb00638.xCitations: 92AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL REFERENCES 1Carusi, E.A. 1977. Evidence for blocked 5′ termini in human adenovirus DNA. Virology 76: 380– 395. 2Green, M., and Pina, M. 1964. Biochemical studies on adenovirus multiplication, VI. Properties of highly purified tumorigenic human adenoviruses and their DNA's. Proc. Natl. Acad. Sci. U.S.A. 51: 1251– 1259. 3Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26: 365– 369. 4Horwitz, S.M. 1976. Bidirectional replication of adenovirus type 2 DNA. J. Virol. 18: 307– 315. 5Johansson, M.E., Wadell, G., Jacobson, P.A., and Svensson, L. 1979. Preparation of specific antisera against adenoviruses by affinity bead immunization. J. Immunol. Methods 26: 141– 149. 6Jones, N., and Shenk, T. 1978. Isolation of deletion and substitution mutants of adenovirus type 5. Cell 13: 181– 188. 7Maxam, A.M., and Gilbert, W. 1977. A new method for sequencing DNA. Proc. Natl. Acad. Sci. U.S.A. 74: 560– 564. 8Norrby, E., Bartha, A., Rowe, P., Russell, W.C., Schlesinger, R.W., and Wigand, R. 1976. Adenoviridae. Intervirology 7: 117– 125. 9Padmanabhan, R., and Padmanabhan, R.V. 1977. Specific interaction of protein(s) at or near the termini of adenovirus 2 DNA. Biochem. Biophys. Res. Commun. 75: 955– 964. 10Pettersson, U., and Sambrook, J. 1973. Amount of viral DNA in the genome of cells transformed by adenovirus type 2. J. Mol. Biol. 73: 125– 130. 11Rekosh, D.M.K., Russel, W.C., Bellett, A.J.D., and Robinson, A.J. 1977. Identification of a protein linked to the ends of adenovirus DNA. Cell 11: 283– 295. 12Robinson, A.J., Younghusband, H.B., and Bellett, A.J.D. 1973. A circular DNA-protein complex from adenoviruses. Virology 56: 54– 69. 13Sharp, P.A., Moor, C., and Haverty, J.L. 1976. The infectivity of adenovirus 5 DNA-protein complex. Virology 75: 442– 456. 14Shinagawa, M., and Padmanabhan, R. 1979. Inhibition of a nuclease contamination in the commercial preparations of Escherichia coli alkaline phosphatase. Anal. Biochem. 95: 458– 464. 15Shinagawa, M., and Padmanabhan, R. 1980. Comparative sequence analysis of the inverted terminal repetitions from different adenoviruses. Proc. Natl. Acad. Sci. U.S.A. 77: 3831– 3835. 16Wimmer, E. 1982. Genome-linked proteins of viruses. Cell 28: 3831– 3835. Citing Literature Volume27, Issue9September 1983Pages 817-822 ReferencesRelatedInformation

A common sequence in the inverted terminal repetitions of human and avian adenovimses

The termini of the avian chick embryo lethal orphan (CELO) virus DNA have been sequenced. The results revealed a 63-bp-long inverted terminal repetition (ITR) which shared the sequence ATAATA with all adenovirus termini, thus far analyzed. The CELO virus ITR differed from those of the mammalian adenoviruses in two major aspects: (i) it is not a perfect duplication; (ii) it begins with a 5′-guanylic acid residue instead of the cytidylic acid normally observed in adenoviruses.

Nucleotide sequences of 5' termini of adenovirus 2 early transforming region E1a and E1b messenger ribonucleic acids.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTNucleotide sequences of 5' termini of adenovirus 2 early transforming region Ela and Elb messenger ribonucleic acidsShuichi Hashimoto, William S. M. Wold, Karl H. Brackmann, and Maurice GreenCite this: Biochemistry 1981, 20, 23, 6640–6647Publication Date (Print):November 1, 1981Publication History Published online1 May 2002Published inissue 1 November 1981https://doi.org/10.1021/bi00526a019RIGHTS & PERMISSIONSArticle Views15Altmetric-Citations9LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (4 MB) Get e-Alerts Get e-Alerts

Nucleotide sequences and mapping of novel heterogenous 5'-termini of adenovirus 2 early region 4 mRNA.

The major 5'-termini of human adenovirus type 2 early gene block 4 mRNA were sequenced. Poly(A+) polyribosomal RNA was isolated from Ad2 early infected cells, the 5'-terminal m7GPPP removed and the 5'-OH of the penultimate 2'-0-methylated nucleotide labeled with [gamma-32P]ATP using polynucleotide kinase. Ad2 E4 mRNA was purified by hybridization to the Ad2 EcoRI-C fragment and was digested with RNase T1. The resulting oligonucleotides were resolved by two dimensional paper electrophoresis-homochromatography. Four major and 3-4 minor 5'-terminal sequences were identified and characterized. The sequence of the 5'-terminal structures of the major four termini are: (1) m7GpppUmU(m)UUACACUGp, (2) m7GpppUmU(m)UACACUGp, (3) m7GpppUmU(m)ACACUGp, and (4) m7Gppp(m6)AmC(m)ACUGp. These major 5'-terminal sequences were aligned with nucleotide 325, 326, 327, and 329 from the righthand end of the known Ad2 DNA sequence (1) in the region mapped as the 5'-terminus of E4 mRNA by electron microscopy (2,3) and S1 nuclease-gel (4) mapping. Two potential ribosomal binding sites and an initiator codon were found at 40 to 65 nucleotides and about 80 nucleotides, respectively, from these heterogenous 5'-termini. Ad2 E4 major mRNA species appear to be unique since mRNA molecules initiate at a pyrimidine, perhaps by RNA polymerase stuttering, or they are products of an unusual type of RNA processing.

Nucleotide sequence at the 5' terminus of adenovirus 2 late messenger RNA.

One major late promoter is transcriptionally active for the majority of adenovirus 2 late RNA synthesis. Nucleotide sequences proximal to this promoter are spliced onto the 5' termini of all messenger RNAs generated from it. We report here the complete nucleotide sequence of the capped 5-'terminal undecanucleotide common to these late mRNAs. The sequence m7G5'ppp5'm6AmCUCUCUUCCGp shows striking similarities to the 5'-terminal sequences of the eukaryotic alpha- and beta-globin mRNAs and contains the sequence 5'CUUCCG3' complementary to a six-base sequence near the 3' terminus of most eukaryotic 18 S ribosomal RNAs. On the basis of this sequence and the finding that this same sequence is present at the 5' end of adenovirus 2 late nuclear RNA precursor, it should now be possible to locate the major late promotor within the adenoviral genomic DNA sequence.

Spliced segments at the 5′ terminus of adenovirus 2 late mRNA

An mRNA fraction coding for hexon polypeptide, the major virion structural protein, was purified by gel electrophoresis from extracts of adenovirus 2-infected cells late in the lytic cycle. The mRNA sequences in this fraction were mapped between 51.7 and 61.3 units on the genome by visualizing RNA-DNA hybrids in the electron microscope. When hybrids of hexon mRNA and single-stranded restriction endonuclease cleavage fragments of viral DNA were visualized in the electron microscope,branched forms were observed in which 160 nucleotides of RNA from the 5' terminus were not hydrogen bonded to the single-stranded DNA. DNA sequences complementary to the RNA sequences in each 5' tail were found by electron microscopy to be located at 17, 20, and 27 units on the same strand as that coding for the body of the hexon mRNA. Thus, four segments of viral RNA may be joined together during the synthesis of mature hexon mRNA. A model is presented for adenovirus late mRNA synthesis that involves multiple splicing during maturation of a larger precursor nuclear RNA.

One predominant 5′-undecanucleotide in adenovirus 2 late messenger RNAs

Oligonucleotides containing the 5′ termini of adenovirus 2 mRNA are selectively retained on columns of dihydroxyboryl cellulose. When total late adenovirus 2 mRNA was treated with RNAase T1, a single 5′ terminal oligonucleotide was isolated, although in several states of methylation. This oligonucleotide has the general structure m 7 GS 5′ ppp 5′ A ★ mCmU(C 4,U 3)G. Since at least twelve individual species of mRNA must be present late after infection, this finding was unexpected and its significance is discussed.

Specific interaction of a protein(s) at or near the termini of adenovirus 2 DNA

When human adenovirus type 2 DNA was extracted from the virions, and the sedimentation profile of the DNA-protein complex in a linear sucrose gradient was analyzed by the nitrocellulose filter binding assay, it was found that 90–95% of adenovirus 2 DNA molecules was tightly associated with the protein(s). The analyses of the binding site of the protein(s) on the DNA by using the restriction endonucleases suggested that the protein(s) interact with the DNA at or near the termini of the viral genome. The stability of the linkage between the DNA and the protein(s) was tested in the presence of various reagents. From the available data, it is suggestive that the protein(s) is linked to the DNA covalently.

Evidence for blocked 5′-termini in human adenovirus DNA

DNA molecules from six human adenovirus serotypes, representative of three oncogenic classes, were investigated using enzymes specific for 5′-, and for 3′-termini in DNA. Results obtained with DNA purified by sequential treatment with papain, sodium dodecyl sulfate, and phenol show that the majority of the 5′-termini of all these DNAs is blocked. Depending upon the source of DNA, from 50 to 80% of the 5′-termini are inaccessible to polynucleotide kinase even after extensive alkaline phosphatase treatment. The DNA molecules with 5′-termini inaccessible to phosphorylation are also resistant to λ exonuclease which hydrolyzes DNA starting from 5′-ends. These 5′-termini remain inaccessible to phosphorylation after high temperature phosphatase treatment, or after phosphatase treatment of denatured, single-stranded DNA. However, about 40% of the blocked 5′-termini of adenovirus 2 DNA becomes accessible to phosphatase and polynucleotide kinase after prolonged treatment of the DNA with sodium hydroxide. These findings suggest that a strongly bonded substituent rather than a peculiar DNA conformation accounts for the inaccessible 5′-termini. The 5′-blocking group does not affect the reactivity of the 3′-termini since all of the molecules are attacked by exonuclease III, which hydrolyzes DNA starting from 3′-ends. Finally, in all of the adenovirus serotypes examined, the nucleotide found at the 5′-termini of DNA molecules accessible to labeling is predominantly deoxycytidylate.

Evidence for palindromic sequences near the termini of adenovirus 2 DNA.

When the kinetics of Escherichia coli exonuclease III digestion of adenovirus 2 DNA were studied by DNA polymerase I-catalyzed repair synthesis at 5°C, there was an indication of the formation of hairpin structure in the single-stranded template, exposed by exonuclease III. The hairpin structure results from a sequence with an inverted repetition of the type, a b c d···d′ c′ b′ a′. The location of these sequences was determined to be about 180 nucleotides from each terminus of adenovirus 2 DNA with the use of specific restriction endonucleases. The possible role of this region in the replication of the adenovirus 2 genome is discussed.