Nucleotide sequence of the inverted terminal repetition in adeno-associated virus DNA.

The inverted terminal repetition in adeno-associated virus type 2 DNA has been sequenced. The terminal repetition contain 145 nucleotides of which the first 125 nucleotides can self-base pair to form a T-shaped hairpin structure. Both restriction endonuclease analysis with SmaI and BglI and direct sequence analysis of the SmaI fragments provide evidence for two sequences in the region of the terminal repetition between nucleotides 44 and 81. The two sequences represent an inversion of the first 125 nucleotides of the terminal repetition. Based on these data a model for adeno-associated virus DNA replication is presented which agrees in detail with a general model for eucaryotic DNA replication originally proposed by Cavalier-Smith (T. Cavalier-Smith, Nature [London] 18:672--684, 1976).     

Tandem repeats within the inverted terminal repetition of vaccinia virus DNA

A tandemly repeated sequence within the genome of vaccinia virus is cut to fragments of approximately 70 bp by Hinf I, Taq I or Mbo II. The 70 bp repetition was localized within the much larger (10,300 bp) inverted terminal repetition by restriction analysis of cloned DNA fragments and by hybridization of the purified 70 bp repeat to vaccinia virus DNA restriction fragments. The molar abundance of the 70 bp fragment corresponds to a 30 fold repetition at each end of the genome. The repeating restriction endonuclease sites were mapped by agarose gel electrophoresis of partial Hinf I digests of the terminally labeled cloned DNA fragment. The first of 13 repetitive Hinf I sites occurred approximately 150 bp from the end of the cloned DNA. After an intervening sequence of approximately 435 bp, a second series of 17 repetitive Hinf I sites occurred. The DNA between the two blocks of repetitions has a unique sequence containing single Dde I, Alu I and Sau 3A sites. Tandem repeats within the inverted terminal repetition could serve to accelerate self-annealing of single strands of DNA to form circular structures during replication.     

The nucleotide sequence of the inverted terminal repetition of the tree shrew adenovirus DNA

The termini of the tupaia (tree shrew) adenovirus (TAV) DNA have been sequenced. The inverted terminal repetitions (ITR) are 166 bp long containing the A + T-rich, highly conserved sequence present in all adenovirus DNAs so far analysed. An unusual feature within the TAV ITR is the presence of four sets of a conserved sequence TGACCG which occur at or near the ends of many adenovirus ITR.     

Inverted terminal repetition in adeno-associated virus DNA: independence of the orientation at either end of the genome

Complementary strands of adeno-associated virus DNA labeled with 32P at the 5' ends were separated and then self-annealed to form single-stranded circles stabilized by hydrogen bonds between the complementary sequences in the inverted terminal repetitions. We have previously shown that there are two distinct sequences in the terminal repetition which represent an inversion of the first 125 nucleotides (E. Lusby et al., J. Virol. 34:402-409, 1980; I. S. Spear et al., Virology 24:627-634, 1977). Base pairing between terminal sequences of the same orientation leads to a normal double helical structure. If sequences of the opposite orientation pair, an aberrant secondary structure is formed. HpaII digestion of the self-annealed, single-stranded circles led to labeled terminal fragments that corresponded both to those generated from termini of a normal double helical structure and those generated from an aberrant terminal secondary structure. Thus, the orientation of the terminal repetition at one end of the genome is not influenced by the orientation at the other end.     

Arrangement of nucleotide sequences in adeno-associated virus DNA

There are two types of adeno-associated virus virions which contain complementary single-stranded DNA genomes of about 1.4 × 10⁶ daltons. The purified complementary single polynucleotide chains anneal to form duplex linear monomers, circular monomers, and linear dimers, in addition to other less well-defined structures, as identified by sedimentation and electron microscopy. All duplex species are formed by linear single polynucleotide chains of unit length, thus duplex circles and linear dimers are assumed to be held together by relatively short overlapping hydrogen-bonded regions. The initial linear monomers present after annealing the complementary single strands do not form duplex circles or oligomers when re-exposed to annealing conditions, but DNA which sediments as linear monomers after heating linear dimers at a temperature from 7 to 25 deg. C below the T_m of duplex AAV² DNA does re-form oligomers and circles when exposed to annealing conditions. Denaturation and reannealing of any duplex species leads to the formation of all forms, indicating that the over-all single strand composition of all species is equivalent. Disruption of duplex circles by limited exonucleolytic digestion using either 3′ or 5′ exonucleases leads to the conclusion that the overlap region may have either 3′ or 5′ termini and that the overlap region represents less than 6% of the length of the genome. Exonucleolytic digestion of linear monomers to the extent of 50% leaves polynucleotide chains which cannot reanneal after denaturation, thus AAV DNA is not randomly circularly permuted. Duplex linear monomers which do not form circles when exposed to annealing conditions do form duplex circles after 1% exonuclease III digestion. A model consistent with these data is one in which the linear single polynucleotide chains present in AAV virions consist of two or more permutations. All of these chains contain terminal repetitions and their start points occur within a limited region, representing < 6% of the length of the genome. According to this interpretation AAV DNA would exist within the virion as a linear single polynucleotide chain or is cleaved at a few specific sites during extraction.     

Interaction of the adeno-associated virus Rep protein with a sequence within the A palindrome of the viral terminal repeat.

We have characterized a Rep binding sequence which is within the A stem region of the adeno-associated virus terminal repeat (TR) and compared its affinity with that of the complete hairpinned TR for pure Rep68. Both the A stem and the complete TR substrates produced a complex pattern of protein-DNA complexes in which at least six different bound species could be distinguished. Competition experiments suggested that the dissociation constant for the A stem sequence is approximately 125-fold higher than that for the complete TR. The competition experiments also suggested that the average number of Rep molecules per TR substrate molecule under conditions of saturating substrate is 3.7:1, while for the A stem substrate, the ratio is 10:1. In spite of the apparent difference in protein-to-DNA ratio in the complexes, no major difference was seen in the mobility or the pattern of the protein-DNA complexes with the two kinds of substrates, suggesting that the difference in protein-to-DNA ratio was due to the lower stability of the A stem complex rather than the actual number of Rep molecules per DNA molecule. At least some of the difference in stability of the two kinds of complexes was due to the fact that the dissociation rate of the A stem substrate from the protein-DNA complexes was approximately fourfold faster than that of the complete TR. The dissociation rate curves for both substrates, however, were complex, suggesting that substrate was being released from at least two different kinds of protein-DNA complexes at different rates. In addition, we have analyzed binding to several substitution mutants within the A stem of the TR. A five-base mutant near the terminal resolution site (trs site) had little effect on binding. Two other mutants produced seven- or five-base substitutions within the 25-bp sequence of the A stem that had been identified in the accompanying report (D. M. McCarty, D. J. Pereira, I. Zolotukhin, X. Zhou, J. H. Ryan, and N. Muzyczka, J. Virol. 68:4988-4997, 1994) as essential for binding. Each of these mutants eliminated some but not all of the repeating GAGC motifs in the 25-bp A stem region. Both of these mutants completely abolished binding to the A stem substrate but only partially reduced binding in the context of the complete hairpinned TR. Furthermore, neither mutant altered the pattern of Rep-DNA complexes produced.(ABSTRACT TRUNCATED AT 400 WORDS)     

Nucleotide sequence at the inverted terminal repetition of adenovirus type 2 DNA.

We have determined the nucleotide sequence of the inverted repetition present at the termini of adenovirus type 2 DNA. The terminal repetition is 103 nucleotides long. It is exactly identical in sequence at both termini. Adenovirus types 2 and 5 molecules share a perfect homology within this region.     

Site-specific nicking within the adenovirus inverted terminal repetition.

Site-specific nicking occurs on the l-strand, but not on the r-strand, of the adenovirus left inverted terminal repeat. Nicks are presumably introduced into double- or single-stranded DNA by a cellular endonuclease in an ATP-independent reaction. The consensus nick site has the sequence: (sequence in text).     

Conformation takes precedence over sequence in adeno-associated virus DNA replication.

Deletion of an 11-base symmetrical sequence in the inverted terminal repetition of the adeno-associated virus 2 genome inhibits DNA replication. Substitution of either an 8- or a 12-base symmetrical sequence unrelated to the original 11-base sequence restores DNA replication.     

Comparison of the DNA sequence and secondary structure of the herpes simplex virus L/S junction and the adeno-associated virus terminal repeat

The defective parvovirus Adeno-associated virus (AAV) is absolutely dependent upon coinfection with either Adenovirus or Herpes Simplex Virus (HSV) for its multiplication. We have compared the terminal repeats of HSV-1F strain DNA with the terminal 200 nucleotides of AAV DNA. Our findings demonstrate similarities between portions of the HSV inverted repeats found at the L/S junction and the termini of AAV. By computer analysis we have determined potential secondary folding patterns for both genomes. The following points can be made about the a, b, and c repeats in HSV: (1) Regions b and c are complementary over a significant portion of their length. (2) The ends of a can fold back on themselves to form large secondary structures. Moreover, when the b and c homology is used to align the ends of a, the b/a and c/a junctions are within 1 base of each other. (3) The short direct repeats within a are essentially a large loop with little secondary structure. The potential implications of this structure are discussed and a model for HSV DNA replication is presented.     

A novel 165-base-pair terminal repeat sequence is the sole cis requirement for the adeno-associated virus life cycle.

Adeno-associated virus (AAV) replication is dependent on two copies of a 145-bp inverted terminal repeat (ITR) that flank the AAV genome. This is the primary cis-acting element required for productive infection and the generation of recombinant AAV (rAAV) vectors. We have engineered a plasmid (pDD-2) containing only 165 bp of AAV sequence: two copies of the D element, a unique sequence adjacent to the AAV nicking site, flanking a single ITR. When assayed in vivo, this modified hairpin was sufficient for the replication of the plasmid vector when Rep and adenovirus (Ad) helper functions were supplied in trans. pDD-2 replication intermediates were characteristic of the AAV replication scheme in which linear monomer, dimer, and other higher-molecular-weight replicative intermediates are generated. Compared to infectious AAV clones for replication, the modified hairpin vector replicated more efficiently independent of size. Further analysis demonstrated conversion of the input circular plasmid to a linear substrate with AAV terminal repeat elements at either end as an initial step for replication. This conversion was independent of both Rep and Ad helper genes, suggesting the role of host factors in the production of these molecules. The generation of these substrates suggested resolution of the modified terminal repeat through a Holliday-like structure rather than replication as a mechanism for rescue. Production of replicative intermediates via this plasmid substrate were competent not only for AAV DNA replication but also for encapsidation, infection, integration, and subsequent rescue from the chromosome when superinfected with Ad and wild-type AAV. These studies demonstrate that this novel 165-bp ITR substrate is sufficient in cis for the AAV life cycle and should provide a valuable reagent for further dissecting the cis sequences involved in AAV replication, packaging, and integration. In addition, this novel plasmid vector can be used as a substrate for both rAAV vector production and synthetic plasmid vector delivery.     

DNA nucleotide sequence analysis of the immediate-early gene of pseudorabies virus.

The complete DNA sequence coding for the immediate-early protein (IE180) of pseudorabies virus was determined. The coding region of IE180 is 4380 nucleotides for 1460 amino acid residues. G+C content of the non-coding portion of the IE gene is 70.3% while the G+C content of the coding portion is considerably higher at 80.1%. Correspondingly, codons consisting mainly of Gs and Cs are favoured. Clusters of amino acid homologies are observed among IE180 of pseudorabies virus, ICP4 of herpes simplex virus type-1 and IE140 of varicella-zoster virus, and are organized similarly in all three polypeptides. Functions exhibited by IE180 are assigned, tentatively, to structural domains of the molecule by analogy to the HSV-1 ICP4 polypeptide.     

Factors that bind to adeno-associated virus terminal repeats.

We have identified and characterized a DNA-protein complex that forms with the adeno-associated virus (AAV) terminal repeats. The complex formed only if the terminal palindrome was in the covalently closed or hairpin configuration; little if any binding was detected with the open duplex form of the terminal repeat. This fact suggested that both secondary structure and primary sequence are essential elements of recognition. DNase I protection studies indicated that virtually all of the A-A' palindrome and significant portions of the B-B' and C-C' palindromes are protected. The postulated terminal resolution site of AAV also is protected. Restriction mapping of the sequences necessary for binding indicated that almost all of the terminal palindrome must be present for binding to occur. Hairpins which are similar in size and shape to the AAV termini did not exhibit competition for binding, and the complex formed only if AAV-infected extracts were used. Thus, the binding reaction is specific for AAV sequences. The viral-coded nonstructural proteins Rep78 and Rep68 comigrated with the DNA-protein complex on neutral acrylamide gels, suggesting that one or both of these proteins are components of the complex. The characteristics of the complex suggested that it has a role in AAV DNA replication.     

Evidence of a repetitive sequence in vaccinia virus DNA.

Analysis of vaccinia DNA by reassociation kinetics revealed that 7% of the genome contains a sequence repeated 10 times. This sequence does not contain any host cell DNA, is viral specific, and is found in virions passed at either high or low multiplicities of infection.     

The nucleotide sequence of cloned wheat dwarf virus DNA

Restriction analysis and cloning of virus-specific double-stranded DNA isolated from plants infected with wheat dwarf virus (WDV) indicated that the virus genome, like that of maize streak virus (MSV), consists of a single DNA circle. The complete nucleotide sequence of cloned WDV DNA (2749 nucleotides) has been determined. Comparison of the potential coding regions in WDV DNA with those in the DNA of two strains of MSV suggests that these viruses encode at least two functional proteins, the coat protein read in the virion (+) DNA sense and a composite protein, formed from two open reading regions, in the complementary (-) DNA sense. Although WDV and MSV are serologically unrelated their coat proteins showed 35% direct amino acid sequence and their DNAs showed 46% nucleotide sequence homology. There was too little homology between the DNAs of WDV and those of two geminiviruses with bipartite genomes, cassava latent virus (CLV) and tomato golden mosaic virus (TGMV), to align the sequences. However comparison of the amino acid sequences of predicted proteins of WDV, MSV, TGMV and CLV revealed clear relationships between these viruses and suggested that the monopartite and the bipartite geminiviruses have a common ancestral origin. Four inverted repeat sequences which have the potential to form hairpin structures of deltaG >/= -14 kcal/mol were detected in WDV DNA. The sequence TAATATTAC present in the loop of one of these hairpins is conserved in similar putative structures in MSV DNA and in both DNA components of CLV and TGMV and may function as a recognition sequence for a protein involved in virus DNA replication.     

Influence of nucleotide sequence adjacent to duplex DNA termini on 3'' terminal labeling by terminal transferase.

We have analyzed the effect of base-pairing at the exposed ends of Hinc II fragments of SV40 DNA on the efficiency of ribonucleotide incorporation catalyzed by terminal transferase. Wide variations in the labeling efficiency of individual DNA fragments have been observed. To elucidate the nature of this variation at the molecular level, we have correlated this effect with nucleotide sequence adjacent to the cleavage site. We found that a G:C base pair right at the exposed end drastically reduces the incorporation of ribonucleotides. Furthermore, the higher the number of G:C base pairs adjacent to the exposed end, the greater the reduction in labeling efficiency. From these results, we conclude that the labeling efficiency is determined by the degree of 'terminal breathing' of the DNA molecule at the exposed end of the duplex.     

Rescue of adeno-associated virus from recombinant plasmids: gene correction within the terminal repeats of AAV

We have isolated three types of pBR322-AAV recombinant plasmids that contain deletions within the 145 bp AAV terminal repeats. When the plasmids were transfected into human cells, mutants that contained deletions within the left (type I) or right (type II) terminal repeat were viable. Of four mutants examined that contained deletions in both termini (type III), only one was viable. All of the viable mutants produced AAV virions that contained wild-type AAV DNA. Furthermore, the viable type III deletion could be converted to a nonviable mutant by deleting all copies of an 11 bp sequence from its termini. We conclude that there is an efficient mechanism for correcting deletions within the AAV termini. A model that could account for these observations is also discussed.