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.     

Evidence for two nucleotide sequence orientations within the terminal repetition of adeno-associated virus DNA.

Duplex adeno-associated virus (AAV) DNA, produced by annealing plus and minus virion single strands, has been digested with several bacterial restriction endonucleases. These studies reveal the existence of alternate secondary structures at the termini of duplex AAV DNA. Analysis of the sites of endo R-Hpa II cleavage, the products of complete endo R-Hpa II digestion, and the multiple terminal secondary structures leads to the conclusion that there are two possible nucleotide sequences at each end of AAV DNA. A model that attributes the terminal nucleotide sequence heterogeneity to two possible orientations of the first 120 nucleotides at each end of the DNA is proposed; in one case the sequence is 1 to 120; in the other case the sequence is inverted. An origin of the inversion is suggested based on previously described intermediates in AAV DNA replication.     

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.     

Adeno-associated virus type 2 DNA replication in vivo: mutation analyses of the D sequence in viral inverted terminal repeats.

The adeno-associated virus type 2 (AAV) genome contains inverted terminal repeats (ITRs) of 145 nucleotides. The terminal 125 nucleotides of each ITR form palindromic hairpin (HP) structures that serve as primers for AAV DNA replication. These HP structures also play an important role in integration as well as rescue of the proviral genome from latently infected cells or from recombinant AAV plasmids. Each ITR also contains a stretch of 20 nucleotides, designated the D sequence, that is not involved in HP structure formation. We have recently shown that the D sequence plays a crucial role in high-efficiency rescue, selective replication, and encapsidation of the AAV genome and that a host cell protein, designated the D sequence-binding protein (D-BP), specifically interacts with this sequence (X.-S. Wang, S. Ponnazhagan, and A. Srivastava, J. Virol. 70:1668-1677, 1996). We have now performed mutational analyses of the D sequences to evaluate their precise role in viral DNA rescue, replication, and packaging. We report here that 10 nucleotides proximal to the HP structure in each of the D sequences are necessary and sufficient to mediate high-efficiency rescue, replication, and encapsidation of the viral genome in vivo. In in vitro studies, the same 10 nucleotides were found to be required for specific interaction with D-BP, but viral Rep protein-mediated cleavage at the functional terminal resolution site is independent of these sequences. These data suggest that AAV replication and terminal resolution functions can be uncoupled and that the lack of efficient replication of AAV DNA may not be a consequence of impaired resolution of the viral ITRs. These studies further illustrate that the D sequence-D-BP interaction plays an important role in the AAV life cycle and indicate that it may be possible to develop the next generation of AAV vectors capable of encapsidating larger pieces of DNA.     

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.     

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.     

Regions of the terminal repetitions of the herpes simplex virus type 1 genome. Relationship to immunoglobulin switch-like DNA sequences

Several recombinant clones isolated from a mouse genomic library were previously shown to hybridize with a SmaI fragment located in the terminal repetition of the S component of herpes simplex virus DNA. We report here the nucleotide sequence of the related regions in two mouse clones, TGL19 and TGL35, as well as that of the SmaI fragment of HSV-1. The mouse DNA clones have a core of repetitive sequences 80% homologous to a tandem repeat (reiteration II) in the viral fragment. The regions of homology are in turn related to immunoglobulin class-switch sequences, due mostly to the presence of the pentamer TGGG(G), involved in class-switch recombination. These results suggest that the HSV genome has recombination sequences identical to those of the host cell and provide a possible explanation for the high frequency of recombination events observed in this region of the viral genome.     

Sequences in the 5′ Nontranslated Region of Hepatitis C Virus Required for RNA Replication

Sequences in the 5' and 3' termini of plus-strand RNA viruses harbor cis-acting elements important for efficient translation and replication. In case of the hepatitis C virus (HCV), a plus-strand RNA virus of the family Flaviviridae, a 341-nucleotide-long nontranslated region (NTR) is located at the 5' end of the genome. This sequence contains an internal ribosome entry site (IRES) that is located downstream of an about 40-nucleotide-long sequence of unknown function. By using our recently developed HCV replicon system, we mapped and characterized the sequences in the 5' NTR required for RNA replication. We show that deletions introduced into the 5' terminal 40 nucleotides abolished RNA replication but only moderately affected translation. By generating a series of replicons with HCV-poliovirus (PV) chimeric 5' NTRs, we could show that the first 125 nucleotides of the HCV genome are essential and sufficient for RNA replication. However, the efficiency could be tremendously increased upon the addition of the complete HCV 5' NTR. These data show that (i) sequences upstream of the HCV IRES are essential for RNA replication, (ii) the first 125 nucleotides of the HCV 5' NTR are sufficient for RNA replication, but such replicon molecules are severely impaired for multiplication, and (iii) high-level HCV replication requires sequences located within the IRES. These data provide the first identification of signals in the 5' NTR of HCV RNA essential for replication of this virus.     

In vitro rescue of an integrated hybrid adeno-associated virus/simian virus 40 genome.

In an in vitro simian virus 40 (SV40) DNA replication assay, we have observed excision of a hybrid adeno-associated virus (AAV)/SV40 insert from a plasmid construct. The excision was dependent on the presence of the palindromic AAV terminal repeat and greatly enhanced by the addition of the SV40 T antigen to the reaction. Analysis of the excision product supports a model in which the palindromic terminal sequences of AAV form a cruciform structure (equivalent to a Holliday recombination intermediate), which is cleaved and resealed so that the excision products are linear duplex pBR322 and linear duplex AAV/SV40 insert. Both the excised linear duplex pBR322 and the excised linear duplex AAV/SV40 insert have each terminus covalently crosslinked by one copy of the palindromic region of the AAV terminal repeat region folded on itself. The excision process may be a model system for cellular homologous recombination. The process as observed was either concomitant with or subsequent to DNA replication.     

A novel terminal resolution-like site in the adeno-associated virus type 2 genome.

The adeno-associated virus 2 (AAV) contains a single-stranded DNA genome of which the terminal 145 nucleotides are palindromic and form T-shaped hairpin structures. These inverted terminal repeats (ITRs) play an important role in AAV DNA replication and resolution, since each of the ITRs contains a terminal resolution site (trs) that is the target site for the AAV rep gene products (Rep). However, the Rep proteins also interact with the AAV DNA sequences that lie outside the ITRs, and the ITRs also play a crucial role in excision of the proviral genome from latently infected cells or from recombinant AAV plasmids. To distinguish between Rep-mediated excision of the viral genome during rescue from recombinant AAV plasmids and the Rep-mediated resolution of the ITRs during AAV DNA replication, we constructed recombinant AAV genomes that lacked either the left or the right ITR sequence and one of the Rep-binding sites (RBSs). No rescue and replication of the AAV genome occurred from these plasmids following transfection into adenovirus type 2-infected human KB cells, as expected. However, excision and abundant replication of the vector sequences was clearly detected from the plasmid that lacked the AAV left ITR, suggesting the existence of an additional putative excision site in the left end of the AAV genome. This site was precisely mapped to one of the AAV promoters at map unit 5 (AAV p5) that also contains an RBS. Furthermore, deletion of this RBS abolished the rescue and replication of the vector sequences. These studies suggest that the Rep-mediated cleavage at the RBS during viral DNA replication may, in part, account for the generation of the AAV defective interfering particles.     

Sequence of inverted terminal repetitions from different adenoviruses: demonstration of conserved sequences and homology between SA7 termini and SV40 DNA.

We have established the nucleotide sequence for the inverted terminal repetition of human adenovirus type 3, a subgroup B adenovirus. The repetition, which is 136 bp long, shows a high degree of homology with the known sequence for the inverted repetition of adenovirus type 5 (Steenbergh et al., 1977) a subgroup C adenovirus. Partial sequence information convering 120 bp of the inverted terminal repetitions of human serotype 12, a subgroup A member, and of simian adenovirus type 7 has also been obtained. A comparison of the established sequences shows that the terminal repetitions, in particular the first 50 bp from the ends, contain sequences that have been well conserved in adenovirus evolution. For instance, only six mismatched base pairs were detected among the first 50 bp in the repetitions of simian adenovirus type 7 and human adenovirus type 5, although the homology between simian adenovirus 7 and human subgroup C adenoviruses was estimated to be only 30%. A 14 bp sequence located 9-22 nucleotides from the ends is present in DNAs from all the human serotypes examined as well as in simian adenovirus 7 DNA. Furthermore, the simian adenovirus 7 repetition contains a 21 bp sequence which is present in SV40 DNA, close to the origin of DNA replication.     

Template requirements for in vivo replication of adenovirus DNA.

The adenovirus (Ad) DNA origin of replication was defined through an analysis of the DNA sequences necessary for the replication of plasmid DNAs with purified viral and cellular proteins. Results from several laboratories have shown that the origin consists of two functionally distinct domains: a 10-base-pair sequence present in the inverted terminal repetition (ITR) of all human serotypes and an adjacent sequence constituting the binding site for a cellular protein, nuclear factor I. To determine whether the same nucleotide sequences are necessary for origin function in vivo, we developed an assay for the replication of plasmid DNAs transfected into Ad5-infected cells. The assay is similar to that described by Hay et al. (J. Mol. Biol. 175:493-510, 1984). With this assay, plasmid DNA replication is dependent upon prior infection of cells with virus and only occurs with linear DNA molecules containing viral terminal sequences at each end. Replicated DNA is resistant to digestion with lambda-exonuclease, suggesting that a protein is covalently bound at both termini. A plasmid containing only the first 67 base pairs of the Ad2 ITR replicates as well as plasmids containing the entire ITR. Deletions or point mutations which reduce the binding of nuclear factor I to DNA in vitro reduce the efficiency of plasmid replication in vivo. A point mutation within the 10-base-pair conserved sequence has a similar effect upon replication. These results suggest that the two sequence domains of the Ad origin identified by in vitro studies are in fact important for viral DNA replication in infected cells. In addition, we found that two separate point mutations which lie outside these two sequence domains, and which have little or no effect upon DNA replication in vitro, also reduce the apparent efficiency of plasmid replication in vivo. Thus, there may be elements of the Ad DNA origin of replication which have not yet been identified by in vitro studies.     

Analysis of the terminal repeat binding abilities of mutant adeno-associated virus replication proteins.

The adeno-associated virus (AAV) Rep78 and Rep68 proteins play essential roles in viral DNA replication, trans activation of viral gene expression, and suppression of oncogene-mediated cellular transformation. By using an extensive set of linker insertion and deletion mutations in the replication gene, we mapped the regions of the Rep78 protein that mediate binding to the AAV origin of replication in vitro. Deletions that removed amino acid codons 25 to 62, 88 to 113, 125 to 256, and 346 to 400 abolished binding. Alterations in several other regions of the protein affected the binding affinity of the mutant proteins. All of the mutant proteins that support AAV DNA replication or p40 trans activation bound to the terminal repeat sequence, thus verifying the importance of binding for these functions. Several mutant rep genes that failed to suppress oncogene-mediated cellular transformation produced proteins that were capable of binding to the AAV terminal repeat sequences.     

Structure of the 3' hairpin termini of four rodent parvovirus genomes: nucleotide sequence homology at origins of DNA replication.

The nucleotide sequences of the 3' termini of the DNA from four autonomous rodent parvoviruses have been determined. The terminus of each genome exists as a Y-shaped hairpin structure involving 115 or 116 nucleotides. The sequence of this region of DNA is highly conserved and shows no evidence of internal sequence heterogeneity, a characteristic which is observed in the terminal nucleotide sequence of the helper-dependent, adeno-associated viruses (Berns et al., 1978a). The implications of these results with respect to the models of parvovirus DNA replication are discussed.     

The adeno-associated virus rep gene inhibits replication of an adeno-associated virus/simian virus 40 hybrid genome in cos-7 cells.

A hybrid adeno-associated virus (AAV)/simian virus 40 (SV40) genome is described. In this construct SV40 regulatory sequences, including the early promoter/enhancers and origin of DNA replication, were substituted for the AAV p5 promoter, which normally controls expression of the AAV rep gene. The hybrid genome was phenotypically indistinguishable from wild-type AAV in human cells in the presence or absence of helper virus. Upon transfection into cos-7 cells, which constitutively produced the SV40 tumor antigen, the genome replicated as a plasmid when the SV40 origin was used, although with a low efficiency compared with that of a non-AAV/SV40 replicon. The low level of replication was due to an inhibitory effect of an AAV rep gene product and was specific for replicons containing AAV sequences. Target AAV sequences required for inhibition by rep appeared to reside in the terminal repetitions since deletion of these sequences allowed efficient replication in the presence of the rep gene. The possible role for negative autoregulation of AAV DNA replication in latent infection and helper-dependent replication by AAV is discussed.     

Adeno-associated virus DNA replication in vitro: activation by a maltose binding protein/Rep 68 fusion protein.

The adeno-associated virus (AAV) nonstructural protein Rep 68 is required for viral DNA replication. An in vitro assay has been developed in which addition of Rep 68 to an extract from uninfected HeLa cells supports AAV DNA replication. In this paper, we report characterization of the replication process when a fusion of the maltose binding protein and Rep 68, expressed in Escherichia coli, was used in the assay. Replication was observed when the template was either linear double-stranded AAV DNA or a plasmid construct containing intact AAV DNA. When the recombinant plasmid construct was used as the template, there was replication of pBR322 DNA as well as the AAV DNA; however, linear pBR322 DNA was not replicated. When the plasmid construct was the template, replication appeared to initiate on the intact plasmid and led to separation of the AAV sequences from those of the vector, a process which has been termed rescue. There was no evidence that replication could initiate on the products of rescue. Rep 68 can make a site-specific nick 124 nucleotides from the 3' end of AAV DNA; the site of the nick has been called the terminal resolution site. Our data are most consistent with initiation occurring at the terminal resolution site and proceeding toward the 3' terminus. When the template was the plasmid construct, either elongation continued past the junction into pBR322 sequences or the newly synthesized sequence hairpinned, switched template strands, and replicated the AAV DNA. Replication was linear for 4 h, during which time 70% of the maximal synthesis took place. An additional finding was that the Rep fusion could resolve AAV dimer length duplex intermediates into monomer duplexes without DNA synthesis.     

Adeno-associated virus general transduction vectors: analysis of proviral structures.

We used two kinds of adeno-associated virus (AAV) vectors to transduce the neomycin resistance gene into human cells. The first of these (dl52-91) retains the AAV rep genes; the second (dl3-94) retains only the AAV terminal repeats and the AAV polyadenylation signal (428 base pairs). Both vectors could be packaged into AAV virions and produced proviral structures that were essentially the same. Thus, the AAV sequences that are required in cis for packaging (pac), integration (int), rescue (res), and replication (ori) of viral DNA are located within a 284-base-pair sequence that includes the terminal repeat. Most of the G418r cell lines (73%) contained proviruses which could be rescued (Res+) when the cells were superinfected with the appropriate helper viruses. Some produced high yields of viral DNA; other rescued at a 50-fold lower level. Most of the lines that were Res+ (79%) contained a tandem repeat of the AAV genome (2 to 20 copies) which was integrated randomly with respect to cellular DNA. Junctions between two consecutive AAV copies in a tandem array contained either one or two copies of the AAV terminal palindrome. Junctions between AAV and cellular sequences occurred predominantly at or within the AAV terminal repeat, but in some cases at internal AAV sequences. Two lines were seen that contained free episomal copies of AAV DNA. Res+ clones contained deleted proviruses or tandem repeats of a deleted genome. Occasionally, flanking cellular DNA was also amplified. There was no superinfection inhibition of AAV DNA integration. Our results suggest that AAV sequences are amplified by DNA replication either before or after integration and that the mechanism of replication is different from the one used during AAV lytic infections. In addition, we have described a new AAV general transduction vector, dl3-94, which provides the maximum amount of room for insertion of foreign DNA and integrates at a high frequency (80%).     

A recombinant plasmid from which an infectious adeno-associated virus genome can be excised in vitro and its use to study viral replication.

A recombinant plasmid carrying an infectious adeno-associated viral genome was constructed that differs in several key respects from previously described recombinants. First, the vector is pEMBL8(+), which allows isolation of viral plus and minus strands. Second, the inserted viral sequences contain two XbaI cleavage sites that flank the viral coding domain. These inserts do not affect replication of the virus, and they allow nonviral sequences to be easily inserted between the cis-acting terminal repeats of adeno-associated virus. Third, the viral genome is flanked by PvuII cleavage sites that allow the entire, infectious viral chromosome to be excised from plasmid sequences in vitro. Viral DNA was replicated more efficiently within adenovirus-infected 293 cells if it was excised from the vector with PvuII before transfection. Presumably, the increased efficiency reflects bypass of the excision step which must normally precede replication when a recombinant plasmid enters the nucleus. The ability to bypass the excision step was exploited to search for a viral function required specifically for excision of the viral genome from the integrated state. None of the mutants tested identified a gene product required for excision that was not also essential for replication. The ability to produce pure populations of viral plus and minus strands was used to demonstrate that both strands are infectious.     

Two major DNA variants present in serially propagated stocks of the WR strain of vaccinia virus.

Two major DNA variants have been isolated from serially propagated stocks of the WR strain of vaccinia virus. Restriction enzyme mapping of the two variants with HindIII, AvaI, XhoI, SstII, and SmaI revealed a 6.3-megadalton deletion in the smaller DNA variant. The deletion was mapped at ca. 6.8 megadaltons in from the left terminus, just beyond the inverted terminal repeat. The additional DNA present in the larger variant was found to represent unique viral sequences that were transcribed both in vitro and in vivo. One-step growth curves in HeLa cells revealed no difference in rat of replication or burst size when progeny was scored on CV-1 monolayers.