scholarly journals Rescue of the Adeno-Associated Virus Genome from a Plasmid Vector: Evidence for Rescue by Replication

2003 ◽  
Vol 77 (21) ◽  
pp. 11480-11490 ◽  
Author(s):  
Peter Ward ◽  
Per Elias ◽  
R. Michael Linden
Keyword(s):  
Cultured Cells ◽  
Plasmid Vector ◽  
Virus Genome ◽  
Helper Virus ◽  
Repeat Sequence ◽  
Inverted Terminal Repeat ◽  
Chromosome 19 ◽  
Adeno Associated Virus ◽  
Inverted Terminal Repeat Sequence ◽  
Simplex Virus

ABSTRACT In cultured cells, adeno-associated virus (AAV) replication requires coinfection with a helper virus, either adenovirus or herpesvirus. In the absence of helper virus coinfection AAV can integrate its genome site specifically into the AAVS1 region of chromosome 19. Upon subsequent infection with a helper virus, the AAV genome is released from chromosome 19 by a process termed rescue, and productive replication ensues. The AAV genome cloned into a plasmid vector can also serve to initiate productive AAV replication. When such constructs are transfected into cells and those cells are simultaneously or subsequently infected with a helper virus, the AAV genome is released from the plasmid. This process is thought to serve as a model for rescue from the human genomic site. In this report we present a model for rescue of AAV genomes by replication. A hallmark of this model is the production of a partially single-stranded and partially double-stranded molecule. We show that the AAV2 Rep 68 protein, together with the UL30/UL42 herpes simplex virus type 1 DNA polymerase and the UL29 single-strand DNA binding protein ICP8, is sufficient to efficiently and precisely rescue AAV from a plasmid in a way that is dependent on the AAV inverted terminal repeat sequence.

scholarly journals Adeno-Associated Virus Serotype-Specific Inverted Terminal Repeat Sequence Role in Vector Transgene Expression

2020 ◽  
Vol 31 (3-4) ◽  
pp. 151-162 ◽  
Author(s):  
Lauriel F. Earley ◽  
Laura M. Conatser ◽  
Victoria M. Lue ◽  
Amanda L. Dobbins ◽  
Chengwen Li ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Repeat Sequence ◽  
Terminal Repeat ◽  
Inverted Terminal Repeat ◽  
Adeno Associated Virus ◽  
Inverted Terminal Repeat Sequence ◽  
Virus Serotype ◽  
Terminal Repeat Sequence

scholarly journals Herpes simplex virus co-infection facilitates rolling circle replication of the adeno-associated virus genome

2021 ◽  
Vol 17 (6) ◽  
pp. e1009638
Author(s):  
Anita Felicitas Meier ◽  
Kurt Tobler ◽  
Remo Leisi ◽  
Anouk Lkharrazi ◽  
Carlos Ros ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Helper Virus ◽  
Inverted Terminal Repeat ◽  
Genome Replication ◽  
Rolling Circle Replication ◽  
Adeno Associated Virus ◽  
Rolling Circle ◽  
Simplex Virus ◽  
Hsv 1

Adeno-associated virus (AAV) genome replication only occurs in the presence of a co-infecting helper virus such as adenovirus type 5 (AdV5) or herpes simplex virus type 1 (HSV-1). AdV5-supported replication of the AAV genome has been described to occur in a strand-displacement rolling hairpin replication (RHR) mechanism initiated at the AAV 3’ inverted terminal repeat (ITR) end. It has been assumed that the same mechanism applies to HSV-1-supported AAV genome replication. Using Southern analysis and nanopore sequencing as a novel, high-throughput approach to study viral genome replication we demonstrate the formation of double-stranded head-to-tail concatemers of AAV genomes in the presence of HSV-1, thus providing evidence for an unequivocal rolling circle replication (RCR) mechanism. This stands in contrast to the textbook model of AAV genome replication when HSV-1 is the helper virus.

scholarly journals Integrating Adenovirus–Adeno-Associated Virus Hybrid Vectors Devoid of All Viral Genes

1999 ◽  
Vol 73 (11) ◽  
pp. 9314-9324 ◽  
Author(s):  
André Lieber ◽  
Dirk S. Steinwaerder ◽  
Cheryl A. Carlson ◽  
Mark A. Kay
Keyword(s):  
First Generation ◽  
Cultured Cells ◽  
Inverted Terminal Repeat ◽  
Stable Gene ◽  
Adeno Associated Virus ◽  
Transgene Integration ◽  
Repeat Sequences ◽  
Hybrid Vectors ◽  
Viral Genes

ABSTRACT Recently, we demonstrated that inverted repeat sequences inserted into first-generation adenovirus (Ad) vector genomes mediate precise genomic rearrangements resulting in vector genomes devoid of all viral genes that are efficiently packaged into functional Ad capsids. As a specific application of this finding, we generated adenovirus–adeno-associated virus (AAV) hybrid vectors, first-generation Ad vectors containing AAV inverted terminal repeat sequences (ITRs) flanking a reporter gene cassette inserted into the E1 region. We hypothesized that the AAV ITRs present within the hybrid vector genome could mediate the formation of rearranged vector genomes (ΔAd.AAV) and stimulate transgene integration. We demonstrate here that ΔAd.AAV vectors are efficiently generated as by-products of first-generation adenovirus-AAV vector amplification. ΔAd.AAV genomes contain only the transgene flanked by AAV ITRs, Ad packaging signals, and Ad ITRs. ΔAd.AAV vectors can be produced at a high titer and purity. In vitro transduction properties of these deleted hybrid vectors were evaluated in direct comparison with first-generation Ad and recombinant AAV vectors (rAAVs). The ΔAd.AAV hybrid vector stably transduced cultured cells with efficiencies comparable to rAAV. Since cells transduced with ΔAd.AAV did not express cytotoxic viral proteins, hybrid viruses could be applied at very high multiplicities of infection to increase transduction rates. Southern analysis and pulsed-field gel electrophoresis suggested that ΔAd.AAV integrated randomly as head-to-tail tandems into the host cell genome. The presence of two intact AAV ITRs was crucial for the production of hybrid vectors and for transgene integration. ΔAd.AAV vectors, which are straightforward in their production, represent a promising tool for stable gene transfer in vitro and in vivo.

scholarly journals Cotransfection with adenovirus DNA enhances transcription from linear DNA containing eucaryotic promoters.

1987 ◽  
Vol 7 (3) ◽  
pp. 1063-1069
Author(s):  
M B Vasudevachari ◽  
V Natarajan ◽  
N P Salzman
Keyword(s):  
Rna Synthesis ◽  
Chloramphenicol Acetyltransferase ◽  
Repeat Sequence ◽  
Early Gene ◽  
Inverted Terminal Repeat ◽  
Adenovirus E1a ◽  
293 Cells ◽  
Inverted Terminal Repeat Sequence ◽  
Inverted Terminal Repeats ◽  
Terminal Repeats

Linear DNAs, containing a copy of the adenovirus serotype 2 (Ad2) inverted terminal repeat sequence at each end, replicate in 293 cells when cotransfected with Ad2 DNA (Hay et al., J. Mol. Biol. 175:493-510, 1984). We have linked either the Ad2 IVa2 promoter (IVa2) or major late promoter (MLP) to the chloramphenicol acetyltransferase gene and inserted this DNA into such a plasmid (pARKR) between its two inverted terminal repeats. These recombinant plasmids were linearized and then used to transfect 293 cells in the presence or absence of Ad2 helper DNA. Synthesis of IVa2 and MLP RNAs, and production of chloramphenicol acetyltransferase was increased dramatically when the Ad2 DNA was included. However, unlike the patterns of temporal regulation which are seen during a cycle of virus replication when these genes are contained within the virion, there was no obvious difference in the timing of RNA synthesis from plasmid IVa2 or MLP after cotransfection. When linearized plasmids containing IVa2 and MLP sequences but lacking inverted terminal repeats at their ends (replication deficient plasmids) were used for transfection, an increase in RNA synthesis from IVa2 or MLP was also observed and similarly required cotransfection with Ad2 DNA. When HeLa cells, which do not constitutively express the adenovirus E1a gene, were cotransfected with linearized plasmids and adenovirus DNA that lacks the E1a region (H5dl312), a stimulation of transcription was also observed, although it was less than the level observed with wild-type DNA. The results of the present study demonstrate that an early gene product(s) besides E1a functions in trans to regulate transcription.

scholarly journals Identification of Rep-Associated Factors in Herpes Simplex Virus Type 1-Induced Adeno-Associated Virus Type 2 Replication Compartments

2010 ◽  
Vol 84 (17) ◽  
pp. 8871-8887 ◽  
Author(s):  
Armel Nicolas ◽  
Nathalie Alazard-Dany ◽  
Coline Biollay ◽  
Loredana Arata ◽  
Nelly Jolinon ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Virus Type ◽  
Herpes Simplex Virus Type ◽  
Particle Production ◽  
Helper Virus ◽  
Adeno Associated Virus ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Adeno-associated virus (AAV) is a human parvovirus that replicates only in cells coinfected with a helper virus, such as adenovirus or herpes simplex virus type 1 (HSV-1). We previously showed that nine HSV-1 factors are able to support AAV rep gene expression and genome replication. To elucidate the strategy of AAV replication in the presence of HSV-1, we undertook a proteomic analysis of cellular and HSV-1 factors associated with Rep proteins and thus potentially recruited within AAV replication compartments (AAV RCs). This study resulted in the identification of approximately 60 cellular proteins, among which factors involved in DNA and RNA metabolism represented the largest functional categories. Validation analyses indicated that the cellular DNA replication enzymes RPA, RFC, and PCNA were recruited within HSV-1-induced AAV RCs. Polymerase δ was not identified but subsequently was shown to colocalize with Rep within AAV RCs even in the presence of the HSV-1 polymerase complex. In addition, we found that AAV replication is associated with the recruitment of components of the Mre11/Rad50/Nbs1 complex, Ku70 and -86, and the mismatch repair proteins MSH2, -3, and -6. Finally, several HSV-1 factors were also found to be associated with Rep, including UL12. We demonstrated for the first time that this protein plays a role during AAV replication by enhancing the resolution of AAV replicative forms and AAV particle production. Altogether, these analyses provide the basis to understand how AAV adapts its replication strategy to the nuclear environment induced by the helper virus.

scholarly journals Packaging of Human Chromosome 19-Specific Adeno-Associated Virus (AAV) Integration Sites in AAV Virions during AAV Wild-Type and Recombinant AAV Vector Production

2003 ◽  
Vol 77 (8) ◽  
pp. 4881-4887 ◽  
Author(s):  
Daniela Hüser ◽  
Stefan Weger ◽  
Regine Heilbronn
Keyword(s):  
Human Chromosome ◽  
Helper Virus ◽  
Productive Infection ◽  
Wild Type ◽  
Chromosome 19 ◽  
Aav Vector ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Site Specific Integration ◽  
Human Chromosome 19

ABSTRACT Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus on human chromosome 19, called AAVS1. During the development of a sensitive real-time PCR assay for site-specific integration, AAV-AAVS1 junctions were reproducibly detected in highly purified AAV wild-type and recombinant AAV vector stocks. A series of controls documented that the junctions were packaged in AAV capsids and were newly generated during a single round of AAV production. Cloned junctions displayed variable AAV sequences fused to AAVS1. These data suggest that packaged junctions represent footprints of AAV integration during productive infection. Apparently, AAV latency established by site-specific integration and the helper virus-dependent, productive AAV cycle are more closely related than previously thought.

scholarly journals Characterization of Adeno-Associated Virus Genomes Isolated from Human Tissues

2005 ◽  
Vol 79 (23) ◽  
pp. 14793-14803 ◽  
Author(s):  
Bruce C. Schnepp ◽  
Ryan L. Jensen ◽  
Chun-Liang Chen ◽  
Philip R. Johnson ◽  
K. Reed Clark
Keyword(s):  
Cultured Cells ◽  
Rolling Circle Amplification ◽  
Chromosome 1 ◽  
Inverted Terminal Repeat ◽  
Human Tissues ◽  
Wild Type ◽  
Tissue Samples ◽  
Adeno Associated Virus ◽  
Rolling Circle

ABSTRACT Infection with wild-type adeno-associated virus (AAV) is common in humans, but very little is known about the in vivo biology of AAV. On a molecular level, it has been shown in cultured cells that AAV integrates in a site-specific manner on human chromosome 19, but this has never been demonstrated directly in infected human tissues. To that end, we tested 175 tissue samples for the presence of AAV DNA, and when present, examined the specific form of the viral DNA. AAV was detected in 7 of 101 tonsil-adenoid samples and in 2 of 74 other tissue samples (spleen and lung). In these nine samples, we were unable to detect AAV integration in the AAVS1 locus using a sensitive PCR assay designed to amplify specific viral-cellular DNA junctions. Additionally, we used a second complementary assay, linear amplification-mediated-PCR (LAM-PCR) to widen our search for integration events. Analysis of individual LAM-PCR products revealed that the AAV genomes were arranged predominantly in a head-to-tail array, with deletions and extensive rearrangements in the inverted terminal repeat sequences. A single AAV-cellular junction was identified from a tonsil sample and it mapped to a highly repetitive satellite DNA element on chromosome 1. Given these data, we entertained the possibility that instead of integrated forms, AAV genomes were present as extrachromosomal forms. We used a novel amplification assay (linear rolling-circle amplification) to show that the majority of wild-type AAV DNA existed as circular double-stranded episomes in our tissues. Thus, following naturally acquired infection, AAV DNA can persist mainly as circular episomes in human tissues. These findings are consistent with the circular episomal forms of recombinant AAV vectors that have been isolated and characterized from in vivo transduced tissues.

scholarly journals Adeno-Associated Virus Type 2 p5 Promoter: a Rep-Regulated DNA Switch Element Functioning in Transcription, Replication, and Site-Specific Integration

2007 ◽  
Vol 81 (8) ◽  
pp. 3721-3730 ◽  
Author(s):  
Mary Murphy ◽  
Janette Gomos-Klein ◽  
Marko Stankic ◽  
Erik Falck-Pedersen
Keyword(s):  
Virus Type ◽  
Transcriptional Control ◽  
Helper Virus ◽  
Inverted Terminal Repeat ◽  
Sequence Element ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Site Specific Integration ◽  
Rep Proteins

ABSTRACT The large Rep proteins, p68 and p78, function as master controllers of the adeno-associated virus type 2 (AAV2) life cycle, involved in transcriptional control, in latency, in rescue, and in viral DNA replication. The p5 promoter may be the nucleic acid complement to the large Rep proteins. It drives expression of the large Rep proteins, it undergoes autoregulation by Rep, it undergoes induction by helper virus, it is a target substrate for Rep-mediated site-specific integration (RMSSI), and it can function as a replicative origin. To better understand the relationship between each of the p5 functions, we have determined the effects of p5 promoter mutations (p5 integration efficiency element, or p5IEE) on transcription, integration, and replication using RMSSI transfection protocols in HeLa cells. The data demonstrate that the organization of the p5 promoter provides a unique platform for regulated AAV2 template transcription and subsequent repression by Rep through direct and indirect mechanisms. The elements of the p5IEE that define its function as a promoter also define its function as a highly optimized substrate for Rep-mediated site-specific integration and replication. The p5 Rep binding element (RBE) is essential in RMSSI and Rep-dependent replication; however, replacement of the p5 RBE with either the AAV2 inverted terminal repeat or the AAVS1 RBE sequence elements neither enhances nor severely compromises RMSSI activity of p5IEE. The RBE by itself or in combination with the YY1+1 initiator/terminal resolution sequence element does not mediate efficient site-specific integration. We found that replication and integration were highly sensitive to sequence manipulations of the p5 TATA/RBE/YY1+1 core structure in a manner that reflects the function of these elements in transcription. The data presented support a model where, depending on the state of the cell (Rep expression and helper virus influences), the p5IEE operates as a transcription/integration switch sequence element.

scholarly journals Involvement of Cellular Double-Stranded DNA Break Binding Proteins in Processing of the Recombinant Adeno-Associated Virus Genome

2001 ◽  
Vol 75 (24) ◽  
pp. 12279-12287 ◽  
Author(s):  
Lorena Zentilin ◽  
Alessandro Marcello ◽  
Mauro Giacca
Keyword(s):  
Homologous Recombination ◽  
Cultured Cells ◽  
Virus Genome ◽  
Nonhomologous End Joining ◽  
Transduction Efficiency ◽  
End Joining ◽  
Adeno Associated Virus ◽  
Vector Genome ◽  
Recombination Pathway

ABSTRACT Unlike postmitotic tissues in vivo, transduction of cultured cells is poor with recombinant adeno-associated virus (rAAV). The ability of rAAV to transduce cells is greatly enhanced by a variety of agents that induce DNA damage and is elevated in cells defective in the ataxia telangiectasia gene product (ATM), showing increased genomic instability. Here we show that DNA double-stranded break (DSB) repair pathways are involved in the regulation of rAAV transduction efficiency. By quantitative chromatin immunoprecipitation, we found that Ku86 and Rad52 proteins associate with viral DNA inside transduced cells. Both proteins are known to competitively recognize hairpin structures and DNA termini and to promote repair of DSBs, the former by facilitating nonhomologous end joining and the latter by initiating homologous recombination. We found that rAAV transduction is increased in Ku86-defective cells while it is inhibited in Rad52 knockout cells. These results suggest that binding of Rad52 to the rAAV genome might be involved in processing of the vector genome through a homologous recombination pathway.

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