Rescue and Replication Signals of the Adeno-associated Virus 2 Genome

Wang, Xu-Shan; Ponnazhagan, Selvarangan; Srivastava, Arun

doi:10.1006/jmbi.1995.0398

Cited by 62 publications

(82 citation statements)

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“…While the origin of the conventional pathway has been well characterized, cis-acting elements that direct replication of circular viral genomes are unknown. Previous studies of cis-acting elements mediating liner AAV replication used duplex wt genomes in the context of a recombinant plasmid (44,45). These studies have helped to identify the major ITR elements that direct AAV replication along the conventional pathway (44)(45)(46).…”

Section: Discussionmentioning

confidence: 99%

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A cis -Acting Element That Directs Circular Adeno-Associated Virus Replication and Packaging

Musatov

Roberts

Pfaff

et al. 2002

J Virol

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A novel pathway of adeno-associated virus (AAV) replication marked by the assembly of circular monomer duplex intermediates (cAAV) has been recently discovered. In the present report we identify a single AD domain of the inverted terminal repeat as a minimal origin of cAAV replication. A small internal palindrome (BB), necessary for optimal Rep-inverted terminal repeat interaction, does not contribute to the efficiency of cAAV replication, while the terminal resolution site is an essential cis-acting element. Furthermore, recombinant cAAV vectors that encompass only the AD domain replicate exclusively in a circular form and no detectable linear duplex replicative intermediates are generated, suggesting that both pathways of AAV replication are independent and can be separated. In addition, we show that cAAVs are efficient templates for encapsidation of single-stranded DNA genomes, an observation that assigns a biological role for these novel replication species. Together, these findings shed new light on the current model of AAV replication and packaging.Adeno-associated virus type 2 (AAV) is a parvovirus nonpathogenic to humans with a genome of approximately 4.7 kb (39). The AAV genome consists of two open reading frames that encode regulatory (Rep) and structural capsid (Cap) proteins flanked by 145-bp inverted terminal repeats (ITR) (39). These ITRs are the only cis-acting elements necessary for virus replication and encapsidation. Recombinant AAVs (rAAV) that do not contain any endogenous coding regions efficiently propagate when Rep and Cap are provided in trans (2, 3). In nature, a secondary infection with helper virus, e.g., adenovirus, is necessary to trigger a productive infection. AAV genomes then undergo replication followed by assembly of infectious virions containing single-stranded DNA (ssDNA) of either positive or negative polarity (2, 4). Adenovirus genes implicated in AAV replication have been identified and include E1A, E1B, E4orf6, E2A, and VA RNA (7,20,21,31,32). Similar to provirus in latently infected cells, AAV genomes can be efficiently rescued from a recombinant cis plasmid following transient transfection into human cells (33-35). The necessary helper functions can be delivered either by adenovirus infection or by transfecting a plasmid encoding a minimal set of adenovirus helper genes (10, 16).Events of AAV lytic infection are described by a commonly accepted self-priming strand displacement model (1). The first 125 nucleotides of AAV termini include elements capable of forming a T-shaped duplex structure (AЈ-BЈ-B-CЈ-C-A) and are followed by a unique 20-bp D-sequence (3, 45). The Rep gene encodes four proteins that are synthesized from the same open reading frame via the use of alternate promoters and splicing (2, 39). Two of these proteins (Rep78 and Rep68) possess site-specific and strand-specific endonuclease activity.They bind to the Rep-binding site (Rbs) mapped to the tetrameric GAGC repeat of the A stem of the ITR and cleave it at the terminal resolution site (trs), positioned b...

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Section: Discussionmentioning

confidence: 99%

“…The first 125 nucleotides of AAV termini include elements capable of forming a T-shaped duplex structure (AЈ-BЈ-B-CЈ-C-A) and are followed by a unique 20-bp D-sequence (3,45). The Rep gene encodes four proteins that are synthesized from the same open reading frame via the use of alternate promoters and splicing (2,39).…”

mentioning

confidence: 99%

A cis -Acting Element That Directs Circular Adeno-Associated Virus Replication and Packaging

Musatov

Roberts

Pfaff

et al. 2002

J Virol

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“…Replication of AAV also can occur after subsequent infection with a helper virus, typically either adenovirus (Ad) or herpesvirus. [6][7][8] The current method of production of rAAV requires transfection of multiple ers of rAAV expressing GFP. It was noted that the titer of rAdAAVrep-cap was lower than the titer of control AdCMVLacZ.…”

Section: Introductionmentioning

confidence: 99%

Recombinant adenovirus expressing adeno-associated virus cap and rep proteins supports production of high-titer recombinant adeno-associated virus

et al. 2001

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It has been difficult to produce a chimeric vector containing both Ad and AAV rep and cap, and to grow such chimeric vectors in 293 cells. By recombination in vitro in a bacterial host, we were able to produce recombinant plasmid AdAAV (pAdAAVrep-cap), which could be used to generate recombinant AdAAV (rAdAAVrep-cap) after transfection into 293 cells. A recombinant adenovirus, rAdAAVGFP, in which the green fluorescent protein (GFP) gene is flanked by the AAV terminal repeats cloned into the E1-deleted site of Ad was also generated. Co-infection of rAdAAVrep-cap together with rAdAAVGFP into 293 cells resulted in production of high tit-

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“…[33][34][35][36][37][38] Another long-term colleague, Xu-Shan Wang, MD, and others attempted to simply delete the D sequences from the viral genome, but quickly learned that the D-sequence deletion was incompatible with genome encapsidation, thereby identifying the D sequence as the ''packaging signal'' for the AAV genome. [39][40][41] Another colleague, Giridhara Rao Jayandharan, PhD, and others identified the presence of a putative negative regulatory element in the D sequence in the 5¢ ITR, to which the NF-jB-repressing factor binds and inhibits transcription. 42 Yet another long-term colleague, Chen Ling, PhD, and others eventually developed one D sequence-deleted genome that led to the generation of the generation X (''GenX'') AAV2 vectors in 2015, 43 and documented significantly enhanced transgene expression in human cell lines in vitro as well as in murine hepatocytes in vivo.…”

Section: Solution To the Genome Puzzlementioning

confidence: 99%

Rescue and Replication Signals of the Adeno-associated Virus 2 Genome

Cited by 62 publications

References 0 publications

A cis -Acting Element That Directs Circular Adeno-Associated Virus Replication and Packaging

A cis -Acting Element That Directs Circular Adeno-Associated Virus Replication and Packaging

Recombinant adenovirus expressing adeno-associated virus cap and rep proteins supports production of high-titer recombinant adeno-associated virus

Adeno-Associated Virus: The Naturally Occurring Virus Versus the Recombinant Vector

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