Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1

Musayev, F.N.; Zárate‐Pérez, Francisco; Bishop, Clayton; Burgner, John W.; Escalante, Carlos

doi:10.1074/jbc.m115.669960

Cited by 26 publications

(26 citation statements)

References 55 publications

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“…Numerous groundbreaking studies, although not based on high-throughput sequencing, have demonstrated the preferred integration of wtAAV2 into AAVS1--located at human chromosome 19--and have uncovered the molecular mechanism underlying this integration preference, identifying the multifunctional, nonstructural wtAAV Rep proteins by AAV2's inverted terminal repeats and AAVS1 (Rep-binding sites and terminal resolution sequence motifs) as key factors. [5][6][7][8] Consistent with this, several pubvectors gained AAVS1 targeting upon administration of Rep protein. [9][10][11][12] As yet, only two large-scale wtAAV integration analyses performed on primary cells have been published.…”

Section: Reply To "Wild-type Aav Insertions In Hepatocellular Carcinomentioning

confidence: 68%

Reply to “Wild-type AAV Insertions in Hepatocellular Carcinoma Do Not Inform Debate Over Genotoxicity Risk of Vectorized AAV”

2016

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Section: Reply To "Wild-type Aav Insertions In Hepatocellular Carcinomentioning

confidence: 68%

Reply to “Wild-type AAV Insertions in Hepatocellular Carcinoma Do Not Inform Debate Over Genotoxicity Risk of Vectorized AAV”

2016

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“…The conserved N-terminal region of the MVC NS proteins encodes a histidine- hydrophobic-histidine (HUH) endonuclease motif (35,36), also conserved in other parvoviral nonstructural proteins (13,36), which is required for site-specific DNA cleavage and ligation during rolling-hairpin replication (12,37). The active site of the MVC HUH motif is predicted to be H122, L123, and H124, which are present in all the MVC NS isoforms (Fig.…”

Section: Discussionmentioning

confidence: 99%

Minute Virus of Canines NP1 Protein Governs the Expression of a Subset of Essential Nonstructural Proteins via Its Role in RNA Processing

Fasina

Stupps

Figueroa-Cuilan

et al. 2017

J Virol

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Parvoviruses use a variety of means to control the expression of their compact genomes. The bocaparvovirus minute virus of canines (MVC) encodes a small, genus-specific protein, NP1, which governs access to the viral capsid gene via its role in alternative polyadenylation and alternative splicing of the single MVC premRNA. In addition to NP1, MVC encodes five additional nonstructural proteins (NS) that share an initiation codon at the left end of the genome and which are individually encoded by alternative multiply spliced mRNAs. We found that three of these proteins were encoded by mRNAs that excise the NP1-regulated MVC intron immediately upstream of the internal polyadenylation site, (pA)p, and that generation of these proteins was thus regulated by NP1. Splicing of their progenitor mRNAs joined the amino termini of these proteins to the NP1 open reading frame, and splice site mutations that prevented their expression inhibited virus replication in a host celldependent manner. Thus, in addition to controlling capsid gene access, NP1 also controls the expression of three of the five identified NS proteins via its role in governing MVC pre-mRNA splicing. IMPORTANCEThe Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. Minute virus of canine (MVC) is an autonomous parvovirus in the genus Bocaparvovirus. It has a single promoter that generates a single pre-mRNA. NP1, a small genus-specific MVC protein, participates in the processing of this pre-mRNA and so controls capsid gene access via its role in alternative internal polyadenylation and splicing. We show that NP1 also controls the expression of three of the five identified NS proteins via its role in governing MVC pre-mRNA splicing. These NS proteins together are required for virus replication in a host cell-dependent manner.KEYWORDS gene expression, parvovirus P arvoviruses use multiple mechanisms to maximize the coding potential of their compact genomes, including alternative transcription initiation, alternative splicing, alternative polyadenylation, and alternative translation initiation mechanisms (1-3).Infection with minute virus of canines (MVC), a member of the genus Bocaparvovirus (4), can cause abortion and stillbirth in pregnant dogs, as well as mild gastroenteritis and respiratory disease in puppies (5-8). MVC generates a single pre-mRNA from a promoter at the left-hand end of the genome (P6) that is processed via alternative splicing and alternative polyadenylation into multiple nonstructural and capsidencoding transcripts (9, 10). As with other parvoviruses, an open reading frame (ORF) in the left half of the genome encodes nonstructural (NS) proteins, while an ORF in the right half encodes the capsid proteins VP1 and VP2 (2). The bocaparvoviruses also

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“…Thus, when a small fluorescent DNA molecule binds a protein, the larger complex will rotate more slowly than the DNA molecule, changing the plane of the polarized light and increasing the anisotropy value. We have used this technique to measure the binding affinity of AAV Rep68 for different DNA substrates (Yoon-Robarts et al , 2004; Musayev et al , 2015; Bardelli et al , 2016). The non-structural AAV Rep proteins carry out most of the DNA transactions that are required to complete the virus life cycle.…”

Section: Introductionmentioning

confidence: 99%

“…These include DNA replication, transcriptional regulation, site-specific integration and packaging of DNA into preformed capsids (Im and Muzyczka, 1990; Weitzman et al , 1994; Wonderling et al , 1995). The large AAV Rep proteins (Rep78/Rep68) contain an N-terminal origin binding domain (OBD) that specifically binds the Rep binding sites (RBS) and displays nuclease activity (Hickman et al , 2004; Musayev et al , 2015). The RBS sites consist of two or more 5′-GCTC-3′ repeats and are found at the viral origin of replication, in several promoters and at the AAVS1 integration site (Weitzman et al , 1994; McCarty et al , 1994).…”

Section: Introductionmentioning

confidence: 99%

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Determination of Adeno-associated Virus Rep DNA Binding Using Fluorescence Anisotropy

Zárate‐Pérez¹,

Santosh²,

Bardelli³

et al. 2017

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Quantitative measurement of proteins binding to DNA is a requisite to fully characterize the structural determinants of complex formation necessary to understand the DNA transactions that regulate cellular processes. Here we describe a detailed protocol to measure binding affinity of the adeno-associated virus (AAV) Rep68 protein for the integration site AAVS1 using fluorescent anisotropy. This protocol can be used to measure the binding constants of any DNA binding protein provided the substrate DNA is fluorescently labeled.

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Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1

Cited by 26 publications

References 55 publications

Reply to “Wild-type AAV Insertions in Hepatocellular Carcinoma Do Not Inform Debate Over Genotoxicity Risk of Vectorized AAV”

Reply to “Wild-type AAV Insertions in Hepatocellular Carcinoma Do Not Inform Debate Over Genotoxicity Risk of Vectorized AAV”

Minute Virus of Canines NP1 Protein Governs the Expression of a Subset of Essential Nonstructural Proteins via Its Role in RNA Processing

Determination of Adeno-associated Virus Rep DNA Binding Using Fluorescence Anisotropy

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