Viral and Cellular Components of AAV2 Replication Compartments

Vogel, Rebecca; Seyffert, Michael; Pereira, Bruna de Andrade; Fraefel, Cornel

doi:10.2174/1874357901307010098

Cited by 10 publications

(8 citation statements)

References 216 publications

(360 reference statements)

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“…Understanding the roles of host factors is important for AAV biology and for rAAV vector production, especially in non-native host systems like baculovirus-insect cells where the lack of certain host factors could be limiting productivity. A number of host proteins associated with AAV have been identified using different approaches like co-immunoprecipitation and subcellular fractionation [18–21] however, for the majority of proteins, little is known about their role in the AAV life cycle. Protein complexes shown to be part of the AAV replication complex include DNA polymerase D, proliferating cell nuclear antigen (PCNA), replication factor C (RFC), the single-stranded DNA binding protein RPA and minichromosome maintenance complex (MCM) [18–20].…”

Section: Introductionmentioning

confidence: 99%

“…Protein complexes shown to be part of the AAV replication complex include DNA polymerase D, proliferating cell nuclear antigen (PCNA), replication factor C (RFC), the single-stranded DNA binding protein RPA and minichromosome maintenance complex (MCM) [18–20]. Another major group of factors interacting with AAV Rep proteins and DNA is the DNA damage response proteins including the MRN complex [22,23], however their significance in the AAV life cycle seems complex and is only partially understood [21,24]. …”

Section: Introductionmentioning

confidence: 99%

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Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae

et al. 2017

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The yeast Saccharomyces cerevisiae has been successfully employed to establish model systems for a number of viruses. Such model systems are powerful tools to study the virus biology and in particular for the identification and characterization of host factors playing a role in the viral infection cycle. Adeno-associated viruses (AAV) are heavily studied due to their use as gene delivery vectors. AAV relies on other helper viruses for successful replication and on host factors for several aspects of the viral life cycle. However the role of host and helper viral factors is only partially known. Production of recombinant AAV (rAAV) vectors for gene delivery applications depends on knowledge of AAV biology and the limited understanding of host and helper viral factors may be precluding efficient production, particularly in heterologous systems. Model systems in simpler eukaryotes like the yeast S. cerevisiae would be useful tools to identify and study the role of host factors in AAV biology. Here we show that expression of AAV2 viral proteins VP1, VP2, VP3, AAP, Rep78, Rep52 and an ITR-flanked DNA in yeast leads to capsid formation, DNA replication and encapsidation, resulting in formation of infectious particles. Many of the AAV characteristics observed in yeast resemble those in other systems, making it a suitable model system. Future findings in the yeast system could be translatable to other AAV host systems and aid in more efficient production of rAAV vectors.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae

et al. 2017

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“…Importantly, in HSV-1 infected cells, the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is degraded through ICP0-dependent proteasomal degradation [ 75 , 76 ]. In HSV-1 and AAV2 co-infected cells, the degradation of DNA-PKcs is delayed in an AAV2-dependent manner, and DNA-PKcs in fact is recruited into AAV2 RCs [ 77 , 78 ]. The modulation of the HSV-1 induced DDR by AAV2 may be due to the AAV2 genome (i.e.…”

Section: Discussionmentioning

confidence: 99%

Novel Mutant AAV2 Rep Proteins Support AAV2 Replication without Blocking HSV-1 Helpervirus Replication

et al. 2017

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As their names imply, parvoviruses of the genus Dependovirus rely for their efficient replication on the concurrent presence of a helpervirus, such as herpesvirus, adenovirus, or papilloma virus. Adeno-associated virus 2 (AAV2) is such an example, which in turn can efficiently inhibit the replication of each helpervirus by distinct mechanisms. In a previous study we have shown that expression of the AAV2 rep gene is not compatible with efficient replication of herpes simplex virus 1 (HSV-1). In particular, the combined DNA-binding and ATPase/helicase activities of the Rep68/78 proteins have been shown to exert opposite effects on the replication of AAV2 and HSV-1. While essential for AAV2 DNA replication these protein activities account for the Rep-mediated inhibition of HSV-1 replication. Here, we describe a novel Rep mutant (Rep-D371Y), which displayed an unexpected phenotype. Rep-D371Y did not block HSV-1 replication, but still supported efficient AAV2 replication, at least when a double-stranded AAV2 genome template was used. We also found that the capacity of Rep-D371Y to induce apoptosis and a Rep-specific DNA damage response was significantly reduced compared to wild-type Rep. These findings suggest that AAV2 Rep-helicase subdomains exert diverging activities, which contribute to distinct steps of the AAV2 life cycle. More important, the novel AAV2 mutant Rep-D371Y may allow deciphering yet unsolved activities of the AAV2 Rep proteins such as DNA second-strand synthesis, genomic integration or packaging, which all involve the Rep-helicase activity.

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“…Given reported mutation rates for HAdV-F41 and other adenoviruses 7,8 , the SNP differences between the case and other HAdV-F41 sequences are likely to have arisen before the outbreak. Importantly, compared with RefSeq sequences, no new or unique amino acid substitutions were noted in the E1a, E2a and E4 HAdV-F41 proteins, which are known to be critical for AAV2 replication 9 .…”

Section: Whole Genome Sequencingmentioning

confidence: 97%

Genomic Investigations of Acute Hepatitis of Unknown Aetiology in Children

Morfopoulou

Buddle

Montaguth

et al. 2022

Preprint

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Since the first reports of hepatitis of unknown aetiology occurring in UK children, over 1000 cases have been reported worldwide, including 268 cases in the UK, with the majority younger than 6 years old. Using genomic, proteomic and immunohistochemical methods, we undertook extensive investigation of 28 cases and 136 control subjects. In five cases who underwent liver transplantation, we detected high levels of adeno-associated virus 2 (AAV2) in the explanted livers. AAV2 was also detected at high levels in blood from 10/11 non-transplanted cases. Low levels of Adenovirus (HAdV) and Human Herpesvirus 6B (HHV-6B), both of which enable AAV2 lytic replication, were also found in the five explanted livers and blood from 15/17 and 6/9 respectively, of the 23 non-transplant cases tested. In contrast, AAV2 was detected at low titre in 6/100 whole bloods from child controls from cohorts with presence or absence of hepatitis and/or adenovirus infection. Our data show an association of AAV2 at high titre in blood or liver tissue, with unexplained hepatitis in children infected in the recent HAdV-F41 outbreak. We were unable to find evidence by electron microscopy, immunohistochemistry or proteomics of HAdV or AAV2 viral particles or proteins in explanted livers, suggesting that hepatic pathology is not due to direct lytic infection by either virus. The potential that AAV2, although not previously associated with disease, may, together with HAdV-F41 and/or HHV-6, be causally implicated in the outbreak of unexplained hepatitis, requires further investigation.

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Viral and Cellular Components of AAV2 Replication Compartments

Cited by 10 publications

References 216 publications

Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae

Generation of infectious recombinant Adeno-associated virus in Saccharomyces cerevisiae

Novel Mutant AAV2 Rep Proteins Support AAV2 Replication without Blocking HSV-1 Helpervirus Replication

Genomic Investigations of Acute Hepatitis of Unknown Aetiology in Children

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