Quantitative proteomic analysis of lentiviral vectors using 2‐DE

Denard, Jérôme; Rundwasser, Stéphanie; Laroudie, Nicolas; Gonnet, Florence; Naldini, Luigi; Radrizzani, Marina; Galy, Anne; Merten, O.‐W.; Danos, Olivier; Svinartchouk, Fédor

doi:10.1002/pmic.200800747

Cited by 25 publications

(26 citation statements)

References 53 publications

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“…The use of iTRAQ allowed the identification of ϳ1,000 host cell proteins associated to viral particles, among which 105 were previously identified by others (42,43,46,47), including (i) Tsg101, AIP-1/Alix, CHMP, and Vps proteins required for virus budding, (ii) clathrin, whose involvement in virus morphogenesis and infectivity has been described recently (70,71), and (iii) transmembrane proteins acquired during the budding process such as adhesion and MHC molecules. Besides the absolute number of host-derived proteins identified in virions, more differences were found between the protein composition of WT and nef-deleted viruses by iTRAQ than by DIGE.…”

Section: Discussionmentioning

confidence: 99%

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Comparative Proteomic Analysis of HIV-1 Particles Reveals a Role for Ezrin and EHD4 in the Nef-Dependent Increase of Virus Infectivity

Brégnard

Zamborlini

Leduc

et al. 2013

J Virol

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gNef is a human immunodeficiency virus type 1 (HIV-1) auxiliary protein that plays an important role in virus replication and the onset of acquired immunodeficiency. Although known functions of Nef might explain its contribution to HIV-1-associated pathogenesis, how Nef increases virus infectivity is still an open question. In vitro, Nef-deleted viruses have a defect that prevents efficient completion of early steps of replication. We have previously shown that this restriction is not due to the absence of Nef in viral particles. Rather, a loss of function in virus-producing cells accounts for the lower infectivity of nef-deleted viruses compared to wild-type (WT) viruses. Here we used DiGE and iTRAQ to identify differences between the proteomes of WT and nef-deleted viruses. We observe that glucosidase II is enriched in WT virions, whereas Ezrin, ALG-2, CD81, and EHD4 are enriched in nef-deleted virions. Functional analysis shows that glucosidase II, ALG-2, and CD81 have no or only Nef-independent effect on infectivity. In contrast, Ezrin and EHD4 are involved in the ability of Nef to increase virus infectivity (referred to thereafter as Nef potency). Indeed, simultaneous Ezrin and EHD4 depletion in SupT1 and 293T virus-producing cells result in an ϳ30 and ϳ70% decrease of Nef potency, respectively. Finally, while Ezrin behaves as an inhibitory factor counteracted by Nef, EHD4 should be considered as a cofactors required by Nef to increase virus infectivity.

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Section: Discussionmentioning

confidence: 99%

“…Extensive proteomic analysis has demonstrated the presence of host cell-derived proteins in virions (42)(43)(44)(45)(46)(47). Given the ability of Nef to interfere with the endosomal network, we hypothesized that it might modify the virus proteome.…”

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confidence: 99%

Comparative Proteomic Analysis of HIV-1 Particles Reveals a Role for Ezrin and EHD4 in the Nef-Dependent Increase of Virus Infectivity

Brégnard

Zamborlini

Leduc

et al. 2013

J Virol

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“…Two-dimensional gel electrophoresis (2-DE) analysis, Coomassie blue staining, and identification of proteins separated by 2-DE were performed as previously described (10). Tryptic monoisotopic peptide masses were searched by using Aldente software (version 11 February 2008) or absence (B) of rAAV-6 (10E12 physical particles) and analyzed by 2-DE.…”

Section: Methodsmentioning

confidence: 99%

Human Galectin 3 Binding Protein Interacts with Recombinant Adeno-Associated Virus Type 6

Denard

Beley

Kotin

et al. 2012

J Virol

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i Recombinant adeno-associated viruses (rAAVs) hold enormous potential for human gene therapy. Despite the well-established safety and efficacy of rAAVs for in vivo gene transfer, there is still little information concerning the fate of vectors in blood following systemic delivery. We screened for serum proteins interacting with different AAV serotypes in humans, macaques, dogs, and mice. We report that serotypes rAAV-1, -5, and -6 but not serotypes rAAV-2, -7, -8, -9, and -10 interact in human sera with galectin 3 binding protein (hu-G3BP), a soluble scavenger receptor. Among the three serotypes, rAAV-6 has the most important capacities for binding to G3BP. rAAV-6 also bound G3BP in dog sera but not in macaque and mouse sera. In mice, rAAV-6 interacted with another protein of the innate immune system, C-reactive protein (CRP). Furthermore, interaction of hu-G3BP with rAAV-6 led to the formation of aggregates and hampered transduction when the two were codelivered into the mouse. Based on these data, we propose that species-specific interactions of AAVs with blood proteins may differentially impact vector distribution and efficacy in different animal models.T he recombinant adeno-associated vector (rAAV) platform, derived from a nonpathogenic dependovirus, has many attributes suitable for in vivo gene transfer: rAAV vectors are capable of transducing a wide range of cell types, including dividing and nondividing cells; rAAV genomes persist as episomal chromatin in the nucleus of transduced cells (38); and stable, persistent expression has been reported for many transgenes in different tissues and species (6,12,36,39). rAAVs have proven to be efficient in preclinical studies in animal models (16,28), and results from clinical trials are promising (7,47).In the case of systemic diseases, clinical relevance requires widespread distribution of the vector in order to target entire organs. This is particularly true for myopathies, where all striated muscles of the skeletal musculature and, frequently, cardiac muscles have to be treated. In this case, vascular delivery would be the optimal route for rAAV administration. Intravascular injection of a number of rAAV serotypes has proven efficient in murine models of muscular dystrophies (11,17,18,34,35). However, translating this approach to large animal models and humans is still challenging. Acquired immunity and neutralizing antibodies present in a large fraction of the human population might obviously be restrictive for rAAV gene delivery (5,20,27,29,30). Moreover, recent studies have demonstrated that serum might also contain other factors neutralizing rAAV vectors (40), indicating that detailed characterization of rAAV's molecular interactions in the bloodstream is obviously important in order to improve vector efficacy.We looked for serum proteins, other than immunoglobulins, which could interact with rAAVs in the bloodstreams of different species. By using a multidisciplinary approach involving proteomics, binding assays, electron microscopy (EM), and in vivo stud...

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“…The number of IN molecules contained in a lentivirus particle is limited with estimates between 20 and 250 molecules [28, 29]. IN-fusion proteins may be present at lower levels because of potentially inefficient IN-fusion protein expression and packaging into new particles in producer cells.…”

Section: Discussionmentioning

confidence: 99%

Lentiviral Protein Transduction with Genome-Modifying HIV-1 Integrase-I-PpoI Fusion Proteins: Studies on Specificity and Cytotoxicity

Turkki

Schenkwein

Timonen

et al. 2014

BioMed Research International

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Rare-cutting endonucleases, such as the I-PpoI, can be used for the induction of double strand breaks (DSBs) in genome editing and targeted integration based on homologous recombination. For therapeutic approaches, the specificity and the pattern of off-target effects are of high importance in these techniques. For its applications, the endonuclease needs to be transported into the target cell nucleus, where the mechanism of transport may affect its function. Here, we have studied the lentiviral protein transduction of the integrase (IN)-PpoI fusion protein using the cis-packaging method. In genome-wide interaction studies, IN-fusion proteins were verified to bind their target sequence containing 28S ribosomal RNA (rRNA) genes with a 100-fold enrichment, despite the well-documented behavior of IN to be tethered into various genomic areas by host-cell factors. In addition, to estimate the applicability of the method, DSB-induced cytotoxic effects with different vector endonuclease configurations were studied in a panel of cells. Varying the amount and activity of endonuclease enabled the adjustment of ratio between the induced DSBs and transported DNA. In cell studies, certain cancerous cell lines were especially prone to DSBs in rRNA genes, which led us to test the protein transduction in a tumour environment in an in vivo study. In summary, the results highlight the potential of lentiviral vectors (LVVs) for the nuclear delivery of endonucleases.

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Quantitative proteomic analysis of lentiviral vectors using 2‐DE

Cited by 25 publications

References 53 publications

Comparative Proteomic Analysis of HIV-1 Particles Reveals a Role for Ezrin and EHD4 in the Nef-Dependent Increase of Virus Infectivity

Comparative Proteomic Analysis of HIV-1 Particles Reveals a Role for Ezrin and EHD4 in the Nef-Dependent Increase of Virus Infectivity

Human Galectin 3 Binding Protein Interacts with Recombinant Adeno-Associated Virus Type 6

Lentiviral Protein Transduction with Genome-Modifying HIV-1 Integrase-I-PpoI Fusion Proteins: Studies on Specificity and Cytotoxicity

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