RESUMO
Adeno-associated virus (AAV) type 5 represents the genetically most distant AAV serotype and the only one isolated directly from human tissue. Seroepidemiological evidence suggests herpes simplex virus (HSV) as a helper virus for human AAV5 infections, underlining the in vivo relevance of the AAV-herpesvirus relationship. In this study we analysed, for the first time, HSV helper functions for productive AAV5 replication, and compared these to AAV2. Using a combination of HSV strains and plasmids for individual genes, the previously defined HSV helper functions for AAV2 replication were shown to induce AAV5 gene expression, DNA replication and production of infectious progeny. The helper functions comprise the replication genes for ICP8 (UL29), helicase-primase (UL5/8/52), and DNA polymerase (UL30/42). HSV immediate-early genes for ICP0 and ICP4 further enhanced AAV5 replication, mainly by induction of rep gene expression. In the presence of HSV helper functions, AAV5 Rep co-localized with ICP8 in nuclear replication compartments, and HSV alkaline exonuclease (UL12) enhanced AAV5 replication, similarly to AAV2. UL12, in combination with ICP8, was shown to induce DNA strand exchange on partially double-stranded templates to resolve and repair concatemeric HSV replication intermediates. Similarly, concatemeric AAV replication intermediates appeared to be processed to yield AAV unit-length molecules, ready for AAV packaging. Taken together, our findings show that productive AAV5 replication is promoted by the same combination of HSV helper functions as AAV2.
Assuntos
Replicação do DNA/fisiologia , Dependovirus/fisiologia , Simplexvirus/fisiologia , Replicação Viral/genética , Animais , Linhagem Celular Tumoral , Chlorocebus aethiops , DNA Helicases/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Dependovirus/genética , Expressão Gênica , Células HeLa , Humanos , Simplexvirus/genética , Células Vero , Proteínas Virais/genéticaRESUMO
Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 induced polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability.
Assuntos
Proteínas Culina/metabolismo , Canais de Potássio/metabolismo , Divisão Celular , Citoplasma/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa/metabolismo , Humanos , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Canais de Potássio/genética , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Transporte Proteico , Técnicas do Sistema de Duplo-Híbrido , UbiquitinaçãoRESUMO
Phage display technology (PD) is a powerful technique for the generation of tumortargeting antibodies. However, there are a number of different selection methods established in different laboratories around the world. Cellbased PD panning methods using primary tumor cells are particularly heterogeneous between laboratories, which can lead to inconsistent results. Therefore, the present study evaluated different cellbased PD selection methods regarding their potential to generate acute myeloid leukemia (AML) blastbinding antibodies. In addition to this evaluation, the present study improved the PD procedure by optimizing selection as well as depletion strategies. To the best of our knowledge, the current study demonstrated for the first time that antigen diversity during the depletion step is of importance for the enrichment of tumortargeting phage antibodies. It is demonstrated that medium levels of depletion antigen diversity led to the most promising antibody candidates. In addition, it was determined that purification of blast cells from patients with AML by immunomagnetic separation ameliorated the selection of AMLbinding phages during panning. Furthermore, suggesting a common designrelated mechanism using a 'singlepot' PD library, such as the wellknown Tomlinson singlechain fragment variable (scFv) library, the present study identified specific binding consensus phage particles in independent panning procedures. By means of these optimized strategies, four promising AML blastbinding phage particles were isolated and soluble scFvFc (scFv cloned to a fragment crystallizable of an IgG2a mouse antibody) fusion proteins were produced. These scFvFc antibodies bound the surface of AML blasts and were successfully internalized into their cytoplasm, indicating that they are potential immunoconjugate candidates for AML immunotherapy.