RESUMO
Endothelial cells (EC) are targets in gene therapy and regenerative medicine, but they are inefficiently transduced with adeno-associated virus (AAV) vectors of various serotypes. To identify barriers hampering efficient transduction and to develop an optimized AAV variant for EC transduction, we screened an AAV serotype 2-based peptide display library on primary human macrovascular EC. Using a new high-throughput selection and monitoring protocol, we identified a capsid variant, AAV-VEC, which outperformed the parental serotype as well as first-generation targeting vectors in EC transduction. AAV vector uptake was improved, resulting in significantly higher transgene expression levels from single-stranded vector genomes detectable within a few hours post-transduction. Notably, AAV-VEC transduced not only proliferating EC but also quiescent EC, although higher particle-per-cell ratios had to be applied. Also, induced pluripotent stem cell-derived endothelial progenitor cells, a novel tool in regenerative medicine and gene therapy, were highly susceptible toward AAV-VEC transduction. Thus, overcoming barriers by capsid engineering significantly expands the AAV tool kit for a wide range of applications targeting EC.
Assuntos
Capsídeo/química , Dependovirus/genética , Engenharia Genética/métodos , Vetores Genéticos/química , Células Endoteliais da Veia Umbilical Humana/metabolismo , Transdução Genética/métodos , Sequência de Aminoácidos , Capsídeo/metabolismo , Diferenciação Celular , Dependovirus/metabolismo , Genes Reporter , Terapia Genética/métodos , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Biblioteca de PeptídeosRESUMO
Adeno-associated virus (AAV) vectors are currently the leading candidates for virus-based gene therapies because of their broad tissue tropism, non-pathogenic nature and low immunogenicity. They have been successfully used in clinical trials to treat hereditary diseases such as haemophilia B (ref. 2), and have been approved for treatment of lipoprotein lipase deficiency in Europe. Considerable efforts have been made to engineer AAV variants with novel and biomedically valuable cell tropisms to allow efficacious systemic administration, yet basic aspects of AAV cellular entry are still poorly understood. In particular, the protein receptor(s) required for AAV entry after cell attachment remains unknown. Here we use an unbiased genetic screen to identify proteins essential for AAV serotype 2 (AAV2) infection in a haploid human cell line. The most significantly enriched gene of the screen encodes a previously uncharacterized type I transmembrane protein, KIAA0319L (denoted hereafter as AAV receptor (AAVR)). We characterize AAVR as a protein capable of rapid endocytosis from the plasma membrane and trafficking to the trans-Golgi network. We show that AAVR directly binds to AAV2 particles, and that anti-AAVR antibodies efficiently block AAV2 infection. Moreover, genetic ablation of AAVR renders a wide range of mammalian cell types highly resistant to AAV2 infection. Notably, AAVR serves as a critical host factor for all tested AAV serotypes. The importance of AAVR for in vivo gene delivery is further highlighted by the robust resistance of Aavr(-/-) (also known as Au040320(-/-) and Kiaa0319l(-/-)) mice to AAV infection. Collectively, our data indicate that AAVR is a universal receptor involved in AAV infection.
Assuntos
Dependovirus/fisiologia , Infecções por Parvoviridae/metabolismo , Infecções por Parvoviridae/virologia , Receptores de Superfície Celular/metabolismo , Receptores Virais/metabolismo , Tropismo Viral , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Linhagem Celular , Dependovirus/classificação , Dependovirus/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Feminino , Deleção de Genes , Terapia Genética/métodos , Especificidade de Hospedeiro , Humanos , Masculino , Camundongos , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptores Virais/antagonistas & inibidores , Receptores Virais/deficiência , Receptores Virais/genética , Tropismo Viral/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Rede trans-Golgi/efeitos dos fármacosRESUMO
Conditionally replicating adenoviruses (CRAds) selectively replicate in and thereby kill cancer cells. The CRAd AdDelta24 with pRb-binding-deficient E1A kills cancer cells efficiently. Arming CRAds with genes encoding prodrug-converting enzymes could allow for enhanced anticancer efficacy by the combined effects of oncolytic replication and local prodrug activation. Here, we investigated combination treatment of human colon cancer cell lines with AdDelta24-type CRAds and gene-directed enzyme prodrug therapy (GDEPT) using two different enzyme/prodrug systems, that is, thymidine kinase/ganciclovir (TK/GCV) and carboxylesterase (CE)/CPT-11. On all three cell lines tested, GDEPT with TK/GCV made CRAd treatment less efficacious. In contrast, expression of a secreted form of CE (sCE2) combined with CPT-11 treatment markedly enhanced the efficacy of AdDelta24 virotherapy. Based on this observation, we constructed an AdDelta24 variant expressing sCE2. In the absence of CPT-11, this new CRAd Ad5-Delta24.E3-sCE2 was similarly effective as its parent in killing human colon cancer cells. Low concentrations of CPT-11 inhibited Ad5-Delta24.E3-sCE2 propagation. Nevertheless, CPT-11 specifically augmented the cytotoxicity of Ad5-Delta24.E3-sCE2 against all three-colon cancer cell lines. Hence, the positive contribution of sCE2/CPT-11 GDEPT to colon cancer cytotoxicity outweighed its negative influence on CRAd propagation. Therefore, CRAd-sCE2/CPT-11 combination therapy appears useful for more effective treatment of colon cancer.
Assuntos
Adenoviridae/genética , Carboxilesterase/genética , Neoplasias do Colo/terapia , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Pró-Fármacos/administração & dosagem , Adenoviridae/fisiologia , Antivirais/uso terapêutico , Carboxilesterase/uso terapêutico , Linhagem Celular Tumoral , Neoplasias do Colo/virologia , Terapia Combinada , Efeito Citopatogênico Viral , Ganciclovir/uso terapêutico , Expressão Gênica , Engenharia Genética , Vetores Genéticos/genética , Humanos , Pró-Fármacos/uso terapêutico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/uso terapêutico , Timidina Quinase/uso terapêutico , Transdução Genética/métodos , Replicação ViralRESUMO
Replication of Alfalfa mosaic virus (AMV) RNAs depends on the virus-encoded proteins P1 and P2. P1 contains methyltransferase- and helicase-like domains, and P2 contains a polymerase-like domain. Coimmunoprecipitation experiments revealed an interaction between in vitro translated-P1 and P2 and showed that these proteins are present together in fractions with RNA-dependent RNA polymerase activity. A deletion analysis in the yeast two-hybrid system showed that in P1 the C-terminal sequence of 509 amino acids with the helicase domain was necessary for the interaction. In P2, the sequence of the N-terminal 241 aa was required for the interaction. In infected protoplasts, P1 and P2 colocalized at a membrane structure that was identified as the tonoplast (i.e., the membrane that surrounds the vacuoles) by using a tonoplast intrinsic protein as a marker in immunofluorescence studies. While P1 was exclusively localized on the tonoplast, P2 was found both at the tonoplast and at other locations in the cell. As Brome mosaic virus replication complexes have been found to be associated with the endoplasmic reticulum (M. A. Restrepo-Hartwig and P. Ahlquist, J. Virol. 70:8908-8916, 1996), viruses in the family Bromoviridae apparently select different cellular membranes for the assembly of their replication complexes.
Assuntos
Vírus do Mosaico da Alfafa/fisiologia , Membranas Intracelulares/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Vacúolos/enzimologia , Proteínas Virais/metabolismo , Vírus do Mosaico da Alfafa/enzimologia , Vírus do Mosaico da Alfafa/genética , Membranas Intracelulares/virologia , Metiltransferases/genética , Metiltransferases/metabolismo , Plantas/virologia , Testes de Precipitina , Protoplastos/virologia , RNA Helicases/genética , RNA Helicases/metabolismo , RNA Polimerase Dependente de RNA/genética , Técnicas do Sistema de Duplo-Híbrido , Vacúolos/virologia , Proteínas Virais/genética , Replicação ViralRESUMO
In this study we have performed a mutational analysis of the cowpea mosaic comovirus (CPMV) genome-linked protein VPg to discern the structural requirements necessary for proper functioning of VPg. Either changing the serine residue linking VPg to RNA at a tyrosine or a threonine or changing the position of the serine from the N-terminal end to position 2 or 3 abolished virus infectivity. Some of the mutations affected the cleavage between the VPg and the 58K ATP-binding protein in vitro, which might have contributed to the lethal phenotype. RNA replication of some of the mutants designed to replace VPg with the related cowpea severe mosaic comovirus was completely abolished, whereas replication of others was not affected or only mildly affected, showing that amino acids that are not conserved between the comoviruses can be critical for the function of VPg. The replicative proteins of one of the mutants failed to accumulate in typical cytopathic structures and this might reflect the involvement of VPg in protein-protein interactions with the other replicative proteins.
Assuntos
Comovirus/genética , Proteínas do Core Viral/genética , Sequência de Aminoácidos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Homologia de Sequência de Aminoácidos , Serina/genética , Proteínas do Core Viral/fisiologiaRESUMO
Replication of cowpea mosaic virus (CPMV) is associated with small membranous vesicles that are induced upon infection. The effect of CPMV replication on the morphology and distribution of the endomembrane system in living plant cells was studied by expressing green fluorescent protein (GFP) targeted to the endoplasmic reticulum (ER) and the Golgi membranes. CPMV infection was found to induce an extensive proliferation of the ER, whereas the distribution and morphology of the Golgi stacks remained unaffected. Immunolocalization experiments using fluorescence confocal microscopy showed that the proliferated ER membranes were closely associated with the electron-dense structures that contain the replicative proteins encoded by RNA1. Replication of CPMV was strongly inhibited by cerulenin, an inhibitor of de novo lipid synthesis, at concentrations where the replication of the two unrelated viruses alfalfa mosaic virus and tobacco mosaic virus was largely unaffected. These results suggest that proliferating ER membranes produce the membranous vesicles formed during CPMV infection and that this process requires continuous lipid biosynthesis.
Assuntos
Comovirus/patogenicidade , Retículo Endoplasmático/ultraestrutura , Fabaceae/virologia , Complexo de Golgi/ultraestrutura , Membranas Intracelulares/metabolismo , Nicotiana/virologia , Plantas Medicinais , Plantas Tóxicas , Comovirus/metabolismo , Comovirus/ultraestrutura , Fabaceae/ultraestrutura , Lipídeos/biossíntese , Microscopia Confocal , Nicotiana/ultraestruturaRESUMO
The RNA genome of tobacco rattle virus (TRV) is bipartite. RNA 2 of the nematode-transmissible TRV isolate PPK20 encodes the viral coat protein (cp) and proteins with molecular weights of 29,400 and 32,800 (29.4K and 32.8K proteins). When this isolate was serially passaged in tobacco by using phenol-extracted RNA as the inoculum in each transfer, defective interfering (DI) RNAs rapidly accumulated. A number of these DI RNAs were cloned. Six DI RNAs had single internal deletions in RNA 2 that removed most of the cp gene, the 29.4K gene, and the 5' half of the 32.8K gene. The borders of the deletions in these DI RNAs were found to be flanked in the genomic RNA 2 by short nucleotide repeats or sequences resembling the 5' end of TRV genomic and subgenomic RNAs. Two DI RNAs were found to be recombinants containing a 5' sequence derived from RNA 2 and a 3' sequence derived from RNA 1. When serial passage of TRV isolate PPK20 was carried out by using leaf homogenates as inocula in each transfer, accumulation of a DI RNA (designated D7) with a functional cp gene was observed. The deletion in D7 covered the 3' end of the cp gene, the 29.4K gene, and the 5' half of the 32.8K gene. An infectious cDNA clone of D7 RNA was made. In mixed infections, D7 RNA rapidly outcompeted RNA 2 but did not compete with RNA 1. The deletion in D7 RNA abolished the nematode transmissibility of the PPK20 isolate. These results may explain the observation that many laboratory isolates of tobraviruses have lost their nematode transmissibility and contain RNA 2 molecules of widely different lengths.