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Fiber shaft-chimeric adenovirus vectors lacking the KKTK motif efficiently infect liver cells in vivo.

ABSTRACT: The molecular mechanisms governing the infectivity of adenovirus (Ad) toward specific cell and tissue types in vivo remain poorly understood. The direct Ad binding to hepatic heparan sulfate proteoglycans via the KKTK motif within the fiber shaft domain was suggested to be the major mechanism of Ad liver cell infection in vivo. Here, we describe the generation and in vitro and in vivo infectivity studies of Ad5-based vectors possessing long Ad31- or Ad41-derived fiber shaft domains, which lack the KKTK motif. We found that all the critical early steps of Ad infection, including attachment to the cellular receptor, internalization, and virus genome transfer into the nucleus, occurred with similar levels of efficiency for fiber shaft-chimeric vectors and unmodified Ad5. Upon intravenous delivery into mice, fiber shaft-chimeric vectors accumulated in liver tissue, transduced liver cells, and induced the production of proinflammatory cytokines (tumor necrosis factor alpha and interleukin-6) and the chemokine monocyte chemoattractant protein 1 at levels indistinguishable from those observed for Ad5. Thus, our data provide evidence that the Ad5 fiber shaft amino acid sequence does not play any substantial role in determining adenovirus infectivity toward hepatic cells in vivo. The data obtained contribute to improving our understanding of the molecular mechanisms determining Ad infectivity and biodistribution in vivo and may aid in designing novel Ad-based vectors for gene therapy applications.

SUBMITTER: Di Paolo NC

PROVIDER: S-EPMC2168974 | biostudies-literature | 2007 Nov

REPOSITORIES: biostudies-literature

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Fiber shaft-chimeric adenovirus vectors lacking the KKTK motif efficiently infect liver cells in vivo.

Di Paolo Nelson C NC Kalyuzhniy Oleksandr O Shayakhmetov Dmitry M DM

Journal of virology 20070912 22

The molecular mechanisms governing the infectivity of adenovirus (Ad) toward specific cell and tissue types in vivo remain poorly understood. The direct Ad binding to hepatic heparan sulfate proteoglycans via the KKTK motif within the fiber shaft domain was suggested to be the major mechanism of Ad liver cell infection in vivo. Here, we describe the generation and in vitro and in vivo infectivity studies of Ad5-based vectors possessing long Ad31- or Ad41-derived fiber shaft domains, which lack t ...[more]

PMID: 17855526

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Project description:Following receptor binding and internalization, intracellular trafficking of adenovirus (Ad) among subgroups B and C is different, with significant amounts of Ad serotype 7 (Ad7) (subgroup B) virions found in cytoplasm during the initial hours of infection while Ad5 (subgroup C) virions rapidly translocate to the nucleus. To evaluate the role of the fiber in these differences, we examined intracellular trafficking of Ad5, Ad7, and Ad5f7 (a chimeric vector composed of the Ad5 capsid with the fiber replaced by the Ad7 fiber) by conjugating Ad capsids directly with Cy3 fluorescent dye, permitting the trafficking of the capsids to be examined by fluorescence microscopy. The human lung carcinoma cell line A549 was infected with Cy3-conjugated viruses for 10 min followed by a 1-h incubation. Ad5 virions rapidly translocated to the nucleus (within 1 h of infection), while Ad7 virions were widely distributed in the cytoplasm at the same time point. Interestingly, chimeric Ad5f7 virions behaved similarly to Ad7 but not Ad5. In this regard, the percentages of nuclear localization of Ad5, Ad7, and Ad5f7 at 1 h following infection were 72% +/- 4%, 32% +/- 6%, and 38% +/- 2%, respectively. Consistent with these observations, fluorescence in situ hybridization demonstrated that most of the Ad5 DNA was detected at the nucleus after 1 h, but at the same time point, DNA of Ad7 and Ad5f7 was distributed in both the nucleus and cytoplasm. Quantification of the kinetics of Ad genomic DNA delivery to the nucleus using a fluorogenic probe-based PCR assay (TaqMan PCR) demonstrated that the percentages of nuclear association of Ad5 DNA and Ad5f7 DNA at 1 h postinfection were 80% +/- 13% and 43% +/- 1%, respectively. Although it has been generally accepted that Ad fiber protein mediates attachment of virions to cells and that fibers dissociate during endocytic uptake, these data suggest that in addition to mediating binding to the cell surface, fiber likely modulates intracellular trafficking as well.

Project description:ObjectiveTo evaluate the effects of fiber-modified hexon-chimeric recombinant oncolytic adenovirus targeting cancer associated fibroblasts (CAFs) on the gastric CAFs and the transplantation tumor mice model of gastric carcinoma (GC).ResultsCompared with BJ cells and GPFs, the reproduction and infectivity of P9, P9-4C or GP adenoviruses were markedly higher in gastric CAFs. In addition, P9, P9-4C or GP had a significantly relatively more killing effect on gastric CAFs compared with GPFs, and have less oncolytic effect in BJ cells. Furthermore, in transplantation tumor mice model of GC we found significantly higher hexon protein expression in tumor tissues, more decreasing tumor growth and increasing inhibitory rates after treatment of P9, P9-4C or GP adenoviruses compared with Ad adenovirus.Materials and methodsBased on the construction of the recombinant oncolytic adenoviruses pRCAdHVR48-SDF1p-Ad/EGFP (Ad, as control) with the E1A gene transcription regulated by stromal-derived factor 1 (SDF1) promoter and the hexon replaced by hexon-chimeric (H5HVR48) gene, three fiber-modified hexon-chimeric oncolytic adenovirus through the modification fiber protein by insertion of different short peptides specifically binding to fibroblast activation protein (FAP), including pRCAdHVR48-SDF1p-FAP-P9/EGFP (P9), pRCAdHVR48-SDF1p-FAP-P9-4C/EGFP (P9-4C), pRCAdHVR48-SDF1p-FAP-GP/EGFP (GP), and their corresponding replication-defective adenovirus in parallel were reconstructed. Then the reproduction, infectivity and killing ability of the four above recombinant adenoviruses were evaluated in gastric CAFs compared with gastric para-mucosa fibroblasts (GPFs) and neonatal human foreskin fibroblasts (BJ). Furthermore, transplantation tumor mice model of GC was established, and then treated by the four above recombinant adenoviruses. Tumor size and tumor growth inhibitory rates were calculated, and histomorphology by HE staining and hexon expressions by immunohistochemistry were evaluated in tumor tissues.ConclusionsThe fiber-modified hexon-chimeric recombinant oncolytic adenovirus targeting CAFs can relatively specifically kill gastric CAFs and inhibit GC cells growth in vivo.

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Fiber shaft-chimeric adenovirus vectors lacking the KKTK motif efficiently infect liver cells in vivo.

Publications

Fiber shaft-chimeric adenovirus vectors lacking the KKTK motif efficiently infect liver cells in vivo.

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