An improved self-deleting retroviral vector derived from avian leukemia and sarcoma virus.

We have previously developed a self-deleting avian leukosis and sarcoma virus (ALSV)- based retroviral vector carrying an additional attachment (att) sequence. Resulting proviruses underwent deletion of viral sequences and were flanked either by two LTRs (LTRs proviruses) or by the additional att sequence and the 3' LTR (att proviruses). Herein, we have tried to increase (1) the self-deleting properties of this vector, either by raising the selection pressure applied on target cells or by optimizing the size of the internal att sequence, (2) the titer of the vector by deleting or inverting some viral sequences. Moreover, a new type of provirus flanked by att sequences at each end was isolated. Finally, under specific conditions, 100% of proviruses had internal sequences deleted, and as many as 92-100% of proviruses were no longer mobilizable by a replication-competent virus. The inactivation procedure achieved here might improve the biosafety of retroviral vectors.

A novel self-deleting retroviral vector carrying an additional sequence recognized by the viral integrase (IN).

During retroviral integration, the viral integrase recognizes the attachment (att) sequence (formed by juxtaposition of two LTRs ends) as the substrate of integration. We have developed a self-deleting Avian Leukosis and Sarcoma Viruses (ALSVs)-based retroviral vector carrying an additional copy of the att sequence, between neo and puro genes. We observed that: (i) the resulting NP3Catt vector was produced at neo and puro titers respectively smaller and higher than that of the parental vector devoid of the att sequence; (ii) 61% of NP3Catt proviruses were flanked by LTRs; most of them were deleted of internal sequences, probably during the reverse transcription step; (iii) 31% of clones were deleted of the whole 5' part of their genome and were flanked, in 5', by the additional att sequence and, in 3', by an LTR. Integration of these last proviruses was often imprecise with respect to the viral ends. At total, 77% of proviruses had lost the packaging signal and were not mobilizable by a replication-competent virus and 92% had lost the selectable gene in a single round of replication. Although still to improve, the att vector could be considered as an interesting new safe retroviral vector for gene transfer experiments.

High-affinity uranyl-specific antibodies suitable for cellular imaging.

Monoclonal antibodies (mAbs) have proved to be valuable models for the study of protein-metal interactions, and previous reports have described very specific antibodies to chelated metal ions, including uranyl. We raised specific mAbs against UO2(2+)-DCP-BSA (DCP, 1,10-phenanthroline-2,9-dicarboxylic acid) to generate new sets of antibodies that might cross-react with various complexed forms of uranyl in different environments for further application in the field of toxicology. Using counter-screening with UO2(2+)-DCP-casein, we selected two highly specific mAbs against uranyl-DCP ( K D 10-100 pM): U04S and U08S. Competitive assays in the presence of different metal ions (UO2(2+), Fe (3+), Zn2+, Cu2+, and Ca2+) showed that uranyl in solution can act as a good competitor, suggesting some antibody ability to cross-react with chelating groups other than DCP in the UO2(2+) equatorial coordination plane. Interestingly, one of the antibodies could be used for revealing uranyl cations in cell samples. Fluorescence activated cell sorting analyses after immunolabeling revealed the interaction of uranyl with human kidney cells HK2. The intracellular accumulation of uranyl could be directly visualized by metal-immunostaining using fluorescent-labeled mAb. Our results suggest that U04S mAb epitopes mostly include the uranyl fraction and its paratopes can accommodate a wide variety of chelating groups.

Gene transfer into mammalian cells using a self-deleting avian leukemia and sarcoma virus-based vector.

OBJECTIVES: We have previously described an avian leukemia and sarcoma virus-based vector containing an additional att sequence in an internal position that is capable of self-deleting most of its 5' viral sequences during one cycle of replication in avian cells [Virus Res 2008;135:72-82; Arch Virol 2008;153:2233-2243]. Herein, our aim was to test the infectivity and self-deleting properties of this avian retroviral vector in human cells. METHODS: Human Hela cells transiently expressing the cellular receptor for avian leukemia and sarcoma viruses (tva) were infected with the avian vector. Molecular analyses of thirteen clones were performed. RESULTS: Data showed that more than 77% of proviruses had lost the 5' part of their genome including the selectable gene. At least 61% of these proviruses were flanked on the left by the additional att sequence and on the right by the LTR. None of the thirteen proviruses was able to express a full-length genomic RNA. CONCLUSION: This study demonstrates that the self-deleting properties of the avian vector in avian cells may be also applicable to human cells.

Structural consequences of binding of UO2(2+) to apotransferrin: can this protein account for entry of uranium into human cells?

It has been established that transferrin binds a variety of metals. These include toxic uranyl ions which form rather stable uranyl-transferrin derivatives. We determined the extent to which the iron binding sites might accommodate the peculiar topographic profile of the uranyl ion and the consequences of its binding on protein conformation. Indeed, metal intake via endocytosis of the transferrin/transferrin receptor depends on the adequate coordination of the metal in its site, which controls protein conformation and receptor binding. Using UV-vis and Fourier transform infrared difference spectroscopy coupled to a microdialysis system, we showed that at both metal binding sites two tyrosines are uranyl ligands, while histidine does not participate with its coordination sphere. Analysis by circular dichroism and differential scanning calorimetry (DSC) showed major differences between structural changes associated with interactions of iron or uranyl with apotransferrin. Uranyl coordination reduces the level of protein stabilization compared to iron, but this may be simply related to partial lobe closure. The lack of interaction between uranyl-TF and its receptor was shown by flow cytometry using Alexa 488-labeled holotransferrin. We propose a structural model summarizing our conclusion that the uranyl-TF complex adopts an open conformation that is not appropriate for optimal binding to the transferrin receptor.