Moloney Murine Leukemia Virus Gag-pol Research Articles

Translational readthrough-promoting drugs enhance pseudoknot-mediated suppression of the stop codon at the Moloney murine leukemia virus gag–pol junction.

Translational readthrough-promoting drugs enhance the incorporation of amino acids at stop codons and can thus bypass premature termination during protein synthesis. The polymerase (Pol) proteins of Moloney murine leukemia virus (MoMLV) are synthesized as a large Gag–Pol fusion protein, formed by the readthrough of a stop codon at the end of the gag ORF. The downstream pol ORF lacks its own start codon, and Pol protein synthesis is wholly dependent on translation of the upstream gag gene and the readthrough event for expression. Here, we explored the effects of readthrough-promoting drugs – aminoglycoside antibiotics and the small molecule ataluren – on the efficiency of readthrough of the stop codon in the context of the MoMLV genome. We showed that these compounds increased readthrough of the stop codon at the MoMLV gag–pol junction in vivo above the already high basal level and that the resulting elevated gag–pol readthrough had deleterious effects on virus replication. We also showed that readthrough efficiency could be driven to even higher levels in vitro, and that the combination of the small molecules and the RNA structure at the MoMLV stop codon could achieve extremely high readthrough efficiencies.

General strategy for decoration of enveloped viruses with functionally active lipid-modified cytokines.

Viral particles preferentially incorporate extra- and intracellular constituents of host cell lipid rafts, a phenomenon central to pseudotyping. Based on this mechanism, we have developed a system for the predictable decoration of enveloped viruses with functionally active cytokines that circumvents the need to modify viral proteins themselves. Human interleukin-2 (hIL-2), hIL-4, human granulocyte-macrophage colony-stimulating factor (hGM-CSF), and murine IL-2 (mIL-2) were used as model cytokines and fused at their C terminus to the glycosylphosphatidylinositol (GPI) acceptor sequence of human Fcgamma receptor III (CD16b). We show here that genetically modified cytokines are all well expressed on 293 producer cells. However, only molecules equipped with GPI anchors but not those linked to transmembrane/intracellular regions of type I membrane proteins are efficiently targeted to lipid rafts and consequently to virus-like particles (VLP) induced by Moloney murine leukemia virus Gag-Pol. hIL-4::GPI and hGM-CSF::GPI coexpressed on VLP were found to differentiate monocytes towards dendritic cells. Apart from myeloid-committed cell types, VLP-bound cytokines also act efficiently on lymphocytes. hIL-2::GPI strongly costimulated T-cell receptor (TCR)/CD3 dependent T-cell activation in vitro and mIL-2::GPI-coactivated antigen-specific T cells in vivo. On a molar basis, the functional activity of VLP-bound hIL-2::GPI was found to be comparable to that of soluble hIL-2. VLP decorated with hIL-2::GPI and coexpressing a TCR/CD3 ligand have an IL-2-specific activity of 5 x 10(4) units/mg protein. Virus particles decorated with lipid-modified cytokines might help to improve viral strains for vaccination purposes, the propagation of factor-dependent cell types, as well as gene transfer by viral systems in the future.

Integrating retroviral cassette extends gene delivery of HSV-1 expression vectors to dividing cells.

Retroviral vectors have long been used in a wide variety of gene transfer applications but have certain drawbacks, such as small cargo size, limited tropism, and low titers. HSV expression vectors overcome these disadvantages, but, because they persist in target cells as nonreplicative episomes, they are not retained in all the progeny of dividing cells. Chimeric HSV/AAV products that can mediate transgene integration in human mitotic cells have been constructed, but, to date, genetic modification of dividing cells in animal models using HSV products has not been possible. Here, we report the construction of hybrid HSV/retroviral vectors that exhibit up to 50-fold higher transgene integration efficiency compared to vectors containing only HSV-1 components. Efficient integration of a retroviral transgene cassette encoding pac in human cells required expression of the Moloney murine leukemia virus gag-pol genes, but in murine cells, could also be mediated by endogenous activities, albeit at a lower level. Gene delivery was equally efficient in BHK21, a cell line resistant to retroviral infection, and transgene retention and expression were observed to be stable for least one month in Hs683 human glioma cells. These vectors have wide applications for the genetic modification of many cell types.

Transgene amplification and persistence after delivery of retroviral vector and packaging functions with E1/E4-deleted adenoviruses

Adenovirus DNA is rapidly lost in actively dividing cells. In addition, first-generation (E1-defective) vectors trigger a strong cytotoxicity that impairs the duration of transgene expression. To solve these issues, we have developed a chimeric vector system that uses E1/E4 doubly defective adenoviruses for efficient production of infectious retroviral vectors. The retroviral vector sequences and packaging functions were split into two E1/E3/E4-deleted adenoviral vectors: the Moloney murine leukemia virus gag-pol cistron was expressed from the human EF1 alpha (elongation factor) promoter (AdGAG/POL), whereas the thymidine kinase transgene, embedded in a retroviral vector context, and an amphotropic retroviral envelope cassette were included within a second adenovirus (AdTK/ENV). This chimeric vector system was evaluated with a special emphasis on recombinant retrovirus production in vitro, as well as transgene amplification and persistence in vivo. Retrovirus titers of >10(5) infectious units/mL were routinely obtained in W162 cells coinfected with both recombinant adenoviruses. Long-term transgene persistence (up to 3 months) was demonstrated in vitro in two different cell lines coinfected with AdGAG/POL and AdTK/ENV, and correlated with the detection of specific provirus sequences. A 10- to 50-fold transgene amplification also was demonstrated in an in vivo tumor model infected with the Ad/Rt chimeric vector system. The chimeric vector system described herein combines the efficiency of gene delivery by recombinant adenoviruses with the integrative properties of infectious retroviral vectors. This versatile vector system may open up new avenues for efficient production of oncogenic, but also non-oncogenic, retroviruses from cells of non-murine origin.

Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus

We have constructed hybrid retrovirus packaging cell lines that express the gibbon ape leukemia virus env and the Moloney murine leukemia virus gag-pol proteins. These cells were used to produce a retrovirus vector at over 10(6) CFU/ml, with a host range that included rat, hamster, bovine, cat, dog, monkey, and human cells. The gag-pol and env expression plasmids were separately transfected to reduce the potential for helper virus production, which was not observed. The NIH 3T3 mouse cells from which the packaging lines were made are not infectable by gibbon ape leukemia virus; thus, the generation and spread of possible recombinant viruses in the packaging cells is greatly reduced. These simian virus-based packaging cells extend the host range of currently available murine and avian packaging cells and should be useful for efficient gene transfer into higher mammals.