Abstract
BackgroundMouse mammary tumor virus (MMTV) is a complex, milk-born betaretrovirus, which preferentially infects dendritic cells (DC) in the gastrointestinal tract and then spreads to T and B lymphocytes and finally to the mammary gland. It is not clear how the prototypic betaretrovirus infects mucosal DCs and naïve lymphocytes as these cells are considered to be non-proliferative. Studies of MMTV biology have been hampered by the difficulty of obtaining sufficient virus/vector titers after transfection of a molecular clone in cultured cells. To surmount this barrier we developed a novel MMTV-based vector system with a split genome design containing potent posttranscriptional regulatory functions.ResultsUsing this system, vector particles were produced to markedly greater titers (>1000-fold) than those obtained previously. The titers (>106 transduction units /ml) were comparable to those achieved with lentiviral or gammaretroviral vectors. Importantly, the vector transduced the enhanced green fluorescence protein gene into the chromosomes of non-dividing cells, such as cells arrested at the G2/M phase of the cell cycle and unstimulated hematopoietic progenitor cells, at an efficiency similar to that obtained with the HIV-1-based vector. In contrast to HIV-1, MMTV transductions were not affected by knocking down the expression of a factor involved in nuclear import of the HIV-1 pre-integration complexes, TNPO3. In contrast to HIV-1, the MMTV-based vector did not preferentially integrate in transcription units. Additionally, no preference for integration near transcription start sites, the regions preferentially targeted by gammaretroviral vectors, was observed. The vector derived from MMTV exhibits a random integration pattern.ConclusionsOverall, the betaretroviral vector system should facilitate molecular virology studies of the prototypic betaretrovirus as well as studies attempting to elucidate fundamental cellular processes such as nuclear import pathways. Random integration in cycling and non-cycling cells may be applicable in unbiased gene delivery.
Highlights
Mouse mammary tumor virus (MMTV) is a complex, milk-born betaretrovirus, which preferentially infects dendritic cells (DC) in the gastrointestinal tract and spreads to T and B lymphocytes and to the mammary gland
Description of MMTV-based vector with a split genome design To overcome the difficulty to produce MMTV vector to high titers that would enable in-depth studies of molecular virology of the prototypic betaretrovirus, we developed a novel betaretrovirus vector production system
The plasmid contains the Mtv-1 sequences upstream of the EcoRI site in pol and a sequence derived from the exogenous virus from GR mice [MMTV(GR)] downstream of this site (Figure 1A, the integrase-coding portion of pol is derived from the MMTV(GR))
Summary
Mouse mammary tumor virus (MMTV) is a complex, milk-born betaretrovirus, which preferentially infects dendritic cells (DC) in the gastrointestinal tract and spreads to T and B lymphocytes and to the mammary gland. Studies of the prototypic betaretrovirus have been hampered due to a low virus production from natural virus producers (such as cell lines derived from mammary tumors), as well as insufficient MMTV-based vector production after transfection of molecular clones into cultured cells This is a result of the complex regulation of virus gene expression, which is dependent on the glucocorticoid hormone, stimulating the MMTV promoter, and on a functional analogue of the HIV-1 Rev protein, Rem. Like Rev, binds to its responsive element on viral RNA, the ribonucleoprotein complex is recognized by a cellular factor CRM1, recruited to the nuclear pore complex and exported to the cytoplasm [7,8,9,10]
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