Mouse mammary tumor virus-based vector transduces non-dividing cells, enters the nucleus via a TNPO3-independent pathway and integrates in a less biased fashion than other retroviruses

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.

C3H strain of mouse mammary tumour virus, like GR strain, infects human mammary epithelial cells, albeit less efficiently than murine mammary epithelial cells

“…To investigate whether MMTV(C3H) infects non-murine cells, single-round transduction experiments were carried out initially using MMTV-based vector particles carrying MMTV Env (GR or C3H), ecotropic MLV Env or amphotropic 4070A MLV Env (generated using our recently described novel high-titre MMTV-based vector production system (Konstantoulas & Indik, 2014)). Prior to transduction, virus particle titres were determined by real-time TaqMan reverse transcriptase (RT)-PCR targeting the egfp gene and normalized virus levels were used for the transduction of human (Hs578T) and murine (NMuMG) mammary epithelial cells.…”

“…The construction of the packaging construct (pCMgpRRE17), the EGFP-labelled MMTV vector plasmid (pRRpCeGFPWPRE25) and the Rev expression construct (pLP2) has been outlined previously (Konstantoulas & Indik, 2014). Likewise, the ecotropic MLVEnv-encoding plasmid (pHCMV-EcoEnv; Addgene 15802) (Sena-Esteves et al, 2004), the amphotropic MLVEnv-encoding plasmid (pAlf) (Cosset et al, 1995) and the MMTV(GR)Env-encoding plasmid [pEnv(GR)] (Indik et al, 2005b;Müllner et al, 2008) have all been described previously.…”

“…Here, we used our novel, highly sensitive MMTV-based vector production system (Konstantoulas & Indik, 2014) for transduction of cells of various origins, to determine whether MMTV(C3H) could infect heterologous cells. Additionally, we analysed whether MMTV(C3H) was capable of productively infecting human cells, and finally we directly compared the infectivity of MMTV(C3H) with that of MMTV(GR).…”

Retroviral DNA Integration

“…However, the field now has a much broader appreciation of how different types of retroviruses target potentially unsafe genomic features such as genes and enhancer regions. 243 In this vein, vectors based on α-retroviruses, 386 β-retroviruses, 387 or spumaviruses, 388 each of which targets genes and enhancers to lesser extents than lentiviruses and γ-retroviruses, respectively, might prove safer than MLV-based vectors. The identification of the mechanisms of integration targeting for the lentiviruses and γ-retroviruses has additionally opened up new approaches to vector design.…”

Cellular and viral determinants of retroviral nuclear entry

“…The infection of PFV is reduced by only 2-fold in TNPO3 KD baby hamster kidney (BHK-21) cells with no effect on late stages, including viral assembly and release (Ali et al 2015), indicating that replication of PFV is less dependent on TNPO3. Consistently, beta-retroviruses, such as mouse mammary tumor virus, can import genetic material via a TNPO3-independent pathway in nondividing cells and can integrate randomly when using a split genome due to low viral titer (Konstantoulas and Indik 2014). Since TNPO3 is the binding partner of IN, low viral production is expected in TNPO3-depleted cells.…”