Structural basis for spumavirus GAG tethering to chromatin

Lesbats, Paul; Serrao, Erik; Maskell, Daniel P.; Pye, V.E.; O’Reilly, Nicola; Lindemann, Dirk; Engelman, Alan; Cherepanov, Peter

doi:10.1073/pnas.1621159114

Cited by 48 publications

(77 citation statements)

References 58 publications

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“…Two loop regions within the PFV IN CCD-CCD dimer interface interact with the H2A-H2B heterodimer, specifically with the C-terminal helix of H2B and N-terminal of H2A [73][74][75]. Binding to the nucleosome results in a 7Å deformation of the target DNA and ultimately drives viral integration into heterochromatin regions, Lamin A/B1 rich-regions, and intergenic regions [73][74][75][76]. Transposing the loop regions of the PFV IN CCD that are implicated in the H2B interaction onto MLV IN resulted in viral titers equivalent to those of the catalytically inactive MLV D184N implying that these surface exposed loops in MLV IN are integral to IN stability or sites of secondary interactions.…”

Section: Recognition Nucleosomes By MLV Inmentioning

confidence: 99%

Disrupting MLV integrase:BET protein interaction biases integration into quiescent chromatin and delays but does not eliminate tumor activation in a MYC/Runx2 mouse model

et al. 2019

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Murine leukemia virus (MLV) integrase (IN) lacking the C-terminal tail peptide (TP) loses its interaction with the host bromodomain and extraterminal (BET) proteins and displays decreased integration at promoter/enhancers and transcriptional start sites/CpG islands. MLV lacking the IN TP via an altered open reading frame was used to infect tumorigenesis mouse model (MYC/Runx2) animals to observe integration patterns and phenotypic effects, but viral passage resulted in the restoration of the IN TP through small deletions. Mice subsequently infected with an MLV IN lacking the TP coding sequence (TP -) showed an improved median survival by 15 days compared to wild type (WT) MLV infection. Recombination with polytropic endogenous retrovirus (ERV), Pmv20, was identified in seven mice displaying both fast and slow tumorigenesis, highlighting the strong selection within the mouse to maintain the full-length IN protein. Mapping the genomic locations of MLV in tumors from an infected mouse with no observed recombination with ERVs, TP -16, showed fewer integrations at TSS and CpG islands, compared to integrations observed in WT tumors. However, this mouse succumbed to the tumor in relatively rapid fashion (34 days). Analysis of the top copy number integrants in the TP -16 tumor revealed their proximity to known MLV common insertion site genes while maintaining the MLV IN TPgenotype. Furthermore, integration mapping in K562 cells revealed an insertion preference of MLV IN TPwithin chromatin profile states associated with weakly transcribed heterochromatin with PLOS Pathogens | https://doi.fewer integrations at histone marks associated with BET proteins (H3K4me1/2/3, and H3K27Ac). While MLV IN TPshowed a decreased overall rate of tumorigenesis compared to WT virus in the MYC/Runx2 model, MLV integration still occurred at regions associated with oncogenic driver genes independently from the influence of BET proteins, either stochastically or through trans-complementation by functional endogenous Gag-Pol protein. Author summaryMany different retroviruses, including murine leukemia virus (MLV), are used as vectors for human gene therapy and cancer immunotherapies (CAR-T) because of their stable and efficient delivery of genetic material into the host DNA. However, this process can result in activation and/or disruption of cellular genes, which has resulted in the outgrowth of tumors in previous clinical trials. Of critical importance is the preferred location within the host genome at which the retrovirus integrates. Our study presents a modified MLV virus that has lost the ability to bind to the host BET proteins, the drivers of insertion at promoter/enhancer regions of highly activated genes. This is the first direct study within an animal model to examine whether infection with this type of modified MLV virus affects tumor progression. We found that the modified virus improved the survival of the well-characterized MYC/Runx2 mouse compared to infections with the wild-type MLV. Most modified viral integrants mapped into less...

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Section: Recognition Nucleosomes By MLV Inmentioning

confidence: 99%

Disrupting MLV integrase:BET protein interaction biases integration into quiescent chromatin and delays but does not eliminate tumor activation in a MYC/Runx2 mouse model

et al. 2019

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“…Furthermore, the vectors have a self-inactivating (SIN) design; deletion of the 3'LTR U3 region in the transfer plasmid is copied in in the 5'LTR sequence during packaging, rendering the deleted FVs safe for gene therapy applications. Bet deletion also renders the host cell susceptible to superinfection (multiple rounds of infection), a desirable feature for difficult to transduce cell targets [15,31,46]. PFV vectors were developed independently in the States and in Europe by Russel's [44] and Rethwilm's [47] groups, respectively.…”

Section: Features Of Fv Vectors For Hsc Gene Deliverymentioning

confidence: 99%

“…On studies performed on human HSCs transduced with FV vectors, the vectors seemed to prefer integrating upstream of transcription start sites specifically within CpG islands, with only 4.4% of the integration sites to be 50kb proximal to proto-oncogenes [13,31,41]. Although Integration Sites (IS) proximity to the genes remains largely random, the preference that the FV vectors display makes them relatively safer than GV and LV vectors, as they prefer constitutively lamina associated regions (cLAD) and less often CpGs [15] to integrate.…”

Section: Features Of Fv Vectors For Hsc Gene Deliverymentioning

confidence: 99%

“…In addition, Stem Cell Transplantation (SCT), is the ultimate marker to support the potential of a viral vector to correct a genetic defect in a HSC; the marked or corrected cells of the donor will be present in the blood cells of the recipient host and will provide phenotypic correction for a lifetime. Active transcriptional units [11] Transcriptional start sites and CpG islands [11,32] Constitutively lamina associated regions (cLAD) and less often CpGs [15]…”

Section: Introductionmentioning

confidence: 99%

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FV Vectors as Alternative Gene Vehicles for Gene Transfer in HSCs

Simantirakis

Tsironis

Vassilopoulos

2020

Viruses

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Hematopoietic Stem Cells (HSCs) are a unique population of cells, capable of reconstituting the blood system of an organism through orchestrated self-renewal and differentiation. They play a pivotal role in stem cell therapies, both autologous and allogeneic. In the field of gene and cell therapy, HSCs, genetically modified or otherwise, are used to alleviate or correct a genetic defect. In this concise review, we discuss the use of SFVpsc_huHSRV.13, formerly known as Prototype Foamy Viral (PFV or FV) vectors, as vehicles for gene delivery in HSCs. We present the properties of the FV vectors that make them ideal for HSC delivery vehicles, we review their record in HSC gene marking studies and their potential as therapeutic vectors for monogenic disorders in preclinical animal models. FVs are a safe and efficient tool for delivering genes in HSCs compared to other retroviral gene delivery systems. Novel technological advancements in their production and purification in closed systems, have allowed their production under cGMP compliant conditions. It may only be a matter of time before they find their way into the clinic.

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“…Historically, it was thought that recognition and binding of gRNA by Gag occurred in the cytoplasm or at the plasma membrane. However, given that retroviral RNA synthesis occurs in the nucleus and that the Gag proteins of many retroviruses, including Rous sarcoma virus (RSV) (1,2,(10)(11)(12)(13)(14)(15), feline immunodeficiency virus (16), foamy virus (17)(18)(19)(20)(21)(22)(23), human immunodeficiency virus type 1 (HIV-1) (13), Mason-Pfizer monkey virus (24)(25)(26), mouse mammary tumor virus (13,27), and murine leukemia virus (28), are present in the nucleus (29), it is plausible to hypothesize that retroviral Gag proteins associate with gRNA in the nucleus.…”

mentioning

confidence: 99%

Visualizing Association of the Retroviral Gag Protein with Unspliced Viral RNA in the Nucleus

Maldonado

Rice

Chen

et al. 2020

mBio

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Packaging of genomic RNA (gRNA) by retroviruses is essential for infectivity, yet the subcellular site of the initial interaction between the Gag polyprotein and gRNA remains poorly defined. Because retroviral particles are released from the plasma membrane, it was previously thought that Gag proteins initially bound to gRNA in the cytoplasm or at the plasma membrane. However, the Gag protein of the avian retrovirus Rous sarcoma virus (RSV) undergoes active nuclear trafficking, which is required for efficient gRNA encapsidation (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc Natl Acad Sci U S A 99:3944–3949, 2002, https://doi.org/10.1073/pnas.062652199; R. Garbitt-Hirst, S. P. Kenney, and L. J. Parent, J Virol 83:6790–6797, 2009, https://doi.org/10.1128/JVI.00101-09). These results raise the intriguing possibility that the primary contact between Gag and gRNA might occur in the nucleus. To examine this possibility, we created a RSV proviral construct that includes 24 tandem repeats of MS2 RNA stem-loops, making it possible to track RSV viral RNA (vRNA) in live cells in which a fluorophore-conjugated MS2 coat protein is coexpressed. Using confocal microscopy, we observed that both wild-type Gag and a nuclear export mutant (Gag.L219A) colocalized with vRNA in the nucleus. In live-cell time-lapse images, the wild-type Gag protein trafficked together with vRNA as a single ribonucleoprotein (RNP) complex in the nucleoplasm near the nuclear periphery, appearing to traverse the nuclear envelope into the cytoplasm. Furthermore, biophysical imaging methods suggest that Gag and the unspliced vRNA physically interact in the nucleus. Taken together, these data suggest that RSV Gag binds unspliced vRNA to export it from the nucleus, possibly for packaging into virions as the viral genome. IMPORTANCE Retroviruses cause severe diseases in animals and humans, including cancer and acquired immunodeficiency syndromes. To propagate infection, retroviruses assemble new virus particles that contain viral proteins and unspliced vRNA to use as gRNA. Despite the critical requirement for gRNA packaging, the molecular mechanisms governing the identification and selection of gRNA by the Gag protein remain poorly understood. In this report, we demonstrate that the Rous sarcoma virus (RSV) Gag protein colocalizes with unspliced vRNA in the nucleus in the interchromatin space. Using live-cell confocal imaging, RSV Gag and unspliced vRNA were observed to move together from inside the nucleus across the nuclear envelope, suggesting that the Gag-gRNA complex initially forms in the nucleus and undergoes nuclear export into the cytoplasm as a viral ribonucleoprotein (vRNP) complex.

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Structural basis for spumavirus GAG tethering to chromatin

Cited by 48 publications

References 58 publications

Disrupting MLV integrase:BET protein interaction biases integration into quiescent chromatin and delays but does not eliminate tumor activation in a MYC/Runx2 mouse model

Disrupting MLV integrase:BET protein interaction biases integration into quiescent chromatin and delays but does not eliminate tumor activation in a MYC/Runx2 mouse model

FV Vectors as Alternative Gene Vehicles for Gene Transfer in HSCs

Visualizing Association of the Retroviral Gag Protein with Unspliced Viral RNA in the Nucleus

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