Analysis of retroviral pol gene products with antisera raised against fusion proteins produced in Escherichia coli

Tanese, Naoko; Roth, Monica J.; Goff, Stephen P.

doi:10.1128/jvi.59.2.328-340.1986

Cited by 55 publications

(32 citation statements)

References 59 publications

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“…Immunoblots were developed using goat anti-p30 (CA) (1:2000, 81S-263) and goat anti-Env (1:1000, 80S-019) (Quality Biotech) with bovine anti-goat HRP (80S-035-180) as secondary antibody (1:10,000). For IN western blot, 10 ml of viral supernatant was pelleted at 15,000 x g for 30 min and the proteins were visualized using 1:1 mix (1:1000) of antiserum from Rabbit 3 and Rabbit 4, Bleed 5 with goat anti rabbit HRP (Pierce #31460) as secondary antibody (1: 5,000) [89].…”

Section: Western Blotmentioning

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: Western Blotmentioning

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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“…Western blots were performed using anti-MuLV IN antibody (rabbit 4, bleed 5) (Tanese et al, 1986). (A) Illustration of IN proteins used in this study.…”

Section: Western Blotmentioning

confidence: 99%

Differential multimerization of Moloney murine leukemia virus integrase purified under nondenaturing conditions

Villanueva¹,

Jonsson

Jones

et al. 2003

Virology

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Retroviral integrases (IN) catalyze the integration of the reverse-transcribed viral DNA into the host genome, an essential process leading to virus replication. For Moloney murine leukemia virus (M-MuLV) IN, the limited solubility of the recombinant protein has restricted the development of biophysical and structural analyses. Herein, recombinant M-MuLV IN proteins, either full length or two nonoverlapping domain constructs, were purified under non-denaturing conditions from solubilized bacterial extracts by Ni(2+)-NTA resins. Additionally, WT IN was further purified by heparin chromatography. All of the purified proteins were shown to be active and stable. WT M-MuLV IN chromatographed with a peak corresponding with a dimer by gel filtration chromatography. In contrast, the single point mutant C209A IN migrated predominantly as a tetramer. For both proteins, fractions in equilibrium between dimers and tetramers were competent to assemble concerted two-end integrations and yielded a unique strand-transfer profile in the presence of a 28-mer U5 oligonucleotide substrate, indicative of a distinct conformation within the synaptic complex. This specific target-site selection was not observed with a shorter 20-mer U5 substrate. These studies provide the foundation for biophysical and structural analysis on M-MuLV IN and the mechanism of retroviral integration.

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“…The 1017-bp cDNA from clone 98 was subcloned in the correct reading frame into the TrpE gene of the PATH expression vector. HBlOl bacteria, transformed with the recombinant PATH vector (Tanese et al, 1986), produced the expected hsp70-TrpE fusion protein (Neumann, 1992). Bacterial lysate (1 mg proteidinjection) in complete Freund's adjuvant was injected into rabbits at ten-day intervals, and antiserum collected ten days after the fourth injection.…”

Section: Dna Probe Antigens and Antiseramentioning

confidence: 99%

Regulation of HSP70 gene expression during the life cycle of the parasitic helminth Schistosoma mansoni

Neumann

Ziv

Lantner

et al. 1993

European Journal of Biochemistry

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Analyses of RNA from different developmental stages of Schistosoma mansoni showed stagespecific expression of heat-shock protein 70 (hsp70), which is regulated by a developmental program and by stress. The developmental program, common to hsp70 and other genes (e. g. paramyosin), refers to constitutive expression in miracidia sporocyst and adult worm but not in cercariae, and to the termination of hsp70 gene transcription during sporocystkercaria transformation. Stress induction, specific to hsp70, refers to transient accumulation of high levels of hsp70 mRNA during cercariae/schistosomula transformation and in adult worms after heat shock (42 "C). Cercariae/ schistosomula transformation can be visualized as a physiological stress involving shifts in temperature (23-37 "C) and in salt concentration (from water to isotonic medium), as well as removal of tails from cercariae to yield isolated bodies that transform into schistosomula. It was found that temperature is an important factor, but not sufficient for strong induction of the hsp70 genes of schistosomula. Tail removal is an obligatory step for full induction of the hsp70 genes of schistosomula, in response to a temperature shift from 23 -37 "C. The hsp70 genes in cercariae and isolated tails do not respond to stimuli (salt and temperature increases) that strongly activate the genes in isolated bodies (i. e., schistosomula). We speculate that the hsp70 genes in intact cercariae are not inducible because the tails can produce inhibitory signals that diffuse to the bodies and suppress their hsp70 genes. This hypothesis is useful to explain the termination of hsp70 gene transcription during sporocystkercaria transformation by the inhibitory effect of the growing tail.

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Analysis of retroviral pol gene products with antisera raised against fusion proteins produced in Escherichia coli

Cited by 55 publications

References 59 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

Differential multimerization of Moloney murine leukemia virus integrase purified under nondenaturing conditions

Regulation of HSP70 gene expression during the life cycle of the parasitic helminth Schistosoma mansoni

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