Use of dna end joining activity of a xenopus laevis egg extract for construction of deletions and expression vectors for hiv-1 tat and rev proteins

The Ku70/Ku80 heterodimer, or Ku, is the central component of the nonhomologous end joining (NHEJ) pathway of double strand break (DSB) repair. Because Ku forms a ring through which the DSB threads, it likely becomes topologically attached to DNA during repair. The mechanism for its removal was unknown. Using a method to identify proteins recruited to DSBs in Xenopus laevis egg extract, we show that DSB-containing DNAs accumulate members of the Skp1–Cul1–F-box complex and K48-linked polyubiquitylated proteins in addition to known repair proteins. We demonstrate that Ku80 is degraded in response to DSBs in a ubiquitin-mediated manner. Strikingly, K48-linked polyubiquitylation, but not proteasomal degradation, is required for the efficient removal of Ku80 from DNA. This removal is DNA length dependent, as Ku80 is retained on duplex oligonucleotides. Finally, NHEJ completion and removal of Ku80 from DNA are independent from one another. We propose that DSB-induced ubiquitylation of Ku80 provides a mechanism to efficiently eliminate Ku from DNA for pre- and postrepair processes.

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Tax induces the recruitment of NF-kB to unintegrated HIV-1 DNA to rescue viral gene expression and replication

10.1101/2021.02.16.431555 ◽

2021 ◽

Author(s):

Ishak D. Irwan ◽

Bryan R. Cullen

Keyword(s):

Gene Expression ◽

T Cells ◽

Lentiviral Vectors ◽

Expression Vectors ◽

Viral Gene ◽

Epigenetic Marks ◽

Tax Protein ◽

Human T Cell ◽

Activation Of Transcription ◽

Hiv 1

AbstractWe have previously reported that the normally essential step of integration of the HIV-1 proviral DNA intermediate into the host cell genome becomes dispensable in T cells that express the Human T cell leukemia virus 1 (HTLV-1) Tax protein. The rescue of integrase (IN) deficient HIV-1 replication by Tax results from the strong activation of transcription from the long terminal repeat (LTR) promoter on episomal HIV-1 DNA, an effect that is closely correlated with the recruitment of activating epigenetic marks, such as H3Ac, and depletion of repressive epigenetic marks, such as H3K9me3, from chromatinized unintegrated proviruses. In addition, activation of transcription from unintegrated HIV-1 DNA coincides with the recruitment of NF-kB to the two NF-kB binding sites found in the HIV-1 LTR enhancer. Here we report that the recruitment of NF-kB to unintegrated viral DNA precedes, and is a prerequisite for, Tax-induced changes in epigenetic marks, so that an IN-HIV-1 mutant lacking both LTR NF-kB sites is entirely non-responsive to Tax and fails to undergo the epigenetic changes listed above. We also report that heterologous promoters introduced into IN-HIV-1-based vectors are transcriptionally active even in the absence of Tax. Finally, we failed to reproduce a recent report arguing that heterologous promoters introduced into IN-vectors based on HIV-1 are more active if the HIV-1 promoter and enhancer, located in the LTR U3 region, are deleted, in a so-called self inactivating or SIN lentivector design.ImportanceIntegrase-deficient expression vectors based on HIV-1 are becoming increasingly popular as tools for gene therapy in vivo due to their inability to cause insertional mutagenesis. However, many IN-lentiviral vectors are able to achieve only low levels of gene expression and methods to increase this low level have not been extensively explored. Here we analyze how the HTLV-1 Tax protein is able to rescue the replication of IN-HIV-1 in T cells and describe IN-lentiviral vectors that are able to express a heterologous gene effectively.

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Centromere and Kinetochore Assembly in Xenopus laevis Egg Extract

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot102509 ◽

2018 ◽

Vol 2018 (9) ◽

pp. pdb.prot102509 ◽

Cited By ~ 1

Author(s):

Julio C. Flores Servin ◽

Aaron F. Straight

Keyword(s):

Xenopus Laevis ◽

Kinetochore Assembly ◽

Egg Extract

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Recombinant BCG Expressing HTI Prime and Recombinant ChAdOx1 Boost Is Safe and Elicits HIV-1-Specific T-Cell Responses in BALB/c Mice

Vaccines ◽

10.3390/vaccines7030078 ◽

2019 ◽

Vol 7 (3) ◽

pp. 78 ◽

Cited By ~ 7

Author(s):

Athina Kilpeläinen ◽

Narcís Saubi ◽

Núria Guitart ◽

Alex Olvera ◽

Tomáš Hanke ◽

...

Keyword(s):

T Cell ◽

Miliary Tuberculosis ◽

T Cell Responses ◽

T Cell Response ◽

Expression Vectors ◽

Effective Vaccine ◽

Vaccine Platform ◽

Cell Responses ◽

Hiv 1 ◽

Recombinant Bcg

Despite the availability of anti-retroviral therapy, HIV-1 infection remains a massive burden on healthcare systems. Bacillus Calmette-Guérin (BCG), the only licensed vaccine against tuberculosis, confers protection against meningitis and miliary tuberculosis in infants. Recombinant BCG has been used as a vaccine vehicle to express both HIV-1 and Simian Immunodeficiemcy Virus (SIV) immunogens. In this study, we constructed an integrative E. coli-mycobacterial shuttle plasmid, p2auxo.HTI.int, expressing the HIVACAT T-cell immunogen (HTI). The plasmid was transformed into a lysine auxotrophic Mycobacterium bovis BCG strain (BCGΔLys) to generate the vaccine BCG.HTI2auxo.int. The DNA sequence coding for the HTI immunogen and HTI protein expression were confirmed, and working vaccine stocks were genetically and phenotypically characterized. We demonstrated that the vaccine was stable in vitro for 35 bacterial generations, and that when delivered in combination with chimpanzee adenovirus (ChAd)Ox1.HTI in adult BALB/c mice, it was well tolerated and induced HIV-1-specific T-cell responses. Specifically, priming with BCG.HTI2auxo.int doubled the magnitude of the T-cell response in comparison with ChAdOx1.HTI alone while maintaining its breadth. The use of integrative expression vectors and novel HIV-1 immunogens can aid in improving mycobacterial vaccine stability as well as specific immunogenicity. This vaccine candidate may be a useful tool in the development of an effective vaccine platform for priming protective responses against HIV-1/TB and other prevalent pediatric pathogens.

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Novel role of mortalin in attenuating HIV-1 Tat-mediated astrogliosis

Journal of Neuroinflammation ◽

10.1186/s12974-020-01912-3 ◽

2020 ◽

Vol 17 (1) ◽

Author(s):

Priyanka ◽

Renu Wadhwa ◽

Rituparna Chaudhuri ◽

Tapas Chandra Nag ◽

Pankaj Seth

Keyword(s):

Western Blotting ◽

Neuronal Death ◽

Neuronal Damage ◽

Fetal Brain ◽

Luciferase Assay ◽

Expression Vectors ◽

Neurocognitive Dysfunction ◽

Hiv 1

Abstract Background In human immunodeficiency virus-1 (HIV-1) infection, activation of astrocytes induces imbalance in physiological functions due to perturbed astrocytic functions that unleashes toxicity on neurons. This leads to inflammatory response finally culminating into neurocognitive dysfunction. In neuroAIDS, HIV-1 protein, transactivator of transcription (Tat) is detected in the cerebrospinal fluid of infected patients. Mortalin, a multifunctional protein, has anti-inflammatory role following its activation in various stress conditions. Recent studies demonstrate downregulation of mortalin in neurodegenerative diseases. Here, we explored the mechanisms of mortalin in modulating HIV-1 Tat-mediated neuroinflammation. Methods Expression of mortalin in autopsy section in normal and diseased individuals were examined using immunohistochemistry. To decipher the role of mortalin in HIV-1 Tat-induced activation, human fetal brain-derived astrocytes were transiently transfected with Tat and mortalin using expression vectors. HIV-1 Tat-mediated damage was analyzed using RT-PCR and western blotting. Modulatory role of mortalin was examined by coexpressing it with Tat, followed by examination of mitochondrial morphodynamics using biochemical assay and confocal and electron microscopy. Extracellular ATP release was monitored using luciferase assay. Neuroinflammation in astrocytes was examined using flow cytometry, dye based study, immunocytochemistry, immunoprecipitation, and western blotting. Indirect neuronal damage was also analyzed. Results HIV-1 Tat downregulates the expression of mortalin in astrocytes, and this is corroborated with autopsy sections of HIV-1 patients. We found that overexpression of mortalin with Tat reduced inflammation and also rescued astrocytic-mediated neuronal death. Using bioinformatics, we discovered that binding of mortalin with Tat leads to Tat degradation and rescues the cell from neuroinflammation. Blocking of proteosomal pathway rescued the Tat degradation and revealed the ubiquitination of Tat. Conclusion Overall, our data demonstrated the protective role of mortalin in combating HIV-1 Tat-mediated damage. We also showed that mortalin could degrade Tat through direct binding with HIV-1 Tat. Overexpression of mortalin in the presence of Tat could significantly reduce cytotoxic effects of Tat in astrocytes. Indirect neuronal death was also found to be rescued. Our in vitro findings were validated as we found attenuated expression of mortalin in the autopsy sections of HIV-1 patients.

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A pilot study of raltegravir and cisplatin in head and neck squamous cell carcinoma (HNSCC).

Journal of Clinical Oncology ◽

10.1200/jco.2012.30.15_suppl.tps5602 ◽

2012 ◽

Vol 30 (15_suppl) ◽

pp. TPS5602-TPS5602

Author(s):

Julie E. Bauman ◽

Garth T Olson ◽

Michael Spafford ◽

Michael Nuara ◽

Sagus Sampath ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Pilot Study ◽

Replication Fork ◽

Annexin V ◽

Integrase Inhibitor ◽

Primary Objective ◽

End Joining ◽

Cancer Society ◽

Hiv 1

TPS5602 Background: Metnase is a recently characterized DNA repair component present in anthropoid primates. Metnase, the fusion of a SET histone methylase domain and a Transposase nuclease (Tn) domain, enhances DNA double-stranded break (DSB) repair. The Tn domain trims free DNA ends for optimal end-joining, and is required to re-start stalled replication forks. The SET domain di-methylates H3K36 at the DSB, recruiting non-homologous end-joining repair components. Cisplatin (CDDP), the central chemotherapy in HNSCC, covalently binds DNA leading to intra- and interstrand crosslinks (ICL). In addition to blocking strand transcription and segregation, the ICL stalls DNA replication fork progression leading to collapse and DSB. The role of Metnase in DNA repair, including overcoming the stalled replication fork, makes it a potential therapeutic target. We hypothesize that Metnase inhibition may be a useful adjunct to CDDP. 3D modeling of the Metnase Tn domain identified significant homology with human immunodeficiency virus 1 (HIV-1) integrase. A virtual screen of the Chem Div library identified the HIV-1 integrase inhibitor, raltegravir, as a lead compound for inhibiting the Metnase Tn domain. We designed a pilot study in HNSCC to evaluate potentiation of CDDP DNA damage by raltegravir. Methods: The primary objective of this window-of-opportunity study is to analyze biomarkers of DNA damage, fork arrest, and apoptosis in serial tumor biopsies from patients (pts) with HNSCC undergoing CDDP, with and without raltegravir. Eligible pts: 1) have HNSCC with tumor site amenable to repeat, awake biopsy; 2) are appropriate candidates for CDDP. Pts are treated with 2 doses of CDDP 30 mg/m2. One CDDP dose is administered with raltegravir 400 mg bid for 5 days, starting the day before CDDP. Pts undergo 3 research biopsies, at baseline and 48-72 hours after each CDDP. Serial biopsies will be evaluated for expression changes in γH2AX, Annexin V, pChk1, pChk2, and p53BP1 by immunohistochemistry. Numerical increase in biomarker expression in tumors exposed to CDDP-raltegravir vs. CDDP will justify development of a phase I/II study. Four of 12 pts have enrolled and completed all biopsies. Supported by a grant from the American Cancer Society.

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Promoter-proximal poly(A) sites are processed efficiently, but the RNA products are unstable in the nucleus.

Molecular and Cellular Biology ◽

10.1128/mcb.17.4.2127 ◽

1997 ◽

Vol 17 (4) ◽

pp. 2127-2135 ◽

Cited By ~ 5

Author(s):

J M Scott ◽

M J Imperiale

Keyword(s):

Steady State ◽

Nucleocytoplasmic Transport ◽

Proximity Effects ◽

Expression Vectors ◽

Genome Replication ◽

Intact Cells ◽

A Site ◽

Hiv 1

The presence of two polyadenylation signals in the primary transcript of the human immunodeficiency virus type 1 (HIV-1) provirus leads to a requirement for regulation of 3'-end processing. To ensure that viral genome replication and gene expression occur, polyadenylation must occur at the poly(A) site transcribed from the 3' long terminal repeat (LTR) but not the 5' LTR. Models that have been proposed to explain this regulation include (i) inhibition of the 5' site as a result of proximity to the promoter and (ii) enhancement of the 3' site by U3 sequences that are transcribed upstream of only the 3' poly(A) site. In previous studies designed to investigate these models, a reduction in the levels of steady-state RNA was observed when the HIV-1 poly(A) site was placed within 500 nucleotides of the cap site. Although these findings were interpreted to be the result of promoter proximity effects on 3'-end processing, in vitro studies demonstrated that the HIV-1 poly(A) site was equally functional in promoter-proximal and promoter-distal positions. These results led to the hypothesis that, in vivo, the poly(A) site is fully active at this close distance but the short transcripts produced are highly unstable in the nucleus and undergo rapid degradation, precluding their appearance as abundant mRNAs in the steady-state pool. To investigate the biogenesis of these short RNAs in vivo, experiments were performed to examine directly the nuclear processing rates of the HIV-1 poly(A) site in intact cells. By using recombinant adenoviruses as expression vectors, it is now demonstrated conclusively that the HIV-1 poly(A) site is indeed processed at equivalent levels independent of its distance from the promoter. Although transcripts containing the promoter-proximal poly(A) site are processed efficiently, most undergo degradation in the nucleus instead of nucleocytoplasmic transport.

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Extracts from eggs and oocytes of Xenopus laevis differ in their capacities for nuclear assembly and DNA replication

Journal of Cell Science ◽

10.1242/jcs.97.1.177 ◽

1990 ◽

Vol 97 (1) ◽

pp. 177-184

Author(s):

L.S. Cox ◽

G.H. Leno

Keyword(s):

Dna Replication ◽

Xenopus Laevis ◽

High Speed ◽

De Novo ◽

Soluble Fraction ◽

Sperm Chromatin ◽

Nuclear Assembly ◽

Egg Extract ◽

Egg Extracts ◽

Sperm Nuclei

We describe a cell-free extract derived from the oocytes of Xenopus laevis. The oocyte extract is capable of decondensing sperm chromatin and of replicating single-stranded DNA in a semiconservative, aphidicolin-sensitive manner. In addition, oocyte extract supports the elongation phase of DNA synthesis in nuclei that have been preinitiated for replication. All of these properties are shared by previously described egg extracts. However, oocyte extracts differ from egg extracts in two important ways. First, they cannot support nuclear assembly, as visualised by phase-contrast, fluorescence and electron microscopy. Second, they do not initiate replication on chromatin or nuclei de novo. Crude low-speed supernatants can be partially fractionated into soluble and vesicular components by high-speed centrifugation. Such fractions from eggs can be functionally reconstituted, but the oocyte soluble fraction does not acquire the ability to assemble nuclei, or replicate them, even when supplemented with the egg vesicular fraction. Similarly, oocyte vesicles cannot substitute for egg vesicles on reconstitution with the egg soluble fraction. When the requirement for nuclear assembly is bypassed by using preformed, quiescent nuclei, replication is observed in egg but not oocyte extracts. However, the oocyte extract is not inhibitory for initiation of replication, as it does not prevent replication of sperm nuclei when mixed with egg extract. We suggest that the different capabilities of egg and oocyte extracts could provide the basis of an assay system for identifying factors involved in the initiation of DNA replication.

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