Molecular hydrogen and catalytic combustion in the production of hyperpolarized 83Kr and 129Xe MRI contrast agents

Hyperpolarized (hp) 83Kr is a promising MRI contrast agent for the diagnosis of pulmonary diseases affecting the surface of the respiratory zone. However, the distinct physical properties of 83Kr that enable unique MRI contrast also complicate the production of hp 83Kr. This work presents a radically new approach in the generation of hp 83Kr that can likewise be utilized for the production of hp 129Xe. Molecular nitrogen, typically used as buffer gas in spin exchange optical pumping (SEOP), was replaced by molecular hydrogen without penalty for the achievable hyperpolarization. In this particular study, the highest obtained nuclear spin polarizations were P = 29 % for 83Kr and P = 63 % for 129Xe. The results were reproduced over many SEOP cycles despite the laser induced on-resonance formation of rubidium hydride (RbH). Following SEOP, the H2 was reactively removed via catalytic combustion without measurable losses in hyperpolarized spin state of either 83Kr or 129Xe. Highly spin polarized 83Kr can now be purified for the first time to provide high signal intensity for the advancement of in vivo hp 83Kr MRI. More generally, a chemical reaction appears as a viable alternative to the cryogenic separation process, the primary purification method of hp 129Xe for the past 2 . decades. The inherent simplicity of the combustion process will facilitate hp 129Xe production and should allow for on-demand continuous flow of purified and highly spin polarized 129Xe

Correction: Interactions of prototype foamy virus capsids with host cell polo-like kinases are important for efficient viral DNA integration.

[This corrects the article DOI: 10.1371/journal.ppat.1005860.]

Structural basis for spumavirus GAG tethering to chromatin

The interactions between a retrovirus and host cell chromatin that underlie integration and provirus expression are poorly understood. The prototype foamy virus (PFV) structural protein GAG associates with chromosomes via a chromatin-binding sequence (CBS) located within its C-terminal region. Here, we show that the PFV CBS is essential and sufficient for a direct interaction with nucleosomes and present a crystal structure of the CBS bound to a mononucleosome. The CBS interacts with the histone octamer, engaging the H2A–H2B acidic patch in a manner similar to other acidic patch-binding proteins such as herpesvirus latency-associated nuclear antigen (LANA). Substitutions of the invariant arginine anchor residue in GAG result in global redistribution of PFV and macaque simian foamy virus (SFVmac) integration sites toward centromeres, dampening the resulting proviral expression without affecting the overall efficiency of integration. Our findings underscore the importance of retroviral structural proteins for integration site selection and the avoidance of genomic junkyards

Формирование имиджа компании как работодателя: аннотация к дипломной работе / Баглай Ольга Мирославовна; Факультет философии и социальных наук; Кафедра социальной коммуникации; научный руководитель: Купчинова Татьяна Владимировна

Unlike for other retroviruses, only a few host cell factors that aid the replication of foamy viruses (FVs) via interaction with viral structural components are known. Using a yeast-two-hybrid (Y2H) screen with prototype FV (PFV) Gag protein as bait we identified human polo-like kinase 2 (hPLK2), a member of cell cycle regulatory kinases, as a new interactor of PFV capsids. Further Y2H studies confirmed interaction of PFV Gag with several PLKs of both human and rat origin. A consensus Ser-Thr/Ser-Pro (S-T/S-P) motif in Gag, which is conserved among primate FVs and phosphorylated in PFV virions, was essential for recognition by PLKs. In the case of rat PLK2, functional kinase and polo-box domains were required for interaction with PFV Gag. Fluorescently-tagged PFV Gag, through its chromatin tethering function, selectively relocalized ectopically expressed eGFP-tagged PLK proteins to mitotic chromosomes in a Gag STP motif-dependent manner, confirming a specific and dominant nature of the Gag-PLK interaction in mammalian cells. The functional relevance of the Gag-PLK interaction was examined in the context of replication-competent FVs and single-round PFV vectors. Although STP motif mutated viruses displayed wild type (wt) particle release, RNA packaging and intra-particle reverse transcription, their replication capacity was decreased 3-fold in single-cycle infections, and up to 20-fold in spreading infections over an extended time period. Strikingly similar defects were observed when cells infected with single-round wt Gag PFV vectors were treated with a pan PLK inhibitor. Analysis of entry kinetics of the mutant viruses indicated a post-fusion defect resulting in delayed and reduced integration, which was accompanied with an enhanced preference to integrate into heterochromatin. We conclude that interaction between PFV Gag and cellular PLK proteins is important for early replication steps of PFV within host cells

Modulation of the functional interfaces between retroviral intasomes and the human nucleosome

Retroviral integration into cell chromatin requires the formation of integrase-viral DNA complexes, called intasomes, and their interaction with the target DNA wrapped around nucleosomes. To further study this mechanism we developed an alphaLISA approach using the prototype foamy virus (PFV) intasome and human nucleosome. This system allowed us to monitor the association between both partners and investigate the protein/protein and protein/DNA interactions engaged in the association with chromatin. Using this approach, we next screened the chemical OncoSET library and selected small molecules that could modulate the intasome/nucleosome complex. Molecules were selected as acting either on the DNA topology within the nucleosome or on the integrase/histone tail interactions. Within these compounds, doxorubicin and histone binders calixarenes were characterized using biochemical, structural and cellular approaches. These drugs were shown to inhibit PFV and HIV-1 integration in vitro as well as HIV-1 infection in primary PBMCs cells. Our work provides new information about intasome-nucleosome interaction determinants and paves the way for further unedited antiviral strategies that target the final step of intasome/chromatin anchoring

Intasome architecture and chromatin density modulate retroviral integration into nucleosome

BACKGROUND: Retroviral integration depends on the interaction between intasomes, host chromatin and cellular targeting cofactors as LEDGF/p75 or BET proteins. Previous studies indicated that the retroviral integrase, by itself, may play a role in the local integration site selection within nucleosomal target DNA. We focused our study on this local association by analyzing the intrinsic properties of various retroviral intasomes to functionally accommodate different chromatin structures in the lack of other cofactors. RESULTS: Using in vitro conditions allowing the efficient catalysis of full site integration without these cofactors, we show that distinct retroviral integrases are not equally affected by chromatin compactness. Indeed, while PFV and MLV integration reactions are favored into dense and stable nucleosomes, HIV-1 and ASV concerted integration reactions are preferred into poorly dense chromatin regions of our nucleosomal acceptor templates. Predicted nucleosome occupancy around integration sites identified in infected cells suggests the presence of a nucleosome at the MLV and HIV-1 integration sites surrounded by differently dense chromatin. Further analyses of the relationships between the in vitro integration site selectivity and the structure of the inserted DNA indicate that structural constraints within intasomes could account for their ability to accommodate nucleosomal DNA and could dictate their capability to bind nucleosomes functionally in these specific chromatin contexts. CONCLUSIONS: Thus, both intasome architecture and compactness of the chromatin surrounding the targeted nucleosome appear important determinants of the retroviral integration site selectivity. This supports a mechanism involving a global targeting of the intasomes toward suitable chromatin regions followed by a local integration site selection modulated by the intrinsic structural constraints of the intasomes governing the target DNA bending and dictating their sensitivity toward suitable specific nucleosomal structures and density

In vitro initial attachment of HIV-1 integrase to viral ends: control of the DNA specific interaction by the oligomerization state

HIV-1 integrase (IN) oligomerization and DNA recognition are crucial steps for the subsequent events of the integration reaction. Recent advances described the involvement of stable intermediary complexes including dimers and tetramers in the in vitro integration processes, but the initial attachment events and IN positioning on viral ends are not clearly understood. In order to determine the role of the different IN oligomeric complexes in these early steps, we performed in vitro functional analysis comparing IN preparations having different oligomerization properties. We demonstrate that in vitro IN concerted integration activity on a long DNA substrate containing both specific viral and nonspecific DNA sequences is highly dependent on binding of preformed dimers to viral ends. In addition, we show that IN monomers bound to nonspecific DNA can also fold into functionally different oligomeric complexes displaying nonspecific double-strand DNA break activity in contrast to the well known single strand cut catalyzed by associated IN. Our results imply that the efficient formation of the active integration complex highly requires the early correct positioning of monomeric integrase or the direct binding of preformed dimers on the viral ends. Taken together the data indicates that IN oligomerization controls both the enzyme specificity and activity

The HIV-1 Integrase α4-Helix Involved in LTR-DNA Recognition Is also a Highly Antigenic Peptide Element

Monoclonal antibodies (MAbas) constitute remarkable tools to analyze the relationship between the structure and the function of a protein. By immunizing a mouse with a 29mer peptide (K159) formed by residues 147 to 175 of the HIV-1 integrase (IN), we obtained a monoclonal antibody (MAba4) recognizing an epitope lying in the N-terminal portion of K159 (residues 147–166 of IN). The boundaries of the epitope were determined in ELISA assays using peptide truncation and amino acid substitutions. The epitope in K159 or as a free peptide (pep-a4) was mostly a random coil in solution, while in the CCD (catalytic core domain) crystal, the homologous segment displayed an amphipathic helix structure (α4-helix) at the protein surface. Despite this conformational difference, a strong antigenic crossreactivity was observed between pep-a4 and the protein segment, as well as K156, a stabilized analogue of pep-a4 constrained into helix by seven helicogenic mutations, most of them involving hydrophobic residues. We concluded that the epitope is freely accessible to the antibody inside the protein and that its recognition by the antibody is not influenced by the conformation of its backbone and the chemistry of amino acids submitted to helicogenic mutations. In contrast, the AA →Glu mutations of the hydrophilic residues Gln148, Lys156 and Lys159, known for their interactions with LTRs (long terminal repeats) and inhibitors (

Repressive LTR Nucleosome Positioning by the BAF Complex Is Required for HIV Latency

The SWI/SNF BAF chromatin remodeling complex generates a repressive nucleosome structure at the HIV LTR conducive to establishment and maintenance of HIV latency, while PBAF augments HIV transcription