The Interaction Between Lentiviral Integrase and LEDGF: Structural and Functional Insights

Since its initial description as an HIV-1 integrase (IN) interactor seven years ago, LEDGF has become one of the best-characterized host factors involved in viral replication. Results of intensive studies in several laboratories indicated that the protein serves as a targeting factor for the lentiviral DNA integration machinery, and accounts for the characteristic preference of Lentivirus to integrate within active transcription units. The IN-LEDGF interaction has been put forward as a promising target for antiretroviral drug development and as a potential tool to improve safety of lentiviral vectors for use in gene therapy. Additionally, as a natural ligand of lentiviral IN proteins, LEDGF has been successfully used in structural biology studies of retroviral DNA integration. This review focuses on the structural aspects of the IN-LEDGF interaction and their functional consequences

3′-Processing and strand transfer catalysed by retroviral integrase in crystallo

Retroviral integrase (IN) is responsible for two consecutive reactions, which lead to insertion of a viral DNA copy into a host cell chromosome. Initially, the enzyme removes di- or trinucleotides from viral DNA ends to expose 3′-hydroxyls attached to the invariant CA dinucleotides (3′-processing reaction). Second, it inserts the processed 3′-viral DNA ends into host chromosomal DNA (strand transfer). Herein, we report a crystal structure of prototype foamy virus IN bound to viral DNA prior to 3′-processing. Furthermore, taking advantage of its dependence on divalent metal ion cofactors, we were able to freeze trap the viral enzyme in its ground states containing all the components necessary for 3′-processing or strand transfer. Our results shed light on the mechanics of retroviral DNA integration and explain why HIV IN strand transfer inhibitors are ineffective against the 3′-processing step of integration. The ground state structures moreover highlight a striking substrate mimicry utilized by the inhibitors in their binding to the IN active site and suggest ways to improve upon this clinically relevant class of small molecules

The Development and Characterisation of Everolimus Resistant Breast Cancer Cells

This thesis was submitted for the award of Doctor of Philosophy and was awarded by Brunel University LondonThe mTOR inhibitor and rapalogue everolimus was approved use in 2012, in HR+, HER-2-, post-menopausal patients, who had previously failed aromatase inhibitor treatment. mTOR pathway activation has been associated with resistance to breast cancer therapies, but how cells may become resistant to mTOR therapies themselves in breast cancer is currently not well explored, due to the relative recentness of everolimus approval. Drug resistance across all areas of cancer research is a major clinical issue, often leading to the spread of a patient’s cancer. This project set out to create in vitro breast cancer models that were resistant to everolimus, and thus explore any changes that had developed in these models, help determine the mechanisms behind resistance and discover drugs/drug combinations that could overcome resistance. Cell lines T47D and MDA-MB-361 were subsequently developed into everolimus resistant lines (EveR) over the course of 4-6 months using an on/off exposure routine. The exact mechanism behind the everolimus resistance was not fully determined but EveR cells did show multiple intriguing characteristics. An increase in dormancy and stem-cell like phenotype was noted, as revealed by a decrease in cell cycle progression and an increase in increase ALDH activity. mTORC2 components and signalling was up-regulated although siRNA down-regulation of PKCα did not decrease everolimus resistance, suggesting other mTORC2 targets may be involved. The rapalogue ‘receptor’, FKBP12, was up-regulated which was accompanied by an increased growth inhibition by the rapalogue, temsirolimus, possible due to temsirolimus lower binding affinity for FKBP12 compared to everolimus. No resistance to the dual mTOR/PI3K inhibitor BEZ-235 was observed, in line with similar published work. The combination of vitamin D/calcitriol and everolimus had no added effect compared to everolimus alone, in parental cells, but the addition of 1μM calcitriol did drastically lower EveR cell resistance to everolimus. Future work focusing on the exact nature of calcitriol’s interaction with the mTOR pathway is required to advance calcitriols role as a breast cancer therapeutic. Research with everolimus resistant breast cancer patients has not yet been published on, but the work presented here aims to help guide such studies, when they are carried out in the future

Investigating how the hand interacts with different mobile phones

In this paper we investigate the physical interaction between the hand and three types of mobile device interaction: touchscreen, physical keyboard and stylus. Through a controlled study using video observational analysis, we observed firstly, how the participants gripped the three devices and how these grips were device dependent. Secondly we looked closely at these grips to uncover how participants performed what we call micro-movements to facilitate a greater range of interaction, e.g. reaching across the keyboard. The results extend current knowledge by comparing three handheld device input methods and observing the movements, which the hand makes in five grips. The paper concludes by describing the development of a conceptual design, proposed as a provocation for the opening of dialogue on how we conceive hand usage and how it might be optimized when designed for mobile devices

Seasonal and meteorological associations with depressive symptoms in older adults:a geo-epidemiological study

Background Given increased social and physiological vulnerabilities, older adults may be particularly susceptible to environmental influences on mood. Whereas the impact of season on mood is well described for adults, studies rarely extend to elders or include objective weather data. We investigated the impact of seasonality and meteorological factors on risk of current depressive symptoms in older adults. Methods We used data on 8027 participants from the first wave of The Irish Longitudinal Study of Ageing, a population-representative cohort of adults aged 50+. Depressive symptoms were recorded using the Centre for Epidemiological Studies Depression Scale. Season was defined according to the World Meteorological Organisation. Data on climate over the preceding thirty years, and temperature and rain over the preceding month, were provided by the Irish Meteorological Service and linked using Geographic Information Systems techniques to participant's geo-coded locations at a resolution of one kilometre. Results The highest levels of depressive symptoms were reported in winter and the lowest in spring (mean 6.56 [CI95% 6.09, 7.04] vs. 5.81 [CI95%: 5.40, 6.22]). In fully adjusted linear regression models, participants living in areas with higher levels of rainfall in the preceding and/or current calendar month had greater depressive symptoms (0.04 SE 0.02; p=0.039 per 10 mm additional rainfall per month) while those living in areas with sunnier climates had fewer depressive symptoms (−2.67 SE 0.88; p=0.003 for every additional hour of average annual daily sunshine). Limitations This was a cross-sectional analysis thus causality cannot be inferred; monthly rain and temperature averages were available only on a calendar month basis while monthly local levels of sunshine data were not available. Conclusions Environmental cues may influence mood in older adults and thus have relevance for the recognition and treatment of depression in this age group

Structures of the DfsB protein family suggest a cationic, helical sibling lethal factor peptide

Bacteria have developed a variety of mechanisms for surviving harsh environmental conditions, nutrient stress and overpopulation. Paenibacillus dendritiformis produces a lethal protein (Slf) that is able to induce cell death in neighbouring colonies and a phenotypic switch in more distant ones. Slf is derived from the secreted precursor protein, DfsB, after proteolytic processing. Here, we present new crystal structures of DfsB homologues from a variety of bacterial species and a surprising version present in the yeast Saccharomyces cerevisiae. Adopting a four-helix bundle decorated with a further three short helices within intervening loops, DfsB belongs to a non-enzymatic class of the DinB fold. The structure suggests that the biologically active Slf fragment may possess a C-terminal helix rich in basic and aromatic residues that suggest a functional mechanism akin to that for cationic antimicrobial peptides

A Novel Co-Crystal Structure Affords the Design of Gain-of-Function Lentiviral Integrase Mutants in the Presence of Modified PSIP1/LEDGF/p75

Lens epithelium derived growth factor (LEDGF), also known as PC4 and SFRS1 interacting protein 1 (PSIP1) and transcriptional co-activator p75, is the cellular binding partner of lentiviral integrase (IN) proteins. LEDGF accounts for the characteristic propensity of Lentivirus to integrate within active transcription units and is required for efficient viral replication. We now present a crystal structure containing the N-terminal and catalytic core domains (NTD and CCD) of HIV-2 IN in complex with the IN binding domain (IBD) of LEDGF. The structure extends the known IN–LEDGF interface, elucidating primarily charge–charge interactions between the NTD of IN and the IBD. A constellation of acidic residues on the NTD is characteristic of lentiviral INs, and mutations of the positively charged residues on the IBD severely affect interaction with all lentiviral INs tested. We show that the novel NTD–IBD contacts are critical for stimulation of concerted lentiviral DNA integration by LEDGF in vitro and for its function during the early steps of HIV-1 replication. Furthermore, the new structural details enabled us to engineer a mutant of HIV-1 IN that primarily functions only when presented with a complementary LEDGF mutant. These findings provide structural basis for the high affinity lentiviral IN–LEDGF interaction and pave the way for development of LEDGF-based targeting technologies for gene therapy