Chromosomal integration of LTR-flanked DNA in yeast expressing HIV-1 integrase: down regulation by RAD51

HIV-1 integrase (IN) is the key enzyme catalyzing the proviral DNA integration step. Although the enzyme catalyzes the integration step accurately in vitro, whether IN is sufficient for in vivo integration and how it interacts with the cellular machinery remains unclear. We set up a yeast cellular integration system where integrase was expressed as the sole HIV-1 protein and targeted the chromosomes. In this simple eukaryotic model, integrase is necessary and sufficient for the insertion of a DNA containing viral LTRs into the genome, thereby allowing the study of the isolated integration step independently of other viral mechanisms. Furthermore, the yeast system was used to identify cellular mechanisms involved in the integration step and allowed us to show the role of homologous recombination systems. We demonstrated physical interactions between HIV-1 IN and RAD51 protein and showed that HIV-1 integrase activity could be inhibited both in the cell and in vitro by RAD51 protein. Our data allowed the identification of RAD51 as a novel in vitro IN cofactor able to down regulate the activity of this retroviral enzyme, thereby acting as a potential cellular restriction factor to HIV infection

Long-term observations minus background monitoring of ground-based brightness temperatures from a microwave radiometer network

Abstract. Ground-based microwave radiometers (MWRs) offer the capability to provide continuous, high-temporal-resolution observations of the atmospheric thermodynamic state in the planetary boundary layer (PBL) with low maintenance. This makes MWR an ideal instrument to supplement radiosonde and satellite observations when initializing numerical weather prediction (NWP) models through data assimilation. State-of-the-art data assimilation systems (e.g. variational schemes) require an accurate representation of the differences between model (background) and observations, which are then weighted by their respective errors to provide the best analysis of the true atmospheric state. In this perspective, one source of information is contained in the statistics of the differences between observations and their background counterparts (O–B). Monitoring of O–B statistics is crucial to detect and remove systematic errors coming from the measurements, the observation operator, and/or the NWP model. This work illustrates a 1-year O–B analysis for MWR observations in clear-sky conditions for an European-wide network of six MWRs. Observations include MWR brightness temperatures (TB) measured by the two most common types of MWR instruments. Background profiles are extracted from the French convective-scale model AROME-France before being converted into TB. The observation operator used to map atmospheric profiles into TB is the fast radiative transfer model RTTOV-gb. It is shown that O–B monitoring can effectively detect instrument malfunctions. O–B statistics (bias, standard deviation, and root mean square) for water vapour channels (22.24–30.0 GHz) are quite consistent for all the instrumental sites, decreasing from the 22.24 GHz line centre ( ∼  2–2.5 K) towards the high-frequency wing ( ∼  0.8–1.3 K). Statistics for zenith and lower-elevation observations show a similar trend, though values increase with increasing air mass. O–B statistics for temperature channels show different behaviour for relatively transparent (51–53 GHz) and opaque channels (54–58 GHz). Opaque channels show lower uncertainties (< 0.8–0.9 K) and little variation with elevation angle. Transparent channels show larger biases ( ∼  2–3 K) with relatively low standard deviations ( ∼  1–1.5 K). The observations minus analysis TB statistics are similar to the O–B statistics, suggesting a possible improvement to be expected by assimilating MWR TB into NWP models. Lastly, the O–B TB differences have been evaluated to verify the normal-distribution hypothesis underlying variational and ensemble Kalman filter-based DA systems. Absolute values of excess kurtosis and skewness are generally within 1 and 0.5, respectively, for all instrumental sites, demonstrating O–B normal distribution for most of the channels and elevations angles

The AROME-WMED reanalyses of the first special observation period of the Hydrological cycle in the Mediterranean experiment (HyMeX)

To study key processes of the water cycle, two special observation periods (SOPs) of the Hydrological cycle in the Mediterranean experiment (HyMeX) took place during autumn 2012 and winter 2013. The first SOP aimed to study high precipitation systems and flash flooding in the Mediterranean area. The AROME-WMED (western Mediterranean) model (Fourrié et al., 2015) is a dedicated version of the mesoscale Numerical Weather Prediction (NWP) AROME-France model, which covers the western Mediterranean basin providing the HyMeX operational center with daily real-time analyses and forecasts. These products allowed for adequate decision-making for the field campaign observation deployment and the instrument operation. Shortly after the end of the campaign, a first reanalysis with more observations was performed with the first SOP operational software. An ensuing comprehensive second reanalysis of the first SOP, which included field research observations (not assimilated in real time) and some reprocessed observation datasets, was made with AROME-WMED. Moreover, a more recent version of the AROME model was used with updated background error statistics for the assimilation process. This paper depicts the main differences between the real-time version and the benefits brought by HyMeX reanalyses with AROME-WMED. The first reanalysis used 9 % additional data and the second one 24 % more compared to the real-time version. The second reanalysis is found to be closer to observations than the previous AROME-WMED analyses. The second reanalysis forecast errors of surface parameters are reduced up to the 18 and 24 h forecast range. In the middle and upper troposphere, fields are also improved up to the 48 h forecast range when compared to radiosondes. Integrated water vapor comparisons indicate a positive benefit for at least 24 h. Precipitation forecasts are found to be improved with the second reanalysis for a threshold up to 10 mm (24 h)-1. For higher thresholds, the frequency bias is degraded. Finally, improvement brought by the second reanalysis is illustrated with the Intensive Observation Period (IOP8) associated with heavy precipitation over eastern Spain and southern France

Hydrometeorological multi-model ensemble simulations of the 4 November 2011 flash flood event in Genoa, Italy, in the framework of the DRIHM Project

The e-Science environment developed in the framework of the EU-funded DRIHM project was used to demonstrate its ability to provide relevant, meaningful hydrometeorological forecasts. This was illustrated for the tragic case of 4 November 2011, when Genoa, Italy, was flooded as the result of heavy, convective precipitation that inundated the Bisagno catchment. The Meteorological Model Bridge (MMB), an innovative software component developed within the DRIHM project for the interoperability of meteorological and hydrological models, is a key component of the DRIHM e-Science environment. The MMB allowed three different rainfall-discharge models (DRiFt, RIBS and HBV) to be driven by four mesoscale limited-area atmospheric models (WRF-NMM, WRF-ARW, Meso-NH and AROME) and a downscaling algorithm (RainFARM) in a seamless fashion. In addition to this multi-model configuration, some of the models were run in probabilistic mode, thus giving a comprehensive account of modelling errors and a very large amount of likely hydrometeorological scenarios (> 1500). The multi-model approach proved to be necessary because, whilst various aspects of the event were successfully simulated by different models, none of the models reproduced all of these aspects correctly. It was shown that the resulting set of simulations helped identify key atmospheric processes responsible for the large rainfall accumulations over the Bisagno basin. The DRIHM e-Science environment facilitated an evaluation of the sensitivity to atmospheric and hydrological modelling errors. This showed that both had a significant impact on predicted discharges, the former being larger than the latter. Finally, the usefulness of the set of hydrometeorological simulations was assessed from a flash flood early-warning perspective

LptM promotes oxidative maturation of the lipopolysaccharide translocon by substrate binding mimicry

Insertion of lipopolysaccharide (LPS) into the bacterial outer membrane (OM) is mediated by a druggable OM translocon consisting of a β-barrel membrane protein, LptD, and a lipoprotein, LptE. The β-barrel assembly machinery (BAM) assembles LptD together with LptE at the OM. In the enterobacterium Escherichia coli, formation of two native disulfide bonds in LptD controls translocon activation. Here we report the discovery of LptM (formerly YifL), a lipoprotein conserved in Enterobacteriaceae, that assembles together with LptD and LptE at the BAM complex. LptM stabilizes a conformation of LptD that can efficiently acquire native disulfide bonds, whereas its inactivation makes disulfide bond isomerization by DsbC become essential for viability. Our structural prediction and biochemical analyses indicate that LptM binds to sites in both LptD and LptE that are proposed to coordinate LPS insertion into the OM. These results suggest that, by mimicking LPS binding, LptM facilitates oxidative maturation of LptD, thereby activating the LPS translocon

Ty1 integrase overexpression leads to integration of non-Ty1 DNA fragments into the genome of Saccharomyces cerevisiae

The integrase of the Saccharomyces cerevisiae retrotransposon Ty1 integrates Ty1 cDNA into genomic DNA likely via a transesterification reaction. Little is known about the mechanisms ensuring that integrase does not integrate non-Ty DNA fragments. In an effort to elucidate the conditions under which Ty1 integrase accepts non-Ty DNA as substrate, PCR fragments encompassing a selectable marker gene were transformed into yeast strains overexpressing Ty1 integrase. These fragments do not exhibit similarity to Ty1 cDNA except for the presence of the conserved terminal dinucleotide 5′-TG-CA-3′. The frequency of fragment insertion events increased upon integrase overexpression. Characterization of insertion events by genomic sequencing revealed that most insertion events exhibited clear hallmarks of integrase-mediated reactions, such as 5 bp target site duplication and target site preferences. Alteration of the terminal dinucleotide abolished the suitability of the PCR fragments to serve as substrates. We hypothesize that substrate specificity under normal conditions is mainly due to compartmentalization of integrase and Ty cDNA, which meet in virus-like particles. In contrast, recombinant integrase, which is not confined to virus-like particles, is able to accept non-Ty DNA, provided that it terminates in the proper dinucleotide sequence

Molecular Characterization of HIV-1 CRF01_AE in Mekong Delta, Vietnam, and Impact of T-Cell Epitope Mutations on HLA Recognition (ANRS 12159)

To date, 11 HIV-1 subtypes and 48 circulating recombinant forms have been described worldwide. The underlying reason why their distribution is so heterogeneous is not clear. Host genetic factors could partly explain this distribution. The aim of this study was to describe HIV-1 strains circulating in an unexplored area of Mekong Delta, Vietnam, and to assess the impact of optimal epitope mutations on HLA binding.We recruited 125 chronically antiretroviral-naive HIV-1-infected subjects from five cities in the Mekong Delta. We performed high-resolution DNA typing of HLA class I alleles, sequencing of Gag and RT-Prot genes and phylogenetic analysis of the strains. Epitope mutations were analyzed in patients bearing the HLA allele restricting the studied epitope. Optimal wild-type epitopes from the Los Alamos database were used as reference. T-cell epitope recognition was predicted using the immune epitope database tool according to three different scores involved in antigen processing (TAP and proteasome scores) and HLA binding (MHC score). with a Vietnamese specificity held by two different haplotypes. The percentage of homology between Mekong and B consensus HIV-1 sequences was above 85%. Divergent epitopes had TAP and proteasome scores comparable with wild-type epitopes. MHC scores were significantly lower in divergent epitopes with a mean of 2.4 (±0.9) versus 2 (±0.7) in non-divergent ones (p<0.0001).Our study confirms the wide predominance of CRF01_AE in the Mekong Delta where patients harbor a specific HLA pattern. Moreover, it demonstrates the lower MHC binding affinity among divergent epitopes. This weak immune pressure combined with a narrow genetic diversity favors immune escape and could explain why CRF01_AE is still predominant in Vietnam, particularly in the Mekong area

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 (

Modelling of the effect of ELMs on fuel retention at the bulk W divertor of JET

Effect of ELMs on fuel retention at the bulk W target of JET ITER-Like Wall was studied with multi-scale calculations. Plasma input parameters were taken from ELMy H-mode plasma experiment. The energetic intra-ELM fuel particles get implanted and create near-surface defects up to depths of few tens of nm, which act as the main fuel trapping sites during ELMs. Clustering of implantation-induced vacancies were found to take place. The incoming flux of inter-ELM plasma particles increases the different filling levels of trapped fuel in defects. The temperature increase of the W target during the pulse increases the fuel detrapping rate. The inter-ELM fuel particle flux refills the partially emptied trapping sites and fills new sites. This leads to a competing effect on the retention and release rates of the implanted particles. At high temperatures the main retention appeared in larger vacancy clusters due to increased clustering rate