Disulfide-linked integrase oligomers involving C280 residues are formed in vitro and in vivo but are not essential for human immunodeficiency virus replication

The human immunodeficiency virus type 1 integrase (IN) forms an oligomer that integrates both ends of the viral DNA. The nature of the active oligomer is unclear. Recombinant IN obtained under reducing conditions is always in the form of noncovalent oligomers. However, disulfide-linked oligomers of IN were recently observed within viral particles. We show that IN produced from a baculovirus expression system can form disulfide-linked oligomers. We investigated which residues are responsible for the disulfide bridges and the relationship between the ability to form covalent dimers and IN activity. Only the mutation of residue C280 was sufficient to prevent the formation of intermolecular disulfide bridges in oligomers of recombinant IN. IN activity was studied under and versus nonreducing conditions: the formation of disulfide bridges was not required for the in vitro activities of the enzyme. Moreover, the covalent dimer does not dissociate into individual protomers on disulfide bridge reduction. Instead, IN undergoes a spontaneous multimerization process that yields a homogenous noncovalent tetramer. The C280S mutation also completely abolished the formation of disulfide bonds in the context of the viral particle. Finally, the replication of the mutant virus was investigated in replicating and arrested cells. The infectivity of the virus was not affected by the C280S IN mutation in either dividing or nondividing cells. The disulfide-linked form of the IN oligomers observed in the viral particles is thus not required for viral replication.     

The Escherichia coli RecQ helicase functions as a monomer

The RecQ helicases belong to an important family of highly conserved DNA helicases that play a key role in chromosomal maintenance, and their defects have been shown to lead to several disorders and cancer in humans. In this work, the conformational and functional properties of the Escherichia coli RecQ helicase have been determined using a wide array of biochemical and biophysical techniques. The results obtained clearly indicate that E. coli RecQ helicase is monomeric in solution up to a concentration of 20 microM and in a temperature range between 4 and 37 degrees C. Furthermore, these properties are not affected by the presence of ATP, which is strictly required for the unwinding and translocating activity of the protein, or by its nonhydrolyzable analogue 5'-adenylyl-beta,gamma-imidodiphosphate. Consistent with the structural properties, functional analysis shows that both DNA unwinding activity and single-stranded DNA-stimulated ATPase specific activity were independent of RecQ concentration. The monomeric state was further confirmed by the ATPase-deficient mutants of RecQ protein. The rate of unwinding was unchanged when the wild type RecQ helicase was mixed with the ATPase-deficient mutants, indicating that nonprotein-protein interactions were involved in the unwinding processes. Taken together, these results indicate that RecQ helicase functions as a monomer and provide new data on the structural and functional properties of RecQ helicase that may help elucidate its mechanism action.     

Dispersal in heterogeneous environments drives population dynamics and control of tsetse flies

Spatio-temporally heterogeneous environments may lead to unexpected population dynamics. Knowledge is needed on local properties favouring population resilience at large scale. For pathogen vectors, such as tsetse flies transmitting human and animal African trypanosomosis, this is crucial to target management strategies. We developed a mechanistic spatio-temporal model of the age-structured population dynamics of tsetse flies, parametrized with field and laboratory data. It accounts for density- and temperature-dependence. The studied environment is heterogeneous, fragmented and dispersal is suitability-driven. We confirmed that temperature and adult mortality have a strong impact on tsetse populations. When homogeneously increasing adult mortality, control was less effective and induced faster population recovery in the coldest and temperature-stable locations, creating refuges. To optimally select locations to control, we assessed the potential impact of treating them and their contribution to the whole population. This heterogeneous control induced a similar population decrease, with more dispersed individuals. Control efficacy was no longer related to temperature. Dispersal was responsible for refuges at the interface between controlled and uncontrolled zones, where resurgence after control was very high. The early identification of refuges, which could jeopardize control efforts, is crucial. We recommend baseline data collection to characterize the ecosystem before implementing any measures.     

Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs

Förster resonance energy transfer (FRET) is a powerful method for obtaining information about small-scale lengths between biomacromolecules. Visible fluorescent proteins (VFPs) are widely used as spectrally different FRET pairs, where one VFP acts as a donor and another VFP as an acceptor. The VFPs are usually fused to the proteins of interest, and this fusion product is genetically encoded in cells. FRET between VFPs can be determined by analysis of either the fluorescence decay properties of the donor molecule or the rise time of acceptor fluorescence. Time-resolved fluorescence spectroscopy is the technique of choice to perform these measurements. FRET can be measured not only in solution, but also in living cells by the technique of fluorescence lifetime imaging microscopy (FLIM), where fluorescence lifetimes are determined with the spatial resolution of an optical microscope. Here we focus attention on time-resolved fluorescence spectroscopy of purified, selected VFPs (both single VFPs and FRET pairs of VFPs) in cuvette-type experiments. For quantitative interpretation of FRET–FLIM experiments in cellular systems, details of the molecular fluorescence are needed that can be obtained from experiments with isolated VFPs. For analysis of the time-resolved fluorescence experiments of VFPs, we have utilised the maximum entropy method procedure to obtain a distribution of fluorescence lifetimes. Distributed lifetime patterns turn out to have diagnostic value, for instance, in observing populations of VFP pairs that are FRET-inactive.     

Articulated coralline algae of the genus Amphiroa are highly effective natural inducers of settlement in the tropical abalone Haliotis asinina

The initiation of metamorphosis in marine invertebrates is strongly linked to the environment. Planktonic larvae typically are induced to settle and metamorphose by external cues such as coralline algae (Corallinaceae, Rhodophyta). Although coralline algae are globally abundant, invertebrate larvae of many taxa settle in response to a very limited suite of species. This specificity impacts population structure, as only locations with the appropriate coralline species can attract new recruits. Abalone (Gastropoda, Haliotidae) are among those taxa in which closely related species are known to respond to different coralline algae. Here we identify highly inductive natural cues of the tropical abalone Haliotis asinina. In contrast to reports for other abalone, the greatest proportion of H. asinina larvae are induced to settle and metamorphose (92.8% to 100% metamorphosis by 48 h postinduction) by articulated corallines of the genus Amphiroa. Comparison with field distribution data for different corallines suggests larvae are likely to be settling on the seaward side of the reef crest. We then compare the response of six different H. asinina larval families to five different coralline species to demonstrate that induction by the best inductive cue (Amphiroa spp.) effectively extinguishes substantial intraspecific variation in the timing of settlement.     

Insight into the integrase-DNA recognition mechanism. A specific DNA-binding mode revealed by an enzymatically labeled integrase

Integration catalyzed by integrase (IN) is a key process in the retrovirus life cycle. Many biochemical or structural human immunodeficiency virus, type 1 (HIV-1) IN studies have been severely impeded by its propensity to aggregate. We characterized a retroviral IN (primate foamy virus (PFV-1)) that displays a solubility profile different from that of HIV-1 IN. Using various techniques, including fluorescence correlation spectroscopy, time-resolved fluorescence anisotropy, and size exclusion chromatography, we identified a monomer-dimer equilibrium for the protein alone, with a half-transition concentration of 20-30 mum. We performed specific enzymatic labeling of PFV-1 IN and measured the fluorescence resonance energy transfer between carboxytetramethylrhodamine-labeled IN and fluorescein-labeled DNA substrates. FRET and fluorescence anisotropy highlight the preferential binding of PFV-1 IN to the 3'-end processing site. Sequence-specific DNA binding was not observed with HIV-1 IN, suggesting that the intrinsic ability of retroviral INs to bind preferentially to the processing site is highly underestimated in the presence of aggregates. IN is in a dimeric state for 3'-processing on short DNA substrates, whereas IN polymerization, mediated by nonspecific contacts at internal DNA positions, occurs on longer DNAs. Additionally, aggregation, mediated by nonspecific IN-IN interactions, occurs preferentially with short DNAs at high IN/DNA ratios. The presence of either higher order complex is detrimental for specific activity. Ionic strength favors catalytically competent over higher order complexes by selectively disrupting nonspecific IN-IN interactions. This counteracting effect was not observed with polymerization. The synergic effect on the selection of specific/competent complexes, obtained by using short DNA substrates under high salt conditions, may have important implications for further structural studies in IN.DNA complexes.     

Pleistocene isolation and recent gene flow in Haliotis asinina, an Indo-Pacific vetigastropod with limited dispersal capacity

Haliotis asinina is a broadcast-spawning mollusc that inhabits Indo-Pacific coral reefs. This tropical abalone develops through a nonfeeding larval stage that is competent to settle on specific species of coralline algae after 3-4 days in the plankton. Failure to contact an inductive algae within 10 days of hatching usually results in death. These life cycle characteristics suggest a limited capacity for dispersal and thus gene flow. This makes H. asinina particularly suitable for elucidating phylogeographical structure throughout the Indo-Malay Archipelagoes, and eastern Indian and western Pacific Oceans, all regions of biogeographical complexity and high conservation value. We assayed 482 bp of the mitochondrial cytochrome oxidase II gene in 206 abalone collected from 16 geographically discrete sites across the Indian and Pacific Oceans and Indo-Malay Archipelagoes. DNA sequence variation was analysed via population genetics and phylogenetics, and by nested clade analyses (NCA). Our data resolved clear phylogeographical breaks among major biogeographical regions, with sequence divergences ranging from a high of 3.7% and 3.0% between Indian and Pacific sites and Pacific and Indo-Malay sites, respectively, to a low of 1.1% between Indian and Indo-Malay sites. Despite the apparent limited dispersal capacity of H. asinina, no finer scale phylogeographical structure was resolved within the respective biogeographical regions. However, amova and NCA identified several significant associations between haplotypes and geographical distribution, most notably higher gene flow among geographical populations associated with major ocean currents. Our study provides further evidence that larval dispersal capacity alone is not a good predictor of population genetic structure in marine invertebrates. We infer instead that a combination of historical events (long-term barriers followed by range expansion associated with Pleistocene sea level changes) and contemporary processes (gene flow restricted by life history and oceanography) have shaped observed patterns of H. asinina phylogeography.