Frequency and Env determinants of HIV-1 subtype C strains from antiretroviral therapy-naive subjects that display incomplete inhibition by maraviroc

Background

Entry of human immunodeficiency virus type 1 (HIV-1) into cells involves the interaction of the viral gp120 envelope glycoproteins (Env) with cellular CD4 and a secondary coreceptor, which is typically one of the chemokine receptors CCR5 or CXCR4. CCR5-using (R5) HIV-1 strains that display reduced sensitivity to CCR5 antagonists can use antagonist-bound CCR5 for entry. In this study, we investigated whether naturally occurring gp120 alterations in HIV-1 subtype C (C-HIV) variants exist in antiretroviral therapy (ART)-naïve subjects that may influence their sensitivity to the CCR5 antagonist maraviroc (MVC).

Results

Using a longitudinal panel of 244 R5 Envs cloned from 20 ART-naïve subjects with progressive C-HIV infection, we show that 40% of subjects (n = 8) harbored viruses that displayed incomplete inhibition by MVC, as shown by plateau’s of reduced maximal percent inhibitions (MPIs). Specifically, when pseudotyped onto luciferase reporter viruses, 16 Envs exhibited MPIs below 98% in NP2–CCR5 cells (range 79.7–97.3%), which were lower still in 293-Affinofile cells that were engineered to express high levels of CCR5 (range 15.8–72.5%). We further show that Envs exhibiting reduced MPIs to MVC utilized MVC-bound CCR5 less efficiently than MVC-free CCR5, which is consistent with the mechanism of resistance to CCR5 antagonists that can occur in patients failing therapy. Mutagenesis studies identified strain-specific mutations in the gp120 V3 loop that contributed to reduced MPIs to MVC.

Conclusions

The results of our study suggest that some ART-naïve subjects with C-HIV infection harbor HIV-1 with reduced MPIs to MVC, and demonstrate that the gp120 V3 loop region contributes to this phenotype.

     

An orphaned Mce‐associated membrane protein of Mycobacterium tuberculosis is a virulence factor that stabilizes Mce transporters

Mycobacterium tuberculosis proteins that are exported out of the bacterial cytoplasm are ideally positioned to be virulence factors; however, the functions of individual exported proteins remain largely unknown. Previous studies identified Rv0199 as an exported membrane protein of unknown function. Here, we characterized the role of Rv0199 in M. tuberculosis virulence using an aerosol model of murine infection. Rv0199 appears to be a member of a Mce‐associated membrane (Mam) protein family leading us to rename it OmamA, for orphaned Mam protein A. Consistent with a role in Mce transport, we showed OmamA is required for cholesterol import, which is a Mce4‐dependent process. We further demonstrated a function for OmamA in stabilizing protein components of the Mce1 transporter complex. These results indicate a function of OmamA in multiple Mce transporters and one that may be analogous to the role of VirB8 in stabilizing Type IV secretion systems, as structural similarities between Mam proteins and VirB8 proteins are predicted by the Phyre 2 program. In this study, we provide functional information about OmamA and shed light on the function of Mam family proteins in Mce transporters.     

Membranous structures transfer cell surface proteins across NK cell immune synapses

Intercellular transfer of cell surface proteins is widespread and facilitates several recently discovered means for immune cell communication. Here, we examined the molecular mechanism for intercellular exchange of the natural killer (NK) cell receptor KIR2DL1 and HLA-C, prototypical proteins that swap between NK cells and target cells. Transfer was contact dependent and enhanced for cells expressing cognate receptor/ligand pairs but did not depend on KIR2DL1 signaling. To a lesser extent, proteins transferred independent from specific recognition. Intracellular domains of transferred proteins were not exposed to the extracellular environment and transferred proteins were removed by brief exposure to low pH. By fluorescence microscopy, transferred proteins localized to discrete regions on the recipient cell surface. Higher resolution scanning electron micrographs revealed that transferred proteins were located within specific membranous structures. Transmission electron microscopy of the immune synapse revealed that membrane protrusions from one cell interacted with the apposing cell surface within the synaptic cleft. These data, coupled with previous observations, lead us to propose that intercellular protein transfer is mediated by membrane protrusions within and surrounding the immunological synapse.     

Structure-activity relationships of SERMs optimized for uterine antagonism and ovarian safety

Structure-activity relationship studies are described, which led to the discovery of novel selective estrogen receptor modulators (SERMs) for the potential treatment of uterine fibroids. The SAR studies focused on limiting brain exposure and were guided by computational properties. Compounds with limited impact on the HPO axis were selected using serum estrogen levels as a biomarker for ovarian stimulation.     

Biochemical and structural features of a novel cyclodextrinase from cow rumen metagenome

A novel enzyme, RA.04, belonging to the alpha-amylase family was obtained after expression of metagenomic DNA from rumen fluid (Ferrer et al.: Environ. Microbiol. 2005, 7, 1996-2010). The purified RA.04 has a tetrameric structure (280 kDa) and exhibited maximum activity (5000 U/mg protein) at 70 degrees C and was active within an unusually broad pH range from 5.5 to 9.0. It maintained 80% activity at pH 5.0 and 9.5 and 75 degrees C. The enzyme hydrolyzed alpha-D-(1,4) bonds 13-fold faster than alpha-D-(1,6) bonds to yield maltose and glucose as the main products, and it exhibited transglycosylation activity. Its preferred substrates, in the descending order, were maltooligosaccharides (C3-C7), cyclomaltoheptaose (beta-CD), cyclomaltohexaose (alpha-CD), cyclomaltooctaose (gamma-CD), soluble starch, amylose, pullulan and amylopectin. The biochemical properties and amino acid sequence alignments suggested that this enzyme is a cyclomaltodextrinase. However, despite the similarity in the catalytic module (with Glu359 and Asp331 being the catalytic nucleophile and substrate-binding residues, respectively), the enzyme bears a shorter N-terminal domain that may keep the active site more accessible for both starch and pullulan, compared to the other known CDases. Moreover, RA.04 lacks the well-conserved N-terminal Trp responsible for the substrate preference typical of CDases/MAases/PNases, suggesting a new residue is implicated in the preference for cyclic maltooligosaccharides. This study has demonstrated the usefulness of a metagenomic approach to gain novel debranching enzymes, important for the bread/food industries, from microbial environments with a high rate of plant polymer turnover, exemplified by the cow rumen.     

Generation of reactive oxygen and antioxidant species by hydrodynamically stressed suspensions of Morinda citrifolia

The generation of reactive oxygen species (ROS) by plant cell suspension cultures, in response to the imposition of both biotic and abiotic stress, is well-documented. This study investigated the generation of hydrogen peroxide by hydrodynamically stressed cultures of Morinda citrifolia, over a 5-h period post-stress imposition. Suspensions were exposed to repeated passages through a syringe, under laminar flow conditions, corresponding to cumulative energy dissipation levels of approximately 3-6 J kg-1. Extracellular hydrogen peroxide was detected using a luminol-based chemiluminescence assay. The addition of exogenous hydrogen peroxide facilitated the detection of low levels of hydrogen peroxide in the presence of antioxidants. Immediately after shear exposure, there was evidence of significant antioxidative capacity in the sheared cell cultures, which potentially masked any oxidative burst (OB), but which decreased over the following 40 min. This antioxidative capacity was determined to derive from the shearing process. Trials in which ground cellular debris was added to control suspensions suggested that some of the antioxidative capacity observed in stressed suspensions was directly associated with debris generated by the shearing process. Using UV-vis spectrophotometry and HPLC, stress-related increases in the levels of phenolic compounds were detected in suspension filtrates. Under the stress conditions investigated, maximum hydrogen peroxide levels of 11.5 muM were observed, 5 h after shear exposure. This study emphasizes the importance of considering both oxidative and antioxidative capacities as part of a holistic approach to the determination of the OB in hydrodynamically stressed plant cell suspension cultures.     

DNA sequences surrounding the centromere of chromosome 21.

The mapping and sequencing of two clones that surround the centromere of chromosome 21 are presented. These clones specify the most proximal known low-order repeat on 21p (p21-7D) and the most proximal known single-copy sequence on 21q (pUT-B37 at locus D21S120).     

Unexpected spatial patterns in an insect outbreak match a predator diffusion model

An outbreak of the western tussock moth ( Orgyia vetusta) with a sharp spatial boundary has been hypothesized to be the result of an interaction with mobile parasitoids. We report here a striking correspondence between the qualitative prediction of a general model for a mobile predator and a sessile prey and initial observations of the outbreak of western tussock moth. The model makes the counterintuitive prediction that the prey species will have its highest population density at the edge of an outbreak, which corresponds to observations made of the western tussock moth. This is a striking pattern which should be looked for in other population systems.     

Age modifies the effect of body size on fecundity in Culex quinquefasciatus Say (Diptera: Culicidae)

Fecundity of mosquitoes can vary with many factors and can have a strong effect on population growth. This study reports the effects of body size, blood meal size, and age on the reproductive output of nulliparous Culex quinquefasciatus, a vector of arboviruses and other pathogens. Mated adult female mosquitoes from a colony were reared under standard conditions and fed on chickens at different ages post‐eclosion. Blood meal size and wing length were recorded, as well as the number of eggs in the first‐cycle egg raft. Each of these factors had a significant influence on fecundity considered in a simple regression context. Multiple regression analysis revealed a significant interaction effect between age and body size on fecundity. Up to 13 days of age, fecundity was positively correlated with body size, but in mosquitoes older than 13 days, this relationship was not significantly different from zero. These results are discussed in terms of the known physiology of this and other species.