Rapid microtiter assays for poxvirus topoisomerase, mammalian type IB topoisomerase and HIV-1 integrase: application to inhibitor isolation

We have developed microtiter assays for detecting catalysis by type IB topoisomerases and retroviral integrases. Each assay employs model DNA substrates containing biotin in one strand and digoxigenin in another. In each case action of the enzyme results in the formation of a single DNA strand containing both groups. This allows the reaction product to be quantified by capturing biotinylated product DNA on avidin-coated plates followed by detection using an anti-digoxigenin ELISA. The order of addition of reactants and inhibitors can be varied to distinguish effects of test compounds on different steps in the reaction. These assays were used to screen compound libraries for inhibitors active against mammalian topoisomerase or HIV integrase. We identified (–)-epigallocatechin 3-O-gallate, as a potent inhibitor of religation by mammalian topoisomerase (IC₅₀ of 26 nM), potentially explaining the anti-cancer properties previously attributed to this compound. New integrase inhibitors were also identified. A similar strategy may be used to develop microtiter assays for many further DNA modifying enzymes.     

Anchorage-independent culture maintains prostate stem cells

Freshly isolated mouse prostate epithelial cells regenerate fully differentiated prostate tissue when combined with embryonic urogenital sinus mesenchyme and grafted in vivo. We show here that this regenerative capacity, which has been attributed to a small population of pleuripotential progenitor epithelial cells, is rapidly lost when the cells are placed in monolayer culture but can be maintained by culture in anchorage-independent conditions. Epithelial cells placed in anchorage-independent culture formed proliferating spheres that could be serially passaged and exhibited increased expression of putative stem cell markers as compared to cells grown in monolayer culture. Epithelial cells isolated from the fetal urogenital sinus, the newborn, and adult prostate formed spheres with similar efficiency, while cells isolated from the post-castration prostate exhibited significantly higher sphere-forming abilities. When passaged spheres were recombined with E17 rat urogenital sinus mesenchyme and grafted in vivo, they generated fully differentiated mouse prostate glandular epithelium containing both p63+ basal cells and p63− luminal cells and expressing a variety of prostate-specific and terminal differentiation markers.     

Fungi of the Murine Gut: Episodic Variation and Proliferation during Antibiotic Treatment

Antibiotic use in humans has been associated with outgrowth of fungi. Here we used a murine model to investigate the gut microbiome over 76 days of treatment with vancomycin, ampicillin, neomycin, and metronidazole and subsequent recovery. Mouse stool was studied as a surrogate for the microbiota of the lower gastrointestinal tract. The abundance of fungi and bacteria was measured using quantitative PCR, and the proportional composition of the communities quantified using 454/Roche pyrosequencing of rRNA gene tags. Prior to treatment, bacteria outnumbered fungi by >3 orders of magnitude. Upon antibiotic treatment, bacteria dropped in abundance >3 orders of magnitude, so that the predominant 16S sequences detected became transients derived from food. Upon cessation of treatment, bacterial communities mostly returned to their previous numbers and types after 8 weeks, though communities remained detectably different from untreated controls. Fungal communities varied substantially over time, even in the untreated controls. Separate cages within the same treatment group showed radical differences, but mice within a cage generally behaved similarly. Fungi increased ∼40-fold in abundance upon antibiotic treatment but declined back to their original abundance after cessation of treatment. At the last time point, Candida remained more abundant than prior to treatment. These data show that 1) gut fungal populations change radically during normal mouse husbandry, 2) fungi grow out in the gut upon suppression of bacterial communities with antibiotics, and 3) perturbations due to antibiotics persist long term in both the fungal and bacterial microbiota.     

Conservation of Gene Cassettes among Diverse Viruses of the Human Gut

Viruses are a crucial component of the human microbiome, but large population sizes, high sequence diversity, and high frequencies of novel genes have hindered genomic analysis by high-throughput sequencing. Here we investigate approaches to metagenomic assembly to probe genome structure in a sample of 5.6 Gb of gut viral DNA sequence from six individuals. Tests showed that a new pipeline based on DeBruijn graph assembly yielded longer contigs that were able to recruit more reads than the equivalent non-optimized, single-pass approach. To characterize gene content, the database of viral RefSeq proteins was compared to the assembled viral contigs, generating a bipartite graph with functional cassettes linking together viral contigs, which revealed a high degree of connectivity between diverse genomes involving multiple genes of the same functional class. In a second step, open reading frames were grouped by their co-occurrence on contigs in a database-independent manner, revealing conserved cassettes of co-oriented ORFs. These methods reveal that free-living bacteriophages, while usually dissimilar at the nucleotide level, often have significant similarity at the level of encoded amino acid motifs, gene order, and gene orientation. These findings thus connect contemporary metagenomic analysis with classical studies of bacteriophage genomic cassettes. Software is available at https://sourceforge.net/projects/optitdba/.     

HRP-2 determines HIV-1 integration site selection in LEDGF/p75 depleted cells

Background

The cellular activity of many factors and pathways is required to execute the complex replication cycle of the human immunodeficiency virus type 1 (HIV-1). To reveal these cellular components, several extensive RNAi screens have been performed, listing numerous 'HIV-dependency factors'. However, only a small overlap between these lists exists, calling for further evaluation of the relevance of specific factors to HIV-1 replication and for the identification of additional cellular candidates. TBC1D20, the GTPase-activating protein (GAP) of Rab1, regulates endoplasmic reticulum (ER) to Golgi trafficking, was not identified in any of these screens, and its involvement in HIV-1 replication cycle is tested here.

Findings

Excessive TBC1D20 activity perturbs the early trafficking of HIV-1 envelope protein through the secretory pathway. Overexpression of TBC1D20 hampered envelope processing and reduced its association with detergent-resistant membranes, entailing a reduction in infectivity of HIV-1 virion like particles (VLPs).

Conclusions

These findings add TBC1D20 to the network of host factors regulating HIV replication cycle.     

A New Class of Multimerization Selective Inhibitors of HIV-1 Integrase

The quinoline-based allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are promising candidates for clinically useful antiviral agents. Studies using these compounds have highlighted the role of IN in both early and late stages of virus replication. However, dissecting the exact mechanism of action of the quinoline-based ALLINIs has been complicated by the multifunctional nature of these inhibitors because they both inhibit IN binding with its cofactor LEDGF/p75 and promote aberrant IN multimerization with similar potencies in vitro. Here we report design of small molecules that allowed us to probe the role of HIV-1 IN multimerization independently from IN-LEDGF/p75 interactions in infected cells. We altered the rigid quinoline moiety in ALLINIs and designed pyridine-based molecules with a rotatable single bond to allow these compounds to bridge between interacting IN subunits optimally and promote oligomerization. The most potent pyridine-based inhibitor, KF116, potently (EC₅₀ of 0.024 µM) blocked HIV-1 replication by inducing aberrant IN multimerization in virus particles, whereas it was not effective when added to target cells. Furthermore, KF116 inhibited the HIV-1 IN variant with the A128T substitution, which confers resistance to the majority of quinoline-based ALLINIs. A genome-wide HIV-1 integration site analysis demonstrated that addition of KF116 to target or producer cells did not affect LEDGF/p75-dependent HIV-1 integration in host chromosomes, indicating that this compound is not detectably inhibiting IN-LEDGF/p75 binding. These findings delineate the significance of correctly ordered IN structure for HIV-1 particle morphogenesis and demonstrate feasibility of exploiting IN multimerization as a therapeutic target. Furthermore, pyridine-based compounds present a novel class of multimerization selective IN inhibitors as investigational probes for HIV-1 molecular biology.     

Improved characterization of medically relevant fungi in the human respiratory tract using next-generation sequencing

Background

Fungi are important pathogens but challenging to enumerate using next-generation sequencing because of low absolute abundance in many samples and high levels of fungal DNA from contaminating sources.

Results

Here, we analyze fungal lineages present in the human airway using an improved method for contamination filtering. We use DNA quantification data, which are routinely acquired during DNA library preparation, to annotate output sequence data, and improve the identification and filtering of contaminants. We compare fungal communities and bacterial communities from healthy subjects, HIV+ subjects, and lung transplant recipients, providing a gradient of increasing lung impairment for comparison. We use deep sequencing to characterize ribosomal rRNA gene segments from fungi and bacteria in DNA extracted from bronchiolar lavage samples and oropharyngeal wash. Comparison to clinical culture data documents improved detection after applying the filtering procedure.

Conclusions

We find increased representation of medically relevant organisms, including Candida, Cryptococcus, and Aspergillus, in subjects with increasingly severe pulmonary and immunologic deficits. We analyze covariation of fungal and bacterial taxa, and find that oropharyngeal communities rich in Candida are also rich in mitis group Streptococci, a community pattern associated with pathogenic polymicrobial biofilms. Thus, using this approach, it is possible to characterize fungal communities in the human respiratory tract more accurately and explore their interactions with bacterial communities in health and disease.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0487-y) contains supplementary material, which is available to authorized users.     

Allosteric Inhibition of Human Immunodeficiency Virus Integrase: LATE BLOCK DURING VIRAL REPLICATION AND ABNORMAL MULTIMERIZATION INVOLVING SPECIFIC PROTEIN DOMAINS*

HIV-1 replication in the presence of antiviral agents results in evolution of drug-resistant variants, motivating the search for additional drug classes. Here we report studies of GSK1264, which was identified as a compound that disrupts the interaction between HIV-1 integrase (IN) and the cellular factor lens epithelium-derived growth factor (LEDGF)/p75. GSK1264 displayed potent antiviral activity and was found to bind at the site occupied by LEDGF/p75 on IN by x-ray crystallography. Assays of HIV replication in the presence of GSK1264 showed only modest inhibition of the early infection steps and little effect on integration targeting, which is guided by the LEDGF/p75·IN interaction. In contrast, inhibition of late replication steps was more potent. Particle production was normal, but particles showed reduced infectivity. GSK1264 promoted aggregation of IN and preformed LEDGF/p75·IN complexes, suggesting a mechanism of inhibition. LEDGF/p75 was not displaced from IN during aggregation, indicating trapping of LEDGF/p75 in aggregates. Aggregation assays with truncated IN variants revealed that a construct with catalytic and C-terminal domains of IN only formed an open polymer associated with efficient drug-induced aggregation. These data suggest that the allosteric inhibitors of IN are promising antiviral agents and provide new information on their mechanism of action.