Analysis of Cellular Factors Involved in Adeno-Associated Virus Type 2 Entry

The study of virus-host interactions has not only produced insights relevant to both preventative drug design and the emerging field of gene therapy, but it has also served as a valuable tool to the field of cell biology. The biological roles of many pathogen recognized cell-surface receptors have yet to be determined, yet are being exploited by pathogens to facilitate their entry and infection. Specifically a small family of sugar modified membrane protein known as heparan sulfate proteoglycans, have been shown to be a critical determinant in the efficiency of infection of many important pathogens. Adeno-associated virus type 2 (AAV) is a small (25nm) single stranded DNA, non-enveloped parvovirus that is currently being used as a vector for gene therapy. Valued for its lack of pathogenicity and ability to stably transduce a variety of terminal differentiated and quiescent cell types, the use of AAV during clinical trails has faced many complications. Consequently, the search for knowledge about this vector is more critical now then ever. Although extensive work on the safety of the in vivo application of this vector has been established, the cell-biological basis of AAV’s life cycle remains to be fully elucidated. Binding and entry of AAV requires heparan sulfate proteoglycan (HSPG) as well as two secondary receptors, fibroblast growth factor receptor and αv/5 β5/1 integrins. In this study, we follow the cellular modifications induced upon AAV binding and subsequent internalization into host cells. Earlier reports implicated receptor-mediated endocytosis as the sole pathway for AAV entry. We show that AAV’s primary receptor, HSPG, mediates an alternate entry pathway that is clathrin-independent and caveolar-independent. We show that inhibitors of macropinocytosis, which did not inhibit the clathrin-dependent entry pathways, blocked the entry of AAV and HSPG as well as blocked AAV transduction. In cells that are deficient in cell surface HSPG, transduction by AAV was reduced but unaffected by drugs that block macropinocytosis. Membrane ruffles induced upon AAV binding were enriched in activated PKC alpha, actin and dynamin. Treatment with dynosore, expression of dominant negative dynamin 2, or an HSPG construct lacking the PKC recruitment domain greatly diminished the size of macropinosomes and AAV internalization. Finally, silencing of clathrin heavy chain by siRNA did not affect AAV infection. Our data shows that HSPGs play a critical role in chaperoning AAV into a macropinocytic pathway, which leads to productive infection

Dendritic cells exposed to Human Immunodeficiency Virus-1 transmit a vigorous cytopathic infection to CD4+ T cells

Cameron, P.U., Freudenthal, P.S., Barker, J.M., Gezelter, S., Inaba, K., and Steinman, R.M. Dendritic cells exposed to Human Immunodeficiency Virus-1 transmit a vigorous cytopathic infection to CD4+ T cells. Science 257: 383-387, 1992https://digitalcommons.rockefeller.edu/historical-scientific-reports/1032/thumbnail.jp

Partnerships for Enhanced Engagement in Research (PEER) health: a new programme to facilitate LMIC research capacity

AbstractBackgroundThe Partnership for Enhanced Engagement in Research (PEER) Health programme, funded by the United States Agency for International Development (USAID), is a new research partnership programme that directly supports researchers from developing countries as Principal Investigators in partnership with collaborators at the US National Institutes of Health (NIH). The goals of this programme are threefold: (1) to advance critical evidence to address crucial health challenges facing LMICs; (2) to support collaboration and build on existing relationships between local public health and research institutions, global health practitioners, host country government, NIH researchers, and USAID Missions and Embassy staff; and (3) to build capacity by supporting researchers in LMICs thereby enabling local solutions to context specific challenges.MethodsMore than 180 preproposals from 25 LMICs were submitted to the first PEER Health research solicitation focused on Child Survival. After consultation with USAID Missions to ensure relevance to host country health priorities, about 80 applicants were invited to submit a full proposal. The National Institute of Child Health and Human Development organised a special emphasis panel for the scientific review of PEER Health full proposals to address both scientific merit and research capacity building aspects of the grant proposals, whilst maintaining key elements of the standard NIH peer review model. 40 experienced NIH reviewers, representing a wide variety of scientific disciplines and global public health experience, did the peer review of grants using a novel IT system developed for the PEER Health initiative. The National Academies of Sciences hosted an interagency protocol review to ensure scientific validity and compliance with international human subject guidelines.FindingsSixteen diverse grant partnerships were awarded in ten USAID Child Survival priority countries in Africa and Asia. Research funded under this programme is varied and includes studies from diverse topical areas, including malaria, neonatal sepsis, breast-feeding, tuberculosis, prevention of mother-to-child transmission, among others. Studies include investigation of both facility and community level interventions. NIH partners are largely US based and half are support by the National Institute of Allergy and Infectious Disease (NIAID).InterpretationBroad interest in this novel programme and applications from 25 countries show a crucial need for more directed funding for research strengthening and partnerships with LMIC Principal Investigators.FundingUSAID, in-kind support from NIH

Higher predation risk for insect prey at low latitudes and elevations

Biotic interactions underlie ecosystem structure and function, but predicting interaction outcomes is difficult. We tested the hypothesis that biotic interaction strength increases toward the equator, using a global experiment with model caterpillars to measure predation risk. Across an 11,660-kilometer latitudinal gradient spanning six continents, we found increasing predation toward the equator, with a parallel pattern of increasing predation toward lower elevations. Patterns across both latitude and elevation were driven by arthropod predators, with no systematic trend in attack rates by birds or mammals. These matching gradients at global and regional scales suggest consistent drivers of biotic interaction strength, a finding that needs to be integrated into general theories of herbivory, community organization, and life-history evolution

Plasma Membrane Is the Site of Productive HIV-1 Particle Assembly

Recently proposed models that have gained wide acceptance posit that HIV-1 virion morphogenesis is initiated by targeting the major structural protein (Gag) to late endosomal membranes. Thereafter, late endosome-based secretory pathways are thought to deliver Gag or assembled virions to the plasma membrane (PM) and extracellular milieu. We present several findings that are inconsistent with this model. Specifically, we demonstrate that HIV-1 Gag is delivered to the PM, and virions are efficiently released into the extracellular medium, when late endosome motility is abolished. Furthermore, we show that HIV-1 virions are efficiently released when assembly is rationally targeted to the PM, but not when targeted to late endosomes. Recently synthesized Gag first accumulates and assembles at the PM, but a proportion is subsequently internalized via endocytosis or phagocytosis, thus accounting for observations of endosomal localization. We conclude that HIV-1 assembly is initiated and completed at the PM, and not at endosomal membranes

Global Spore Sampling Project: A global, standardized dataset of airborne fungal DNA

Novel methods for sampling and characterizing biodiversity hold great promise for re-evaluating patterns of life across the planet. The sampling of airborne spores with a cyclone sampler, and the sequencing of their DNA, have been suggested as an efficient and well-calibrated tool for surveying fungal diversity across various environments. Here we present data originating from the Global Spore Sampling Project, comprising 2,768 samples collected during two years at 47 outdoor locations across the world. Each sample represents fungal DNA extracted from 24 m3 of air. We applied a conservative bioinformatics pipeline that filtered out sequences that did not show strong evidence of representing a fungal species. The pipeline yielded 27,954 species-level operational taxonomic units (OTUs). Each OTU is accompanied by a probabilistic taxonomic classification, validated through comparison with expert evaluations. To examine the potential of the data for ecological analyses, we partitioned the variation in species distributions into spatial and seasonal components, showing a strong effect of the annual mean temperature on community composition.publishedVersio

Airborne DNA reveals predictable spatial and seasonal dynamics of fungi.

Fungi are among the most diverse and ecologically important kingdoms in life. However, the distributional ranges of fungi remain largely unknown as do the ecological mechanisms that shape their distributions1,2. To provide an integrated view of the spatial and seasonal dynamics of fungi, we implemented a globally distributed standardized aerial sampling of fungal spores3. The vast majority of operational taxonomic units were detected within only one climatic zone, and the spatiotemporal patterns of species richness and community composition were mostly explained by annual mean air temperature. Tropical regions hosted the highest fungal diversity except for lichenized, ericoid mycorrhizal and ectomycorrhizal fungi, which reached their peak diversity in temperate regions. The sensitivity in climatic responses was associated with phylogenetic relatedness, suggesting that large-scale distributions of some fungal groups are partially constrained by their ancestral niche. There was a strong phylogenetic signal in seasonal sensitivity, suggesting that some groups of fungi have retained their ancestral trait of sporulating for only a short period. Overall, our results show that the hyperdiverse kingdom of fungi follows globally highly predictable spatial and temporal dynamics, with seasonality in both species richness and community composition increasing with latitude. Our study reports patterns resembling those described for other major groups of organisms, thus making a major contribution to the long-standing debate on whether organisms with a microbial lifestyle follow the global biodiversity paradigms known for macroorganisms4,5

Data from: Specificity in the symbiotic association between fungus-growing ants and protective Pseudonocardia bacteria

Fungus-growing ants (tribe Attini) engage in a mutualism with a fungus that serves as the ants' primary food source, but successful fungus cultivation is threatened by microfungal parasites (genus Escovopsis). Actinobacteria (genus Pseudonocardia) associate with most of the phylogenetic diversity of fungus-growing ants; are typically maintained on the cuticle of workers; and infection experiments, bioassay challenges and chemical analyses support a role of Pseudonocardia in defence against Escovopsis through antibiotic production. Here we generate a two-gene phylogeny for Pseudonocardia associated with 124 fungus-growing ant colonies, evaluate patterns of ant–Pseudonocardia specificity and test Pseudonocardia antibiotic activity towards Escovopsis. We show that Pseudonocardia associated with fungus-growing ants are not monophyletic: the ants have acquired free-living strains over the evolutionary history of the association. Nevertheless, our analysis reveals a significant pattern of specificity between clades of Pseudonocardia and groups of related fungus-growing ants. Furthermore, antibiotic assays suggest that despite Escovopsis being generally susceptible to inhibition by diverse Actinobacteria, the ant-derived Pseudonocardia inhibit Escovopsis more strongly than they inhibit other fungi, and are better at inhibiting this pathogen than most environmental Pseudonocardia strains tested. Our findings support a model that many fungus-growing ants maintain specialized Pseudonocardia symbionts that help with garden defence

Data from: Specificity in the symbiotic association between fungus-growing ants and protective Pseudonocardia bacteria

Fungus-growing ants (tribe Attini) engage in a mutualism with a fungus that serves as the ants' primary food source, but successful fungus cultivation is threatened by microfungal parasites (genus Escovopsis). Actinobacteria (genus Pseudonocardia) associate with most of the phylogenetic diversity of fungus-growing ants; are typically maintained on the cuticle of workers; and infection experiments, bioassay challenges and chemical analyses support a role of Pseudonocardia in defence against Escovopsis through antibiotic production. Here we generate a two-gene phylogeny for Pseudonocardia associated with 124 fungus-growing ant colonies, evaluate patterns of ant–Pseudonocardia specificity and test Pseudonocardia antibiotic activity towards Escovopsis. We show that Pseudonocardia associated with fungus-growing ants are not monophyletic: the ants have acquired free-living strains over the evolutionary history of the association. Nevertheless, our analysis reveals a significant pattern of specificity between clades of Pseudonocardia and groups of related fungus-growing ants. Furthermore, antibiotic assays suggest that despite Escovopsis being generally susceptible to inhibition by diverse Actinobacteria, the ant-derived Pseudonocardia inhibit Escovopsis more strongly than they inhibit other fungi, and are better at inhibiting this pathogen than most environmental Pseudonocardia strains tested. Our findings support a model that many fungus-growing ants maintain specialized Pseudonocardia symbionts that help with garden defence