Replication Vesicles are Load- and Choke-Points in the Hepatitis C Virus Lifecycle

Hepatitis C virus (HCV) infection develops into chronicity in 80% of all patients, characterized by persistent low-level replication. To understand how the virus establishes its tightly controlled intracellular RNA replication cycle, we developed the first detailed mathematical model of the initial dynamic phase of the intracellular HCV RNA replication. We therefore quantitatively measured viral RNA and protein translation upon synchronous delivery of viral genomes to host cells, and thoroughly validated the model using additional, independent experiments. Model analysis was used to predict the efficacy of different classes of inhibitors and identified sensitive substeps of replication that could be targeted by current and future therapeutics. A protective replication compartment proved to be essential for sustained RNA replication, balancing translation versus replication and thus effectively limiting RNA amplification. The model predicts that host factors involved in the formation of this compartment determine cellular permissiveness to HCV replication. In gene expression profiling, we identified several key processes potentially determining cellular HCV replication efficiency.     

Surveillance-Activated Defenses Block the ROS–Induced Mitochondrial Unfolded Protein Response

Disturbance of cellular functions results in the activation of stress-signaling pathways that aim at restoring homeostasis. We performed a genome-wide screen to identify components of the signal transduction of the mitochondrial unfolded protein response (UPR^mt) to a nuclear chaperone promoter. We used the ROS generating complex I inhibitor paraquat to induce the UPR^mt, and we employed RNAi exposure post-embryonically to allow testing genes whose knockdown results in embryonic lethality. We identified 54 novel regulators of the ROS–induced UPR^mt. Activation of the UPR^mt, but not of other stress-signaling pathways, failed when homeostasis of basic cellular mechanisms such as translation and protein transport were impaired. These mechanisms are monitored by a recently discovered surveillance system that interprets interruption of these processes as pathogen attack and depends on signaling through the JNK-like MAP-kinase KGB-1. Mutation of kgb-1 abrogated the inhibition of ROS–induced UPR^mt, suggesting that surveillance-activated defenses specifically inhibit the UPR^mt but do not compromise activation of the heat shock response, the UPR of the endoplasmic reticulum, or the SKN-1/Nrf2 mediated response to cytosolic stress. In addition, we identified PIFK-1, the orthologue of the Drosophila PI 4-kinase four wheel drive (FWD), and found that it is the only known factor so far that is essential for the unfolded protein responses of both mitochondria and endoplasmic reticulum. This suggests that both UPRs may share a common membrane associated mechanism.     

Proteoglycans Act as Cellular Hepatitis Delta Virus Attachment Receptors

The hepatitis delta virus (HDV) is a small, defective RNA virus that requires the presence of the hepatitis B virus (HBV) for its life cycle. Worldwide more than 15 million people are co-infected with HBV and HDV. Although much effort has been made, the early steps of the HBV/HDV entry process, including hepatocyte attachment and receptor interaction are still not fully understood. Numerous possible cellular HBV/HDV binding partners have been described over the last years; however, so far only heparan sulfate proteoglycans have been functionally confirmed as cell-associated HBV attachment factors. Recently, it has been suggested that ionotrophic purinergic receptors (P2XR) participate as receptors in HBV/HDV entry. Using the HBV/HDV susceptible HepaRG cell line and primary human hepatocytes (PHH), we here demonstrate that HDV entry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) side chains of cellular heparan sulfate proteoglycans. We furthermore provide evidence that P2XR are not involved in HBV/HDV entry and that effects observed with inhibitors for these receptors are a consequence of their negative charge. HDV infection was abrogated by soluble GAGs and other highly sulfated compounds. Enzymatic removal of defined carbohydrate structures from the cell surface using heparinase III or the obstruction of GAG synthesis by sodium chlorate inhibited HDV infection of HepaRG cells. Highly sulfated P2XR antagonists blocked HBV/HDV infection of HepaRG cells and PHH. In contrast, no effect on HBV/HDV infection was found when uncharged P2XR antagonists or agonists were applied. In summary, HDV infection, comparable to HBV infection, requires binding to the carbohydrate side chains of hepatocyte-associated heparan sulfate proteoglycans as attachment receptors, while P2XR are not actively involved.     

Intestinal Microbiota Composition of Interleukin-10 Deficient C57BL/6J Mice and Susceptibility to Helicobacter hepaticus-Induced Colitis

The mouse pathobiont Helicobacter hepaticus can induce typhlocolitis in interleukin-10-deficient mice, and H. hepaticus infection of immunodeficient mice is widely used as a model to study the role of pathogens and commensal bacteria in the pathogenesis of inflammatory bowel disease. C57BL/6J Il10^−/− mice kept under specific pathogen-free conditions in two different facilities (MHH and MIT), displayed strong differences with respect to their susceptibilities to H. hepaticus-induced intestinal pathology. Mice at MIT developed robust typhlocolitis after infection with H. hepaticus, while mice at MHH developed no significant pathology after infection with the same H. hepaticus strain. We hypothesized that the intestinal microbiota might be responsible for these differences and therefore performed high resolution analysis of the intestinal microbiota composition in uninfected mice from the two facilities by deep sequencing of partial 16S rRNA amplicons. The microbiota composition differed markedly between mice from both facilities. Significant differences were also detected between two groups of MHH mice born in different years. Of the 119 operational taxonomic units (OTUs) that occurred in at least half the cecum or colon samples of at least one mouse group, 24 were only found in MIT mice, and another 13 OTUs could only be found in MHH samples. While most of the MHH-specific OTUs could only be identified to class or family level, the MIT-specific set contained OTUs identified to genus or species level, including the opportunistic pathogen, Bilophila wadsworthia. The susceptibility to H. hepaticus-induced colitis differed considerably between Il10^−/− mice originating from the two institutions. This was associated with significant differences in microbiota composition, highlighting the importance of characterizing the intestinal microbiome when studying murine models of IBD.     

A comparison of the strength of biodiversity effects across multiple functions

In order to predict which ecosystem functions are most at risk from biodiversity loss, meta-analyses have generalised results from biodiversity experiments over different sites and ecosystem types. In contrast, comparing the strength of biodiversity effects across a large number of ecosystem processes measured in a single experiment permits more direct comparisons. Here, we present an analysis of 418 separate measures of 38 ecosystem processes. Overall, 45 % of processes were significantly affected by plant species richness, suggesting that, while diversity affects a large number of processes not all respond to biodiversity. We therefore compared the strength of plant diversity effects between different categories of ecosystem processes, grouping processes according to the year of measurement, their biogeochemical cycle, trophic level and compartment (above- or belowground) and according to whether they were measures of biodiversity or other ecosystem processes, biotic or abiotic and static or dynamic. Overall, and for several individual processes, we found that biodiversity effects became stronger over time. Measures of the carbon cycle were also affected more strongly by plant species richness than were the measures associated with the nitrogen cycle. Further, we found greater plant species richness effects on measures of biodiversity than on other processes. The differential effects of plant diversity on the various types of ecosystem processes indicate that future research and political effort should shift from a general debate about whether biodiversity loss impairs ecosystem functions to focussing on the specific functions of interest and ways to preserve them individually or in combination.     

The curious case of Hermodice carunculata (Annelida: Amphinomidae): evidence for genetic homogeneity throughout the Atlantic Ocean and adjacent basins

Over the last few decades, advances in molecular techniques have led to the detection of strong geographic population structure and cryptic speciation in many benthic marine taxa, even those with long‐lived pelagic larval stages. Polychaete annelids, in particular, generally show a high degree of population divergence, especially in mitochondrial genes. Rarely have molecular studies confirmed the presence of ‘cosmopolitan’ species. The amphinomid polychaete Hermodice carunculata was long considered the sole species within its genus, with a reported distribution throughout the Atlantic and adjacent basins. However, recent studies have indicated morphological differences, primarily in the number of branchial filaments, between the East and West Atlantic populations; these differences were invoked to re‐instate Hermodice nigrolineata, formerly considered a junior synonym of H. carunculata. We utilized sequence data from two mitochondrial (cytochrome c oxidase subunit I, 16S rDNA) markers and one nuclear (internal transcribed spacer) marker to examine the genetic diversity of Hermodice throughout its distribution range in the Atlantic Ocean, including the Mediterranean Sea, the Caribbean Sea, the Gulf of Mexico and the Gulf of Guinea. Our analyses revealed generally low genetic divergences among collecting localities and between the East and West Atlantic, although phylogenetic trees based on mitochondrial data indicate the presence of a private lineage in the Mediterranean Sea. A re‐evaluation of the number of branchial filaments confirmed differences between East and West Atlantic populations; however, the differences were not diagnostic and did not reflect the observed genetic population structure. Rather, we suspect that the number of branchial filaments is a function of oxygen saturation in the environment. Our results do not support the distinction between H. carunculata in the West Atlantic and H. nigrolineata in the East Atlantic. Instead, they re‐affirm the older notion that H. carunculata is a cohesive species with a broad distribution across the Atlantic Ocean.     

Reduced clonal reproduction indicates low potential for establishment of hybrids between wild and cultivated strawberries (Fragaria vesca × F. × ananassa)

The genus Fragaria (Rosaceae) contains 24 species, including hybrid species such as the garden strawberry (Fragaria × ananassa Duch.). Natural hybridization between Fragaria species has repeatedly been reported, and studies on the hybridization potential between F. × ananassa and its wild relatives have become increasingly important with the outlook for genetically modified garden strawberries. In Europe, a candidate species for hybridization with garden strawberries is the common woodland strawberry (Fragaria vesca L.). Although a previous field survey indicated that the potential for hybridization between F. vesca and F. × ananassa is low, it is not clear whether the lack of natural hybrids is caused by known pre- and postzygotic barriers, or whether hybrid plants lack the fitness to establish in natural F. vesca populations. We grew different F. vesca and F. vesca × F. × ananassa hybrid clones with and without competition in a greenhouse and assessed biomass production, clonal reproduction, and sexual reproduction of plants. While some hybrid clones exceeded F. vesca in biomass production, general clonal reproduction was much lower and delayed in hybrids. Furthermore, hybrids were sterile. These results demonstrate a mechanism by which the general lack of F. vesca × F. × ananassa hybrids in natural habitats can be explained, in addition to the known low hybridization potential between garden and woodland strawberries. We conclude that hybrids have a competitive disadvantage against co-occurring F. vesca plants due to inferior and delayed clonal reproduction, and that the potential for hybrid establishment under natural conditions is low.