The Activities of Current Antimalarial Drugs on the Life Cycle Stages of Plasmodium Life Cycle: A Comparative Study with Human and Rodent Parasites. (2012) PLoS Med 9(2): e1001169. doi:10.1371/journal.pmed.1001169
Background: Malaria remains a disease of devastating global impact, killing more than 800,000 people every year—the vast majority being children under the age of 5. While effective therapies are available, if malaria is to be eradicated a broader range of small molecule therapeutics that are able to target the liver and the transmissible sexual stages are required. These new medicines are needed both to meet the challenge of malaria eradication and to circumvent resistance.
Methods and Findings: Little is known about the wider stage-specific activities of current antimalarials that were primarily designed to alleviate symptoms of malaria in the blood stage. To overcome this critical gap, we developed assays to measure activity of antimalarials against all life stages of malaria parasites, using a diverse set of human and nonhuman parasite species, including male gamete production (exflagellation) in Plasmodium falciparum, ookinete development in P. berghei, oocyst development in P. berghei and P. falciparum, and the liver stage of P. yoelii. We then compared 50 current and experimental antimalarials in these assays. We show that endoperoxides such as OZ439, a stable synthetic molecule currently in clinical phase IIa trials, are strong inhibitors of gametocyte maturation/gamete formation and impact sporogony; lumefantrine impairs development in the vector; and NPC-1161B, a new 8-aminoquinoline, inhibits sporogony.
Conclusions: These data enable objective comparisons of the strengths and weaknesses of each chemical class at targeting each stage of the lifecycle. Noting that the activities of many compounds lie within achievable blood concentrations, these results offer an invaluable guide to decisions regarding which drugs to combine in the next-generation of antimalarial drugs. This study might reveal the potential of life-cycle–wide analyses of drugs for other pathogens with complex life cycles.

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Researchers used complete sequencing of microbial genomes to observe speciation in action. They looked at Archaea called Sulfolobus islandicus gathered from a geothermal hot spring from the Mutnovsky Volcano in Kamchatka, Russia, whose physical isolation allows us to pinpoint evolutionary processes to one location. Contrary to the theoretical predictions for microbes, they were able to provide evidence that two novel lineages are in the process of becoming ecologically distinct and evolutionarily independent despite the fact that they recombine. The divergence is not happening uniformly across the genome because certain genomic regions are more prone to become differentiated between species than others. This genomic view of the process of speciation occurring within a single natural microbial population contributes to our understanding of the generation of biodiversity in Archaea and furthers our understanding of speciation across the tree of life.

Patterns of Gene Flow Define Species of Thermophilic Archaea. (2012) PLoS Biol 10(2): e1001265. doi:10.1371/journal.pbio.1001265
Despite a growing appreciation of their vast diversity in nature, mechanisms of speciation are poorly understood in Bacteria and Archaea. Here we use high-throughput genome sequencing to identify ongoing speciation in the thermoacidophilic Archaeon Sulfolobus islandicus. Patterns of homologous gene flow among genomes of 12 strains from a single hot spring in Kamchatka, Russia, demonstrate higher levels of gene flow within than between two persistent, coexisting groups, demonstrating that these microorganisms fit the biological species concept. Furthermore, rates of gene flow between two species are decreasing over time in a manner consistent with incipient speciation. Unlike other microorganisms investigated, we do not observe a relationship between genetic divergence and frequency of recombination along a chromosome, or other physical mechanisms that would reduce gene flow between lineages. Each species has its own genetic island encoding unique physiological functions and a unique growth phenotype that may be indicative of ecological specialization. Genetic differentiation between these coexisting groups occurs in large genomic ”continents,” indicating the topology of genomic divergence during speciation is not uniform and is not associated with a single locus under strong diversifying selection. These data support a model where species do not require physical barriers to gene flow but are maintained by ecological differentiation.

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This study confirms the effects of MV P gene products of a wild MV strain on IFN pathways and examined that of other viral proteins on it. The authors found that N protein acts as an IFN-α/β and γ-antagonist as strong as P gene products. They investigated the mechanisms of MV-N inhibition, and revealed that MV-N blocks the nuclear import of activated STAT without preventing STAT and Jak activation or STAT degradation, and that the nuclear translocation of MV-N is important for the inhibition. The inhibitory effect of the N protein was observed as a common feature of other morbilliviruses. These results suggest that N protein of MV as well as P/V/C proteins is involved in the inhibition of host IFN signaling pathways.

The nucleocapsid protein of measles virus blocks host interferon response. Virology. 05 Jan 2012

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Mycobacterium tuberculosis is one of the most life-threatening pathogens of all time. Despite the development of vaccines and antibiotics, this pathogen is still a major public health problem. The HIV epidemic has also had an important impact on the rise of M. tuberculosis infections since immunodeficient people are highly susceptible. Commonly, M. tuberculosis has been thought to reside in a membrane-bound compartment within its host cells during the entire infection cycle from invasion to cell death.

Using a fluorescence-based method, new research provides evidence that M. tuberculosis is able to rupture its membrane-bound compartment and gain access to the host cytosol, where it can cause cell death. Importantly, the researchers were able to track the dynamics of infection to understand the consequences of M. tuberculosis phagosomal rupture. This revealed that phagosomal rupture results in cell toxicity and host cell death involving necrosis. Together, this data provides a new angle in the worldwide fight against tuberculosis and could lead to new approaches in the development of innovative treatments.

Phagosomal Rupture by Mycobacterium tuberculosis Results in Toxicity and Host Cell Death. (2012) PLoS Pathog 8(2): e1002507. doi:10.1371/journal.ppat.1002507
Survival within macrophages is a central feature of Mycobacterium tuberculosis pathogenesis. Despite significant advances in identifying new immunological parameters associated with mycobacterial disease, some basic questions on the intracellular fate of the causative agent of human tuberculosis in antigen-presenting cells are still under debate. To get novel insights into this matter, we used a single-cell fluorescence resonance energy transfer (FRET)-based method to investigate the potential cytosolic access of M. tuberculosis and the resulting cellular consequences in an unbiased, quantitative way. Analysis of thousands of THP-1 macrophages infected with selected wild-type or mutant strains of the M. tuberculosis complex unambiguously showed that M. tuberculosis induced a change in the FRET signal after 3 to 4 days of infection, indicating phagolysosomal rupture and cytosolic access. These effects were not seen for the strains M. tuberculosisΔRD1 or BCG, both lacking the ESX-1 secreted protein ESAT-6, which reportedly shows membrane-lysing properties. Complementation of these strains with the ESX-1 secretion system of M. tuberculosis restored the ability to cause phagolysosomal rupture. In addition, control experiments with the fish pathogen Mycobacterium marinum showed phagolysosomal translocation only for ESX-1 intact strains, further validating our experimental approach. Most importantly, for M. tuberculosis as well as for M. marinum we observed that phagolysosomal rupture was followed by necrotic cell death of the infected macrophages, whereas ESX-1 deletion- or truncation-mutants that remained enclosed within phagolysosomal compartments did not induce such cytotoxicity. Hence, we provide a novel mechanism how ESX-1 competent, virulent M. tuberculosis and M. marinum strains induce host cell death and thereby escape innate host defenses and favor their spread to new cells. In this respect, our results also open new research directions in relation with the extracellular localization of M. tuberculosis inside necrotic lesions that can now be tackled from a completely new perspective.

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Although the first miRNA was identified 18 years ago, it was only in 2001, with the development of technologies that allowed the efficient cDNA cloning and sequencing of small RNA species, that it became apparent that all multicellular eukaryotes encode numerous members of this class of small regulatory RNAs. Shortly after the identification of the first human miRNAs, the first virally encoded miRNAs were reported in the human herpesvirus Epstein–Barr virus (EBV). Although this initial discovery suggested that viruses in general might use miRNAs to down-regulate cellular factors that inhibit viral replication, the subsequent analysis of a wide range of RNA viruses failed to identify any viral miRNAs. However, virally encoded miRNAs are expressed by many members of the herpesvirus family of nuclear DNA viruses and are also found in a small number of other nuclear DNA viruses, particularly polymaviruses.

Although >250 viral microRNAs (miRNAs) are expressed by a range of nuclear DNA viruses, efforts to identify miRNAs expressed by RNA viruses have so far been in vain. In PNAS, Kincaid et al. report the identification of five miRNAs encoded by the delta retrovirus bovine leukemia virus (BLV) that are expressed in BLV-transformed B cells. It appears likely that these viral miRNAs play an important role in BLV pathogenesis:

MicroRNA expression by an oncogenic retrovirus. PNAS USA, 30 Jan 2012

RNA virus microRNA that mimics a B-cell oncomiR. PNAS USA, 30 Jan 2012; doi: 10.1073/pnas.1116107109

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Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora. PNAS USA 17 January 2012 doi: 10.1073/pnas.1115346109
Syncytins are envelope protein genes of retroviral origin that have been captured for a function in placentation. Two such genes have already been identified in simians, two distinct, unrelated genes have been identified in Muridae, and a fifth gene has been identified in the rabbit. Here, we searched for similar genes in the Laurasiatheria clade, which diverged from Euarchontoglires—primates, rodents, and lagomorphs—shortly after mammalian radiation (100 Mya). In silico search for envelope protein genes with full-coding capacity within the dog and cat genomes identified several candidate genes, with one common to both species that displayed placenta-specific expression, which was revealed by RT-PCR analysis of a large panel of tissues. This gene belongs to a degenerate endogenous retroviral element, with precise proviral integration at a site common to dog and cat. Cloning of the gene for an ex vivo pseudotype assay showed fusogenicity on both dog and cat cells. In situ hybridization on placenta sections from both species showed specific expression at the level of the invasive fetal villi within the placental junctional zone, where trophoblast cells fuse into a syncytiotrophoblast layer to form the maternofetal interface. Finally, we show that the gene is conserved among a series of 26 Carnivora representatives, with evidence for purifying selection and conservation of fusogenic activity. The gene is not found in the Pholidota order and, therefore, it was captured before Carnivora radiation, between 60 and 85 Mya. This gene is the oldest syncytin gene identified to date, and it is the first in a new major clade of eutherian mammals.

See also: Retroviruses push the envelope for mammalian placentation

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Filovirus Entry: A Novelty in the Viral Fusion World. (2012) Viruses 4(2): 258-275; doi:10.3390/v4020258
Fliovirus entry into cells is initiated by the interaction of the viral glycoprotein1 subunit (GP1) with both adherence factors and one or more receptors on the surface of host cells. On epithelial cells, we recently demonstrated that TIM-1 serves as a receptor for this family of viruses, but the cell surface receptors in other cell types remain unidentified. Upon receptor binding, the virus is internalized into endosomes primarily via macropinocytosis, but perhaps by other mechanisms as well. Within the acidified endosome, the heavily glycosylated GP1 is cleaved to a smaller form by the low pH-dependent cellular proteases Cathepsin L and B, exposing residues in the receptor binding site (RBS). Details of the molecular events following cathepsin-dependent trimming of GP1 are currently incomplete; however, the processed GP1 specifically interacts with endosomal/lysosomal membranes that contain the Niemann Pick C1 (NPC1) protein and expression of NPC1 is required for productive infection, suggesting that GP/NPC1 interactions may be an important late step in the entry process. Additional events such as further GP1 processing and/or reducing events may also be required to generate a fusion-ready form of the glycoprotein. Once this has been achieved, sequences in the filovirus GP2 subunit mediate viral/cellular membrane fusion via mechanisms similar to those previously described for other enveloped viruses. This multi-step entry pathway highlights the complex and highly orchestrated path of internalization and fusion that appears unique for filoviruses.

MicrobiologyBytes: Yes folks, it’s that naughty Niemann Pick C1 (NPC1) protein again!
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Identification of the Niemann-Pick C1–like 1 cholesterol absorption receptor as a new hepatitis C virus entry factor. Nature Medicine 08 January 2012 doi:10.1038/nm.2581
Hepatitis C virus (HCV) is a leading cause of liver disease worldwide. With ~170 million individuals infected and current interferon-based treatment having toxic side effects and marginal efficacy, more effective antivirals are crucially needed. Although HCV protease inhibitors were just approved by the US Food and Drug Administration (FDA), optimal HCV therapy, analogous to HIV therapy, will probably require a combination of antivirals targeting multiple aspects of the viral lifecycle. Viral entry represents a potential multifaceted target for antiviral intervention; however, to date, FDA-approved inhibitors of HCV cell entry are unavailable. Here we show that the cellular Niemann-Pick C1–like 1 (NPC1L1) cholesterol uptake receptor is an HCV entry factor amendable to therapeutic intervention. Specifically, NPC1L1 expression is necessary for HCV infection, as silencing or antibody-mediated blocking of NPC1L1 impairs cell culture–derived HCV (HCVcc) infection initiation. In addition, the clinically available FDA-approved NPC1L1 antagonist ezetimibe potently blocks HCV uptake in vitro via a virion cholesterol–dependent step before virion-cell membrane fusion. Moreover, ezetimibe inhibits infection by all major HCV genotypes in vitro and in vivo delays the establishment of HCV genotype 1b infection in mice with human liver grafts. Thus, we have not only identified NPC1L1 as an HCV cell entry factor but also discovered a new antiviral target and potential therapeutic agent.

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Arenavirus entry into the host cell is mediated by the envelope glycoprotein complex, GPC. The virion is endocytosed on binding to a cell-surface receptor, and membrane fusion is initiated in response to physiological acidification of the endosome. As with other class I virus fusion proteins, GPC-mediated membrane fusion is promoted through a regulated sequence of conformational changes leading to formation of the classical postfusion trimer-of-hairpins structure. GPC is, however, unique among the class I fusion proteins in that the mature complex retains a stable signal peptide (SSP) as a third subunit, in addition to the canonical receptor-binding and fusion proteins.

This review describes the properties of the tripartite GPC complex and the evidence that SSP interacts with the fusion subunit to modulate pH-induced activation of membrane fusion. This unusual solution to maintaining the metastable prefusion state of GPC on the virion and activating the class I fusion cascade at acidic pH provides novel targets for antiviral intervention.

The Curious Case of Arenavirus Entry, and Its Inhibition. (2012) Viruses 4(1), 83-101 doi:10.3390/v4010083

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