Architecture of respiratory syncytial virus revealed by electron cryotomography

Human respiratory syncytial virus is a human pathogen that causes severe infection of the respiratory tract. Current information about the structure of the virus and its interaction with host cells is limited. We carried out an electron cryotomographic characterization of cell culture-grown human respiratory syncytial virus to determine the architecture of the virion. The particles ranged from 100 nm to 1,000 nm in diameter and were spherical, filamentous, or a combination of the two. The filamentous morphology correlated with the presence of a cylindrical matrix protein layer linked to the inner leaflet of the viral envelope and with local ordering of the glycoprotein spikes. Recombinant viruses with only the fusion protein in their envelope showed that these glycoproteins were predominantly in the postfusion conformation, but some were also in the prefusion form. The ribonucleocapsids were left-handed, randomly oriented, and curved inside the virions. In filamentous particles, they were often adjacent to an intermediate layer of protein assigned to M2-1 (an envelope-associated protein known to mediate association of ribonucleocapsids with the matrix protein). Our results indicate important differences in structure between the Paramyxovirinae and Pneumovirinae subfamilies within the Paramyxoviridae, and provide fresh insights into host cell exit of a serious pathogen.     

A novel system for identification of inhibitors of rift valley Fever virus replication

Rift Valley fever virus (RVFV) is a human and livestock pathogen endemic to sub-Saharan Africa. We have developed a T7-dependent system for the efficient production of RVFV-like particles (RVF-VLPs) based on the virulent ZH-501 strain of RVFV. The RVF-VLPs are capable of performing a single round of infection, allowing for the study of viral replication, assembly, and infectivity. We demonstrate that these RVF-VLPs are antigenically indistinguishable from authentic RVFV and respond similarly to a wide array of known and previously unknown chemical inhibitors. This system should be useful for screening for small molecule inhibitors of RVFV replication.     

In between: Gypsy in Drosophila melanogaster Reveals New Insights into Endogenous Retrovirus Evolution

Retroviruses are RNA viruses that are able to synthesize a DNA copy of their genome and insert it into a chromosome of the host cell. Sequencing of different eukaryote genomes has revealed the presence of many such endogenous retroviral sequences. The mechanisms by which these retroviral sequences have colonized the genome are still unknown, and the endogenous retrovirus gypsy of Drosophila melanogaster is a powerful experimental model for deciphering this process in vivo. Gypsy is expressed in a layer of somatic cells, and then transferred into the oocyte by an unknown mechanism. This critical step is the start of the endogenization process. Moreover gypsy has been shown to have infectious properties, probably due to its envelope gene acquired from a baculovirus. Recently we have also shown that gypsy maternal transmission is reduced in the presence of the endosymbiotic bacterium Wolbachia. These studies demonstrate that gypsy is a unique and powerful model for understanding the endogenization of retroviruses.     

Origin and diversification of wings: Insights from a neopteran insect

Winged insects underwent an unparalleled evolutionary radiation, but mechanisms underlying the origin and diversification of wings in basal insects are sparsely known compared with more derived holometabolous insects. In the neopteran species Oncopeltus fasciatus, we manipulated wing specification genes and used RNA-seq to obtain both functional and genomic perspectives. Combined with previous studies, our results suggest the following key steps in wing origin and diversification. First, a set of dorsally derived outgrowths evolved along a number of body segments including the first thoracic segment (T1). Homeotic genes were subsequently co-opted to suppress growth of some dorsal flaps in the thorax and abdomen. In T1 this suppression was accomplished by Sex combs reduced, that when experimentally removed, results in an ectopic T1 flap similar to prothoracic winglets present in fossil hemipteroids and other early insects. Global gene-expression differences in ectopic T1 vs. T2/T3 wings suggest that the transition from flaps to wings required ventrally originating cells, homologous with those in ancestral arthropod gill flaps/epipods, to migrate dorsally and fuse with the dorsal flap tissue thereby bringing new functional gene networks; these presumably enabled the T2/T3 wing’s increased size and functionality. Third, “fused” wings became both the wing blade and surrounding regions of the dorsal thorax cuticle, providing tissue for subsequent modifications including wing folding and the fit of folded wings. Finally, Ultrabithorax was co-opted to uncouple the morphology of T2 and T3 wings and to act as a general modifier of hindwings, which in turn governed the subsequent diversification of lineage-specific wing forms.Some 350 million years ago, the development of insect wings was a seminal event in the evolution of insect body design (1, 2). The ability to fly was critical to insects becoming the most diverse and abundant animal group, and the origin of such novelty has been a focus of intense scientific inquiry for more than a century (3, 4). More recently, through studies of genetic model systems such as Drosophila, the mechanisms of wing morphogenesis have been elucidated (5–12). Still lacking however is a comprehensive understanding of transitional steps connecting the morphology of structures observed in the fossil record with that of the modern-day insects, including wing origins and subsequent diversification.The initial stages of insect wing evolution are missing from the fossil record and it is therefore necessary to use indirect evidence from fossils that postdate the origin and initial radiation of pterygotes (2). Larvae of many of those taxa featured dorsally positioned outgrowths on each of the thoracic and abdominal segments (2, 13), apparently serial homologs (i.e., similar structures likely arising from a common set of developmental mechanisms). Diverse lineages independently lost those dorsal appendages on the abdomen while undergoing parallel modifications of wing-like structures on thoracic (T1–T3) segments. Specifically, the T1 winglets were always much smaller in fossils and apparently lacked hinge articulation whereas T2 (fore-) and T3 (hind-) wings were fully operational in adults, featuring muscles, venation, and size that rendered them capable of flapping flight (14). T1 winglets were subsequently repressed in multiple lineages (15–18) whereas T2 and T3 wings acquired morphology similar to modern day Paleoptera (mayflies and dragonflies) and other extinct paleopterous orders. The transition from Paleoptera, which rest with wings extended from the body, to Neoptera, which rest with wings folded flat against the body, required changes in the hinge mechanism, with many orders also evolving a precise mechanical fit between wing margins and the adjacent body wall of the dorsal thorax. Finally, the radiation of Neoptera encompassed a further divergence between the fore- and hindwings in terms of their shape, size, and texture (5, 19, 20). Together, this set of transitions accounts for major features of the diversity and lineage-specific wing morphologies among fossil and extant taxa.To gain insight into genetic mechanisms governing these transitional steps, we used a direct-developing neopteran species, the milkweed bug (Oncopeltus fasciatus; Hemimetabola, Hemiptera). The phylogenetic placement of Oncopeltus, basal to the more derived holometabola (e.g., flies, beetles, bees, butterflies, and so forth that have a pupal stage), is important because holometabolous appendage development occurs from imaginal discs, which are collections of cells that form and commit to appendage identity early in larval development. Oncopeltus and other hemimetabolous insects lack imaginal discs and acquire adult morphology gradually through a series of nymphal stages, similar to early fossil insects. Hence, examination of wing development in a hemimetabolous insect can help resolve the ancestral versus derived status of developmental traits present in holometabola and in general may provide new evolutionary perspectives.Oncopeltus features brightly colored forewings with a stiff proximal region and a more flexible membranous apex (hemelytra), entirely membranous hind wings, and a well-developed dorsal T2 structure (scutellum) that fits precisely against trailing edges of the folded wings. To examine wing developmental mechanisms underlying these features, we combined a candidate gene approach in which we depleted (via RNA interference, RNAi) the expression of Oncopeltus orthologs of wing specification genes, and a global approach (RNA-seq) (21) that characterized all expressed genes in wild-type T2 and T3 wings, ectopic T1 wings, and wild-type T1 body wall. The results provide independent and expanded insights into the origins and fate of T1 wings, the transition from paleoptera to neoptera, and the eventual diversification of T2 and T3 wing morphology.     

Identification of glycosaminoglycan-binding sites within hepatitis C virus envelope glycoprotein E2*

Heparan sulphate is one of the candidate receptors for hepatitis C virus (HCV). Envelope glycoproteins of HCV have been proposed to be responsible for recognition and binding with cell receptors. They are characterized by great genetic polymorphism. In this study the mapping of regions with glycosaminoglycan-binding properties within HCV envelope proteins has been undertaken. We prepared a set of overlapping peptides corresponding to conserved regions of these envelope proteins and analysed them by solid phase heparin-binding assay. The search for established glycosaminoglycan-binding motifs in the HCV envelope proteins showed the absence of the sites corresponding to the glycosaminoglycan-binding patterns in consensus sequence. We identified one highly conserved and two less conserved heparin-binding sequences within the envelope protein E2 based on solid phase assay results. We did not find any differences in binding efficiency of these peptides with heparin, heparan sulphate or dextran sulphate. Our data supported the specific association between HCV envelope protein E2 and cell surface glycosaminoglycans. We hypothesize that identified regions from E2 can contribute to HCV binding to cell surface glycosaminoglycans.     

Mitochondrial copper(I) transfer from Cox17 to Sco1 is coupled to electron transfer

The human protein Cox17 contains three pairs of cysteines. In the mitochondrial intermembrane space (IMS) it exists in a partially oxidized form with two S-S bonds and two reduced cysteines (HCox17(2S-S)). HCox17(2S-S) is involved in copper transfer to the human cochaperones Sco1 and Cox11, which are implicated in the assembly of cytochrome c oxidase. We show here that Cu(I)HCox17(2S-S), i.e., the copper-loaded form of the protein, can transfer simultaneously copper(I) and two electrons to the human cochaperone Sco1 (HSco1) in the oxidized state, i.e., with its metal-binding cysteines forming a disulfide bond. The result is Cu(I)HSco1 and the fully oxidized apoHCox17(3S-S), which can be then reduced by glutathione to apoHCox17(2S-S). The HSco1/HCox17(2S-S) redox reaction is thermodynamically driven by copper transfer. These reactions may occur in vivo because HSco1 can be found in the partially oxidized state within the IMS, consistent with the variable redox properties of the latter compartment. The electron transfer-coupled metallation of HSco1 can be a mechanism within the IMS for an efficient specific transfer of the metal to proteins, where metal-binding thiols are oxidized. The same reaction of copper-electron-coupled transfer does not occur with the human homolog of Sco1, HSco2, for kinetic reasons that may be ascribed to the lack of a specific metal-bridged protein-protein complex, which is instead observed in the Cu(I)HCox17(2S-S)/HSco1 interaction.     

Morphologic Differentiation of Viruses beyond the Family Level

Electron microscopy has been instrumental in the identification of viruses by being able to characterize a virus to the family level. There are a few cases where morphologic or morphogenesis factors can be used to differentiate further, to the genus level. These include viruses in the families Poxviridae, Reoviridae, Retroviridae, Herpesviridae, Filoviridae, and Bunyaviridae.     

Arabidopsis MZT1 homologs GIP1 and GIP2 are essential for centromere architecture

Centromeres play a pivotal role in maintaining genome integrity by facilitating the recruitment of kinetochore and sister-chromatid cohesion proteins, both required for correct chromosome segregation. Centromeres are epigenetically specified by the presence of the histone H3 variant (CENH3). In this study, we investigate the role of the highly conserved γ-tubulin complex protein 3-interacting proteins (GIPs) in Arabidopsis centromere regulation. We show that GIPs form a complex with CENH3 in cycling cells. GIP depletion in the gip1gip2 knockdown mutant leads to a decreased CENH3 level at centromeres, despite a higher level of Mis18BP1/KNL2 present at both centromeric and ectopic sites. We thus postulate that GIPs are required to ensure CENH3 deposition and/or maintenance at centromeres. In addition, the recruitment at the centromere of other proteins such as the CENP-C kinetochore component and the cohesin subunit SMC3 is impaired in gip1gip2. These defects in centromere architecture result in aneuploidy due to severely altered centromeric cohesion. Altogether, we ascribe a central function to GIPs for the proper recruitment and/or stabilization of centromeric proteins essential in the specification of the centromere identity, as well as for centromeric cohesion in somatic cells.In eukaryotes, centromeres play a critical role in accurate chromosome segregation and in the maintenance of genome integrity through their regulated assembly and the maintenance of their cohesion until anaphase. Centromeres consist each of a central core (1) characterized epigenetically by the recruitment of the histone H3 variant CENH3 (CENP-A in animals). Extensive studies are still ongoing to identify the regulatory factors for loading and maintenance of CENH3 at centromeres. In yeast, suppressor of chromosome missegregation protein 3 was identified as a specific chaperone for CENP-A loading (2). In animals, the Mis18 complex, including the CENH3 assembly factor Kinetochore Null 2 (KNL2; also called Mis18BP1), recruits the cell cycle-dependent maintenance and deposition factor of CENP-A, HJURP (Holliday junction recognition protein), to centromeres (3). Recently, two Mis18-complex components, Eic1 and Eic2, were identified in fission yeast (4). Whereas Eic1 promotes CENH3 loading and maintenance, Eic2 is recruited at centromeres independently of its association with Mis18. Together with CENH3, the conserved kinetochore assembly protein CENP-C participates in pericentromeric cohesin recruitment (5). The CENH3 loading machinery changed rapidly during evolution, and a CENH3 chaperone has not been identified in plants thus far. Moreover, nothing is known about a possibly conserved interplay between CENH3 loading and sister chromatid cohesion at centromeres. Recently, the plant homolog of KNL2 was proposed as an upstream component for CENH3 deposition at centromeres (6). Finally, the regulation of centromeric complex positioning at the nuclear envelope environment is still elusive in plants.

Table S1.

Gene accession numbers

Epidermal growth factor receptor and metastatic colorectal cancer: Insights into target therapies

Colorectal cancer(CRC)has high incidence and mortality worldwide.In 2012,CRC was the second most prevalent cancer among males(9%)and the third among females(8%).In recent decades,standard chemotherapies protocols combining 5-fluorouracil,leucovorin,irinotecan and oxaliplatin were important for improve survival in this set of patients.Further,biological drugs throughout epidermal growth factor receptor(EGFR)pathways showed interesting results in metastatic disease(mCRC)control when in association to standard chemotherapy regimens.Cetuximab and panitumumab are two cornerstones for mCRC treatment and are both approved in Europe and United States based on previous results phaseⅢtrials.This paper will briefly summarize those anti-EGFR therapies framework in mCRC and discusses some issues in this regard.     

Evolutionary plasticity of developmental gene regulatory network architecture

Sea stars and sea urchins evolved from a last common ancestor that lived at the end of the Cambrian, approximately half a billion years ago. In a previous comparative study of the gene regulatory networks (GRNs) that embody the genomic program for embryogenesis in these animals, we discovered an almost perfectly conserved five-gene network subcircuit required for endoderm specification. We show here that the GRN structure upstream and downstream of the conserved network kernel has, by contrast, diverged extensively. Mesoderm specification is accomplished quite differently; the Delta-Notch signaling system is used in radically distinct ways; and various regulatory genes have been coopted to different functions. The conservation of the conserved kernel is thus the more remarkable. The results indicate types of network linkage subject to evolutionary change. An emergent theme is that subcircuit design may be preserved even while the identity of genes performing given roles changes because of alteration in their cis-regulatory control systems.     

Purification and application of C-terminally truncated hepatitis C virus E1 proteins expressed in Escherichia coli

AIM: To explore the possibility of expressing hepatitis C virus (HCV) envelope protein 1 (E1) in Escherichia coli (E. coli) and to test the purified recombinant E1 proteins for clinical and research applications. METHODS: C-terminally truncated E1 fragments were expressed in E. coli as hexa-histidine-tagged fusion proteins. The expression products were purified under denaturing conditions using immobilized-metal affinity chromatography. Purified E1 proteins were used to immunize rabbits. Rabbit anti-sera thus obtained were reacted with both E. coli- and mammalian cell-expressed E1 glycoproteins as detected by Western blot. RESULTS: Full-length E1 protein proved difficult to express in E. coli. C-terminally truncated E1 was successfully expressed in E. coli as hexa-histidine-tagged recombinant fusion protein and was purified under denaturing conditions on Ni(2+)-NTA agarose. Rabbit anti-sera raised against purified recombinant E1 specifically reacted with mammalian cell-expressed E1 glycoproteins in Western blot. Furthermore, E. coli-derived E1 protein was able to detect animal antibodies elicited by E1-based DNA immunization. CONCLUSION: These results demonstrate that the prokaryotically expressed E1 proteins share identical epitopes with eukaryotically expressed E1 glycoprotein. The E. coli-derived E1 proteins and corresponding antisera can become useful tools in anti-HCV vaccine research.     

Role of hepatitis B virus DNA integration in human hepatocarcinogenesis

Liver cancer ranks sixth in cancer incidence, and is the third leading cause of cancer-related deaths worldwide. Hepatocellular carcinoma (HCC) is the most common type of liver cancer, which arises from hepatocytes and accounts for approximately 70%-85% of cases. Hepatitis B virus (HBV) frequently causes liver inflammation, hepatic damage and subsequent cirrhosis. Integrated viral DNA is found in 85%-90% of HBV-related HCCs. Its presence in tumors from non-cirrhotic livers of children or young adults further supports the role of viral DNA integration in hepatocarcinogenesis. Integration of subgenomic HBV DNA fragments into different locations within the host DNA is a significant feature of chronic HBV infection. Integration has two potential consequences: (1) the host genome becomes altered (“cis” effect); and (2) the HBV genome becomes altered (“trans” effect). The cis effect includes insertional mutagenesis, which can potentially disrupt host gene function or alter host gene regulation. Tumor progression is frequently associated with rearrangement and partial gain or loss of both viral and host sequences. However, the role of integrated HBV DNA in hepatocarcinogenesis remains controversial. Modern technology has provided a new paradigm to further our understanding of disease mechanisms. This review summarizes the role of HBV DNA integration in human carcinogenesis.     

Insights into Campylobacter jejuni colonization of the mammalian intestinal tract using a novel mouse model of infection

A lack of relevant disease models for Campylobacter jejuni has long been an obstacle to research into this common enteric pathogen. We recently published that mice deficient in Single IgG Interleukin-1 related receptor (SIGIRR), a repressor of MyD88-dependent innate immune signaling, were highly susceptible to enteric infection by murine bacterial pathogens. Subsequently, we successfully employed these mice as an animal model for the human pathogen C. jejuni and gained substantial new insights into infection by this pathogen. The infected mice developed significant intestinal inflammation, primarily via TLR4 stimulation. Furthermore, the resulting gastroenteritis was dependent on C. jejuni pathogenesis as bacterial strains suffering mutations in key virulence factors were attenuated in causing disease. The ability to infect SIGIRR-deficient mice with C. jejuni sheds new light onto how these bacteria colonize the mucus layer of the intestinal tract, invade epithelial cells, and raises new prospects for studying the virulence strategies and pathogenesis of C. jejuni.     

Complete nucleotide sequence of watermelon chlorotic stunt virus originating from oman

Watermelon chlorotic stunt virus (WmCSV) is a bipartite begomovirus (genus Begomovirus, family Geminiviridae) that causes economic losses to cucurbits, particularly watermelon, across the Middle East and North Africa. Recently squash (Cucurbita moschata) grown in an experimental field in Oman was found to display symptoms such as leaf curling, yellowing and stunting, typical of a begomovirus infection. Sequence analysis of the virus isolated from squash showed 97.6-99.9% nucleotide sequence identity to previously described WmCSV isolates for the DNA A component and 93-98% identity for the DNA B component. Agrobacterium-mediated inoculation to Nicotiana benthamiana resulted in the development of symptoms fifteen days post inoculation. This is the first bipartite begomovirus identified in Oman. Overall the Oman isolate showed the highest levels of sequence identity to a WmCSV isolate originating from Iran, which was confirmed by phylogenetic analysis. This suggests that WmCSV present in Oman has been introduced from Iran. The significance of this finding is discussed.     

Structure of the Rift Valley fever virus nucleocapsid protein reveals another architecture for RNA encapsidation

Rift Valley fever virus (RVFV) is a negative-sense RNA virus (genus Phlebovirus, family Bunyaviridae) that infects livestock and humans and is endemic to sub-Saharan Africa. Like all negative-sense viruses, the segmented RNA genome of RVFV is encapsidated by a nucleocapsid protein (N). The 1.93-Å crystal structure of RVFV N and electron micrographs of ribonucleoprotein (RNP) reveal an encapsidated genome of substantially different organization than in other negative-sense RNA virus families. The RNP polymer, viewed in electron micrographs of both virus RNP and RNP reconstituted from purified N with a defined RNA, has an extended structure without helical symmetry. N-RNA species of ∼100-kDa apparent molecular weight and heterogeneous composition were obtained by exhaustive ribonuclease treatment of virus RNP, by recombinant expression of N, and by reconstitution from purified N and an RNA oligomer. RNA-free N, obtained by denaturation and refolding, has a novel all-helical fold that is compact and well ordered at both the N and C termini. Unlike N of other negative-sense RNA viruses, RVFV N has no positively charged surface cleft for RNA binding and no protruding termini or loops to stabilize a defined N-RNA oligomer or RNP helix. A potential protein interaction site was identified in a conserved hydrophobic pocket. The nonhelical appearance of phlebovirus RNP, the heterogeneous ∼100-kDa N-RNA multimer, and the N fold differ substantially from the RNP and N of other negative-sense RNA virus families and provide valuable insights into the structure of the encapsidated phlebovirus genome.