How many for dinner? Recruitment and monitoring by glances in capuchins

Group members present physical and physiological differences according to their age, sex or social status, which could generate motivation differences among individuals during travel. In spite of these divergences of interest among individuals, the group succeeds more often than not in making a collective decision about departure time and which direction to take. To reach a consensus decision, animals should exchange information relating to characteristics of group movement through different communication channels. The main purpose of this study is to understand the function of behaviour patterns displayed during movements of white-faced capuchins (Cebus capucinus). We designed experiments in which we provoked collective movements involving a binary choice. During experiments, a video camera recorded the behaviour of each capuchin, which enabled us to determine which individuals displayed a behavioural pattern during travel and how this behaviour influenced the other group members. We found that looking backwards seemed to permit the recruitment of group mates during collective movement. This behaviour also seemed to allow the quantification of the number of followers, since the emitter modified its locomotion speed according to this number. In this preliminary study, we showed that visual behaviour was used to recruit and monitor group mates during collective movements and provided information on mechanisms involved in maintaining cohesion and coordination among group members during travel.     

Linking structural change with functional regulation-insights from mass spectrometry

A wide range of biophysical approaches has been applied to structural biology, all with the same overall goal-to understand the molecular machines that allow cells to function. While knowledge of the identity and composition of component protein subunits is an important foundation for understanding these macromolecular complexes it has become increasingly clear that knowledge of the exact composition alone is insufficient for understanding dynamic interactions and regulatory mechanisms. In this review we focus on recent developments of mass spectrometry (MS) that allow us to unravel the functional 'secrets' of non-covalent molecular machines.     

Host factors and their relevance to virus infection in plants

Virus infection is established when viral proteins can interact with host factors to execute replication and/or cell-to-cell movement. Even after the virus infection has started, host resistance reactions, if trigged, would suppress further virus propagation. We would like to introduce what we understand about host factors as determinants of infection establishment and as key resistance molecules. Genome-wide information of Arabidopsis is providing us much information about such host factors involved in virus infection.     

Virus trafficking - learning from single-virus tracking

What could be a better way to study virus trafficking than 'miniaturizing oneself' and 'taking a ride with the virus particle' on its journey into the cell? Single-virus tracking in living cells potentially provides us with the means to visualize the virus journey. This approach allows us to follow the fate of individual virus particles and monitor dynamic interactions between viruses and cellular structures, revealing previously unobservable infection steps. The entry, trafficking and egress mechanisms of various animal viruses have been elucidated using this method. The combination of single-virus trafficking with systems approaches and state-of-the-art imaging technologies should prove exciting in the future.     

Nuclear targeting of SV40 and adenovirus

Import o f viral DNA into the nucleus is essential for the successful replication o f DNA tumour viruses. To achieve this goal, viruses have adapted strategies to traverse the barriers between the plasma membrane and the nucleus o f a host cell. Two DNA tumour viruses, simian virus 40 and adenovirus, achieve the nuclear-entry step in slightly different ways. SV40 DNA enters the nucleus through the nuclear pore complexes (NPCs) in apparently intact virions. By contrast, adenovirus particles dissociate near the NPC before the viral DNA is imported into the nucleus. In both cases, karyophilic protein components o f the viruses appear to mediate nuclear entry o f the viral genomes. In this article, we discuss how an understanding o f the cell biology o f virus entry can help us understand the process o f nuclear transport.     

Viruses as probes for systems analysis of cellular signalling, cytoskeleton reorganization and endocytosis

It is well known that mammalian viruses hijack the cellular signalling and internalization machineries to enter and to infect their host cells; however, only in the past six years have researchers started to follow individual virus particles and to investigate the events that they induce in living cells. The relative ease of imaging individual virus particles with time-lapse microscopy, despite being limited by light-diffraction, allows for specific and local kinetic analysis of individual events in signalling, cytoskeleton reorganization and endocytosis. Furthermore, virus infection is an easy-to-use endpoint readout, which is ideally suited for functional genomics approaches. The combined information from these studies will be crucial for the development of models that describe the underlying systems of cellular signalling, cytoskeleton reorganization and membrane trafficking during virus entry.     

Cyanovirin-N inhibits hepatitis C virus entry by binding to envelope protein glycans

Inhibition of viruses at the stage of viral entry provides a route for therapeutic intervention. Because of difficulties in propagating hepatitis C virus (HCV) in cell culture, entry inhibitors have not yet been reported for this virus. However, with the development of retroviral particles pseudotyped with HCV envelope glycoproteins (HCVpp) and the recent progress in amplification of HCV in cell culture (HCVcc), studying HCV entry is now possible. In addition, these systems are essential for the identification and the characterization of molecules that block HCV entry. The lectin cyanovirin-N (CV-N) has initially been discovered based on its potent activity against human immunodeficiency virus. Because HCV envelope glycoproteins are highly glycosylated, we sought to determine whether CV-N has an antiviral activity against this virus. CV-N inhibited the infectivity of HCVcc and HCVpp at low nanomolar concentrations. This inhibition is attributed to the interaction of CV-N with HCV envelope glycoproteins. In addition, we showed that the carbohydrate binding property of CV-N is involved in the anti-HCV activity. Finally, CV-N bound to HCV envelope glycoproteins and blocked the interaction between the envelope protein E2 and CD81, a cell surface molecule involved in HCV entry. These data demonstrate that targeting the glycans of HCV envelope proteins is a promising approach in the development of antiviral therapies to combat a virus that is a major cause of chronic liver diseases. Furthermore, CV-N is a new invaluable tool to further dissect the early steps of HCV entry into host cells.     

Proteomic Analysis of Mamestra Brassicae Nucleopolyhedrovirus Progeny Virions from Two Different Hosts

Mamestra brassicae nucleopolyhedrovirus (MabrNPV) has a wide host range replication in more than one insect species. In this study, a sequenced MabrNPV strain, MabrNPV-CTa, was used to perform proteomic analysis of both BVs and ODVs derived from two infected hosts: Helicoverpa armigera and Spodoptera exigua. A total of 82 and 39 viral proteins were identified in ODVs and BVs, respectively. And totally, 23 and 76 host proteins were identified as virion-associated with ODVs and BVs, respectively. The host proteins incorporated into the virus particles were mainly involved in cytoskeleton, signaling, vesicle trafficking, chaperone and metabolic systems. Some host proteins, such as actin, cyclophilin A and heat shock protein 70 would be important for viral replication. Several host proteins involved in immune response were also identified in BV, and a C-type lectin protein was firstly found to be associated with BV and its family members have been demonstrated to be involved in entry process of other viruses. This study facilitated the annotation of baculovirus genome, and would help us to understand baculovirus virion structure. Furthermore, the identification of host proteins associated with virions produced in vivo would facilitate investigations on the involvement of intriguing host proteins in virus replication.     

Proteomic analysis of plasma membranes isolated from undifferentiated and differentiated HepaRG cells

ABSTRACT: Liver infection with hepatitis B virus (HBV), a DNA virus of the Hepadnaviridae family, leads to severe disease, such as fibrosis, cirrhosis and hepatocellular carcinoma. The early steps of the viral life cycle are largely obscure and the host cell plasma membrane receptors are not known. HepaRG is the only proliferating cell line supporting HBV infection in vitro, following specific differentiation, allowing for investigation of new host host-cell factors involved in viral entry, within a more robust and reproducible environment. Viral infection generally begins with receptor recognition at the host cell surface, following highly specific cell-virus interactions. Most of these interactions are expected to take place at the plasma membrane of the HepaRG cells. In the present study, we used this cell line to explore changes between the plasma membrane of undifferentiated (-) and differentiated (+) cells and to identify differentially-regulated proteins or signaling networks that might potentially be involved in HBV entry. Our initial study identified a series of proteins that are differentially expressed in the plasma membrane of (-) and (+) cells and are good candidates for potential cell-virus interactions. To our knowledge, this is the first study using functional proteomics to study plasma membrane proteins from HepaRG cells, providing a platform for future experiments that will allow us to understand the cell-virus interaction and mechanism of HBV viral infection.     

Structural characterization of CD81-Claudin-1 hepatitis C virus receptor complexes

Tetraspanins are thought to exert their biological function(s) by co-ordinating the lateral movement and trafficking of associated molecules into tetraspanin-enriched microdomains. A second four-TM (transmembrane) domain protein family, the Claudin superfamily, is the major structural component of cellular TJs (tight junctions). Although the Claudin family displays low sequence homology and appears to be evolutionarily distinct from the tetraspanins, CD81 and Claudin-1 are critical molecules defining HCV (hepatitis C virus) entry; we recently demonstrated that CD81-Claudin-1 complexes have an essential role in this process. To understand the molecular basis of CD81-Claudin-1 complex formation, we produced and purified milligram quantities of full-length CD81 and Claudin-1, alone and in complex, in both detergent and lipid contexts. Structural characterization of these purified proteins will allow us to define the mechanism(s) underlying virus-cell interactions and aid the design of therapeutic agents targeting early steps in the viral life cycle.     

Dynamic tradeoffs in the raft-mediated entry of human immunodeficiency virus type 1 into cells

To initiate an infection human immunodeficiency virus type 1 (HIV-1) particles must first bind to receptors on the surface of their host cells, a process that eventually leads to fusion of viral and cellular membranes and release of the viral genome into the cytoplasm. Understanding the molecular mechanisms of these processes may enable the development of new anti-HIV strategies. Disagreement currently prevails on the role in virus entry of microdomains within the cellular plasma membrane known as lipid rafts. Experiments have suggested that lipid rafts, in their interactions with cellular receptors and viral particles, either promote or have minimal effect on viral entry. Here we develop a dynamic model for HIV-1 entry that enables us to identify and quantitatively assess tradeoffs that can arise from the clustering of receptors in rafts. Specifically, receptor clustering can be detrimental to the initiation of viral infection by reducing the probability that a virus particle finds its primary receptor, CD4. However, receptor clustering can also enable a virus particle, once bound, to rapidly form multivalent interactions with receptors and co-receptors that are required for virus-cell membrane fusion. We show how the resolution of such tradeoffs hinges on the level and spatial distribution of receptors and co-receptors on the cell surface, and we discuss implications of these effects for the design of therapeutics that inhibit HIV-1 entry.     

Proteomic Characterization of Bovine Herpesvirus 4 Extracellular Virions

Gammaherpesviruses are important pathogens in human and animal populations. During early events of infection, these viruses manipulate preexisting host cell signaling pathways to allow successful infection. The different proteins that compose viral particles are therefore likely to have critical functions not only in viral structures and in entry into target cell but also in evasion of the host''s antiviral response. In this study, we analyzed the protein composition of bovine herpesvirus 4 (BoHV-4), a close relative of the human Kaposi''s sarcoma-associated herpesvirus. Using mass spectrometry-based approaches, we identified 37 viral proteins associated with extracellular virions, among which 24 were resistant to proteinase K treatment of intact virions. Analysis of proteins associated with purified capsid-tegument preparations allowed us to define protein localization. In parallel, in order to identify some previously undefined open reading frames, we mapped peptides detected in whole virion lysates onto the six frames of the BoHV-4 genome to generate a proteogenomic map of BoHV-4 virions. Furthermore, we detected important glycosylation of three envelope proteins: gB, gH, and gp180. Finally, we identified 38 host proteins associated with BoHV-4 virions; 15 of these proteins were resistant to proteinase K treatment of intact virions. Many of these have important functions in different cellular pathways involved in virus infection. This study extends our knowledge of gammaherpesvirus virions composition and provides new insights for understanding the life cycle of these viruses.     

Modelling microtubule patterns

The cellular cytoskeleton is well studied in terms of its biological and physical properties, making it an attractive subject for systems approaches. Here, we describe the experimental and theoretical strategies used to study the collective behaviour of microtubules and motors. We illustrate how this led to the beginning of an understanding of dynamic cellular patterns that have precise functions.     

杆状病毒与昆虫宿主相互作用的研究进展

杆状病毒与昆虫宿主相互作用是一种基本的分子和生态问题, 不仅在农业上, 而且在真核表达系统、 基因治疗、 蛋白表面展示 系统以及基因工程疫苗等方面都有重要的实际应用。杆状病毒还是一种很有潜力的病毒杀虫剂, 而且对环境来说是安全的。研究这些相互 作用也产生了许多重要和有价值的发现。杆状病毒生命循环中存在两种不同形式的病毒, 即包埋型病毒粒子(occlusion derived virus, ODV) 和出芽型病毒粒子(budded virus, BV)。ODV包裹于多角体中, 主要负责宿主的原发感染; 而BV由感染的宿主细胞释放后引发继发 感染。病毒侵染起始于敏感的昆虫宿主食用了污染包涵体病毒的植物。在宿主中肠的碱性环境中, 多角体溶解释放ODV, ODV与宿主肠道 柱状上皮细胞细胞膜融合, 通过内吞体进入细胞。之后核衣壳从内吞体中逃脱并被转运到细胞核。病毒转录和复制在细胞核进行, 新生 的BV粒子从基底膜出芽引起全身感染。杆状病毒与宿主细胞相互作用包括从病毒结合和进入时的相互作用, 到宿主基因表达调节, 以及 修饰与调节细胞和机体所发生的生理和防御的相互作用的复杂和微妙的机制。本文主要以杆状病毒侵染昆虫宿主的过程为线索, 总结和评 述了杆状病毒与昆虫宿主相互作用方面研究的最新进展, 特别是杆状病毒基因在病毒入侵过程中所起的作用。     

Molecular basis of bacterial outer membrane permeability revisited.

Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.     

Identification of a putative coreceptor on Vero cells that participates in dengue 4 virus infection

Dengue virus infects target cells by attaching to a cell surface receptor through the envelope (E) glycoprotein, located on the surface of the viral membrane. On Vero and BHK cells, heparan sulfate (HS) moieties of proteoglycans are the receptors for dengue virus; however, additional proteins have also been described as putative dengue virus receptors on C6/36, HL60, and BM cells. HS can also act as a receptor for other types of viruses or as an attachment molecule for viruses that require additional host cell molecules to allow viral penetration. In this study we searched for molecules other than HS that could participate in dengue virus infection of Vero cells. Labeled dengue 4 virus bound with high affinity to two molecules of 74 and 44 kDa. Binding of dengue virus to the 74-kDa molecule was susceptible to protease and sodium periodate treatment and resistant to heparinase treatments. Lectins such as concanavalin A and wheat germ agglutinin prevented dengue virus binding to both the 74- and the 44-kDa protein in overlay assays, while phytohemagglutinin P did not affect binding, suggesting that carbohydrate residues (alpha-mannose or N-acetylglucosamine) are important in virus binding to host cells. Protease susceptibility, biotin labeling, and immunofluorescence with a polyclonal antibody raised against the 74-kDa protein consistently identified the protein on the surfaces of Vero cells. Moreover, the antibody against the 74-kDa protein was able to inhibit dengue virus infection. These data suggest that HS might serve as a primary receptor, probably concentrating virus particles on the surfaces of Vero cells, and then other molecules, such as the 74-kDa protein, might participate as coreceptors in viral penetration. The 74-kDa protein possibly constitutes part of a putative receptor complex for dengue virus infection of Vero cells.     

Primate and feline lentivirus vector RNA packaging and propagation by heterologous lentivirus virions

Development of safe and effective gene transfer systems is critical to the success of gene therapy protocols for human diseases. Currently, several primate lentivirus-based gene transfer systems, such as those based on human and simian immunodeficiency viruses (HIV/SIV), are being tested; however, their use in humans raises safety concerns, such as the generation of replication-competent viruses through recombination with related endogenous retroviruses or retrovirus-like elements. Due to the greater phylogenetic distance from primate lentiviruses, feline immunodeficiency virus (FIV) is becoming the lentivirus of choice for human gene transfer systems. However, the safety of FIV-based vector systems has not been tested experimentally. Since lentiviruses such as HIV-1 and SIV have been shown to cross-package their RNA genomes, we tested the ability of FIV RNA to get cross-packaged into primate lentivirus particles such as HIV-1 and SIV, as well as a nonlentiviral retrovirus such as Mason-Pfizer monkey virus (MPMV), and vice versa. Our results reveal that FIV RNA can be cross-packaged by primate lentivirus particles such as HIV-1 and SIV and vice versa; however, a nonlentivirus particle such as MPMV is unable to package FIV RNA. Interestingly, FIV particles can package MPMV RNA but cannot propagate the vector RNA further for other steps of the retrovirus life cycle. These findings reveal that diverse retroviruses are functionally more similar than originally thought and suggest that upon coinfection of the same host, cross- or copackaging may allow distinct retroviruses to generate chimeric variants with unknown pathogenic potential.     

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.     

Identification of Echovirus 1 and coxsackievirus A9 receptor molecules via a novel flow cytometric quantification method

BACKGROUND: Virus-receptor binding is an essential step in every virus infectious process. Many viruses employ more than one receptor molecule or even receptor complexes for attachment. In this study, we investigate the binding of Echovirus 1 (Echo1) and Coxsackievirus A9 (CAV-9) on cell surface molecules. CAV-9 has been reported to utilize integrin alpha v beta(3) in binding to cells, whereas Echo1 has been known to utilize integrin alpha 2 beta(1). METHODS AND RESULTS We directly test whether the presence of these molecules alone was sufficient for virus binding. We devised a novel flow cytometric binding assay that enables us to quantify virus particles bound on host cells and to further determine the extent to which viruses utilize specific receptors. CONCLUSIONS: By quantifying virus particles and possible receptor molecules, we found that Echo1 utilizes mainly integrin alpha 2 beta(1). CAV-9 utilizes integrin alpha v beta(3) to a much lesser extent (40%), indicating that CAV-9 also utilizes other receptor(s).     

Structural Basis for Functional Tetramerization of Lentiviral Integrase

Experimental evidence suggests that a tetramer of integrase (IN) is the protagonist of the concerted strand transfer reaction, whereby both ends of retroviral DNA are inserted into a host cell chromosome. Herein we present two crystal structures containing the N-terminal and the catalytic core domains of maedi-visna virus IN in complex with the IN binding domain of the common lentiviral integration co-factor LEDGF. The structures reveal that the dimer-of-dimers architecture of the IN tetramer is stabilized by swapping N-terminal domains between the inner pair of monomers poised to execute catalytic function. Comparison of four independent IN tetramers in our crystal structures elucidate the basis for the closure of the highly flexible dimer-dimer interface, allowing us to model how a pair of active sites become situated for concerted integration. Using a range of complementary approaches, we demonstrate that the dimer-dimer interface is essential for HIV-1 IN tetramerization, concerted integration in vitro, and virus infectivity. Our structures moreover highlight adaptable changes at the interfaces of individual IN dimers that allow divergent lentiviruses to utilize a highly-conserved, common integration co-factor.