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[en] Glycoproteins gB and gH/gL are required for entry of Epstein-Barr virus (EBV) into cells, but the role of each glycoprotein and how they function together to mediate fusion is unclear. Analysis of the functional homology of gB from the closely related primate gammaherpesvirus, rhesus lymphocryptovirus (Rh-LCV), showed that EBV gB could not complement Rh gB due to a species-specific dependence between gB and gL. To map domains of gB required for this interaction, we constructed a panel of EBV/Rh gB chimeric proteins. Analysis showed that insertion of Rh gB from residues 456 to 807 restored fusion function of EBV gB with Rh gH/gL, suggesting this region of gB is important for interaction with gH/gL. Split YFP bimolecular complementation (BiFC) provided evidence of an interaction between EBV gB and gH/gL. Together, our results suggest the importance of a gB-gH/gL interaction in EBV-mediated fusion with B cells requiring the region of EBV gB from 456 to 807.
[en] In our previous studies, we have stated to build a new strategy for developing defective, pseudoinfectious flaviviruses (PIVs) and applying them as a new type of vaccine candidates. PIVs combined the efficiency of live vaccines with the safety of inactivated or subunit vaccines. The results of the present work demonstrate further development of chimeric PIVs encoding dengue virus 2 (DEN2V) glycoproteins and yellow fever virus (YFV)-derived replicative machinery as potential vaccine candidates. The newly designed PIVs have synergistically functioning mutations in the prM and NS2A proteins, which abolish processing of the latter proteins and make the defective viruses capable of producing either only noninfectious, immature and/or subviral DEN2V particles. The PIV genomes can be packaged to high titers into infectious virions in vitro using the NS1-deficient YFV helper RNAs, and both PIVs and helpers can then be passaged as two-component genome viruses at an escalating scale.
[en] Erns is a pestivirus envelope glycoprotein indispensable for virus attachment and infection of target cells. Unlike the other two envelope proteins E1 and E2, Erns lacks a transmembrane domain and a vast quantity is secreted into the medium of infected cells. The protein is also present in fractions of pure pestivirus virions, raising the important and intriguing question regarding the mechanism of its attachment to the pestivirus envelope. In this study a direct interaction between Erns and E2 glycoproteins was demonstrated in both pestivirus-infected cells and mature virions. By co- and sequential immunoprecipitation we showed that an Erns-E2 heterodimer is assembled very early after translation of the viral polyprotein and before its processing is completed. Our results suggest that Erns is attached to the pestivirus envelope via a direct interaction with E2 and explain the role of Erns in the initial virus-target cell interaction
[en] We investigated the targeting of the γ-actin isoform in skeletal myofibers. For this purpose we used expression vectors to produce green fluorescent protein (GFP-) as well as myc-tagged γ-actin in rat flexor digitorum brevis myofibers. We found that the γ-actin fusion proteins accumulated into Z discs but not beneath the sarcolemma. Instead, the GFP-tagged skeletal muscle-specific α-actin isoform was preferentially incorporated into the pointed ends of thin contractile filaments. The localization pattern of the γ-actin fusion proteins was completely different from that of the dystrophin glycoprotein complex on the sarcolemma. The results emphasize the role of γ-actin as a Z disc component but fail to reveal an actin-based sub-sarcolemmal cytoskeleton in skeletal muscle cells
[en] Venezuelan equine encephalitis virus (VEEV) is a prototypical enveloped ssRNA virus of the family Togaviridae. To better understand alphavirus assembly, we analyzed newly formed nucleocapsid particles (termed pre-viral nucleocapsids) isolated from infected cells. These particles were intermediates along the virus assembly pathway, and ultimately bind membrane-associated viral glycoproteins to bud as mature infectious virus. Purified pre-viral nucleocapsids were spherical with a unimodal diameter distribution. The structure of one class of pre-viral nucleocapsids was determined with single particle reconstruction of cryo-electron microscopy images. These studies showed that pre-viral nucleocapsids assembled into an icosahedral structure with a capsid stoichiometry similar to the mature nucleocapsid. However, the individual capsomers were organized significantly differently within the pre-viral and mature nucleocapsids. The pre-viral nucleocapsid structure implies that nucleocapsids are highly plastic and undergo glycoprotein and/or lipid-driven rearrangements during virus self-assembly. This mechanism of self-assembly may be general for other enveloped viruses.
[en] Both mutant p53 and chemoresistance are poor prognostic factors in cancer. Many studies have examined the influence of these factors on fluoro-2-deoxy-D-glucose (FDG) incorporation. Whilst mutant p53 is associated with increased FDG incorporation, chemoresistance, especially when associated with P-glycoprotein, is associated with decreased FDG incorporation.
[en] The structural analysis of glycoproteins is a challenging endeavor and is under steadily increasing demand, but only a very limited number of labs have the expertise required to accomplish this task. This tutorial is aimed at researchers from the fields of molecular biology and biochemistry that have discovered that glycoproteins are important in their biological research and are looking for the tools to elucidate their structure. It provides brief descriptions of the major and most common analytical techniques used in glycomics and glycoproteomics analysis, including explanations of the rationales for individual steps and references to published literature containing the experimental details necessary to carry out the analyses. Glycomics includes the comprehensive study of the structure and function of the glycans expressed in a given cell or organism along with identification of all the genes that encode glycoproteins and glycosyltransferases. Glycoproteomics which is subset of both glycomics and proteomics is the identification and characterization of proteins bearing carbohydrates as posttranslational modification. This tutorial is designed to ease entry into the glycomics and glycoproteomics field for those without prior carbohydrate analysis experience.
[en] Hemorrhagic fevers caused by arenaviruses are among the most devastating emerging human diseases. Considering the number of individuals affected, the current lack of a licensed vaccine, and the limited therapeutic options, arenaviruses are arguably among the most neglected tropical pathogens and the development of efficacious anti-arenaviral drugs is of high priority. Over the past years significant efforts have been undertaken to identify novel potent inhibitors of arenavirus infection. High throughput screening of small molecule libraries employing pseudotype platforms led to the discovery of several potent and broadly active inhibitors of arenavirus cell entry that are effective against the major hemorrhagic arenaviruses. Mechanistic studies revealed that these novel entry inhibitors block arenavirus membrane fusion and provided novel insights into the unusual mechanism of this process. The success of these approaches highlights the power of small molecule screens in antiviral drug discovery and establishes arenavirus membrane fusion as a robust drug target. These broad screenings have been complemented by strategies targeting cellular factors involved in productive arenavirus infection. Approaches targeting the cellular protease implicated in maturation of the fusion-active viral envelope glycoprotein identified the proteolytic processing of the arenavirus glycoprotein precursor as a novel and promising target for anti-arenaviral strategies.
[en] The membrane-spanning domain (MSD) of human immunodeficiency virus type I (HIV-1) envelope glycoprotein (Env) is critical for its biological activity. Initial studies have defined an almost invariant 'core' structure in the MSD and demonstrated that it is crucial for anchoring Env in the membrane and virus entry. We show here that amino acid substitutions in the MSD 'core' do not influence specific virus-cell attachment, nor CD4 receptor and CXCR4 coreceptor recognition by Env. However, substitutions within the MSD 'core' delayed the kinetics and reduced the efficiency of cell-cell fusion mediated by Env. Although we observed no evidence that membrane fusion mediated by the MSD core mutants was arrested at a hemifusion stage, impaired Env fusogenicity was correlated with minor conformational changes in the V2, C1, and C5 regions in gp120 and the immunodominant loop in gp41. These changes could delay initiation of the conformational changes required in the fusion process.