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Arenaviruses and hantaviruses cause severe human disease. Little is known regarding host proteins required for their propagation. We identified human proteins that interact with the glycoproteins (GPs) of a prototypic arenavirus and hantavirus and show that the lectin endoplasmic reticulum (ER)-Golgi intermediate compartment 53 kDa protein (ERGIC-53), a cargo receptor required for glycoprotein trafficking within the early exocytic pathway, associates with arenavirus, hantavirus, coronavirus, orthomyxovirus, and filovirus GPs. ERGIC-53 binds to arenavirus GPs through a lectin-independent mechanism, traffics to arenavirus budding sites, and is incorporated into virions. ERGIC-53 is required for arenavirus, coronavirus, and filovirus propagation; in its absence, GP-containing virus particles form but are noninfectious, due in part to their inability to attach to host cells. Thus, we have identified a class of pathogen-derived ERGIC-53 ligands, a lectin-independent basis for their association with ERGIC-53, and a role for ERGIC-53 in the propagation of several highly pathogenic RNA virus families., (Copyright © 2013 Elsevier Inc. All rights reserved.) more...

With an estimated 40% of the world population at risk, dengue poses a significant threat to human health, especially in tropical and subtropical regions. Preventative and curative efforts, such as vaccine development and drug discovery, face additional challenges due to the occurrence of four antigenically distinct serotypes of the causative dengue virus (DEN1 to -4). Complex immune responses resulting from repeat assaults by the different serotypes necessitate simultaneous targeting of all forms of the virus. One of the promising targets for drug development is the highly conserved two-component viral protease NS2B-NS3, which plays an essential role in viral replication by processing the viral precursor polyprotein into functional proteins. In this paper, we report the 2.1-A crystal structure of the DEN1 NS2B hydrophilic core (residues 49 to 95) in complex with the NS3 protease domain (residues 1 to 186) carrying an internal deletion in the N terminus (residues 11 to 20). While the overall folds within the protease core are similar to those of DEN2 and DEN4 proteases, the conformation of the cofactor NS2B is dramatically different from those of other flaviviral apoprotease structures. The differences are especially apparent within its C-terminal region, implicated in substrate binding. The structure reveals for the first time serotype-specific structural elements in the dengue virus family, with the reported alternate conformation resulting from a unique metal-binding site within the DEN1 sequence. We also report the identification of a 10-residue stretch within NS3pro that separates the substrate-binding function from the catalytic turnover rate of the enzyme. Implications for broad-spectrum drug discovery are discussed. more...

Hepatitis delta virus (HDV) expresses two essential proteins with distinct functions. The small hepatitis delta antigen (HDAg-S) is expressed throughout replication and is needed to promote that process. The large form (HDAg-L) is farnesylated, is expressed only at later times via RNA editing of the amber/W site, and is required for virion assembly. When HDAg-L is artificially expressed at the onset of replication, it strongly inhibits replication. However, there is controversy concerning whether HDAg-L expressed naturally at later times as a consequence of editing and replication can similarly inhibit replication. Here, by stabilizing the predicted secondary structure downstream from the amber/W site, a replication-competent HDV mutant that exhibited levels of editing higher than those of the wild type was created. This mutant expressed elevated levels of HDAg-L early during replication, and at later times, its replication aborted prematurely. No further increase in amber/W editing was observed following the cessation of replication, indicating that editing was coupled to replication. A mutation in HDAg-L and a farnesyl transferase inhibitor were both used to abolish the ability of HDAg-L to inhibit replication. Such treatments rescued the replication defect of the overediting mutant, and even higher levels of amber/W editing resulted. It was concluded that when expressed naturally during replication, HDAg-L is able to inhibit replication and thereby inhibit amber/W editing and its own synthesis. In addition, the structure adjacent to the amber/W site is suboptimal for editing, and this creates a window of time in which replication can occur in the absence of HDAg-L. more...

The density of carbohydrate epitopes on the surface of tumor cells is a governing factor for immune recognition and antibody-mediated targeting of tumor-associated carbohydrate antigens in cancer immunotherapy. A sensitive cell-suspension ELISA (cs-ELISA) is developed for quantitation of the functionally exposed carbohydrate epitopes on the cell surface. The factors affecting the measurement of tumor-cell surface glycoconjugates are evaluated using three human melanoma cell lines before and after exposure to various cell preservation treatments. The results of cs-ELISA are compared with the quantitative profile obtained by biochemical and flow cytometry assays. Cs-ELISA measures the density of the functionally exposed specific sugar epitopes on the surface of tumor cells, even in the presence of other similar carbohydrate antigens, provided that the monoclonal antibodies to carbohydrate epitopes are monospecific and sensitive, and that the cells are viable and present in optimal density. Of the three melanoma cell lines, M10-v and M101 expressed disialolactosyl residues of GD3 at concentrations of 5-6 pmol/10(6) cells and 2-3 pmol/10(6) cells, respectively. In both cell lines, the cell-surface GD2 was less than 1.0 pmol/10(6) cells. M24 melanoma cells expressed trace quantities (< 0.1 pmol/10(6) cells) of GD3 and GD2. Trypsinization of M10-v and M101 cells significantly reduced the cell-surface expression of GD3, suggesting GD3 loss, but increased the expression of GD2, suggesting crypticity of membrane-bound GD2. Cs-ELISA results showed that cryopreservation with 10% DMSO and irradiation at 15 krad decreased melanoma cell viability and ganglioside expression for M10-v but not M101 and M24. Formalinization did not affect cs-ELISA measurement of cell-surface carbohydrates. Cs-ELISA was used to monitor the quantity of incorporation of exogenous GD3 onto the surface of GD3-deficient M24 cells. Cs-ELISA for assessment of density of cell surface carbohydrate epitopes may be useful to characterize different types of tumors, to develop carbohydrate-based whole cell vaccines from tumor biopsies, to monitor the effects of cell preservation treatments commonly used in a whole cell vaccine preparation, and to evaluate the incorporation of a particular glycolipid (antigen or adjuvant) into glycolipid-deficient cells that are useful for carbohydrate-based active specific immunotherapy. more...