Dataset Information

The primed ebolavirus glycoprotein (19-kilodalton GP1,2): sequence and residues critical for host cell binding.

ABSTRACT: Entry of ebolavirus (EBOV) into cells is mediated by its glycoprotein (GP(1,2)), a class I fusion protein whose structure was recently determined (J. E. Lee et al., Nature 454:177-182, 2008). Here we confirmed two major predictions of the structural analysis, namely, the residues in GP(1) and GP(2) that remain after GP(1,2) is proteolytically primed by endosomal cathepsins for fusion and residues in GP(1) that are critical for binding to host cells. Mass spectroscopic analysis indicated that primed GP(1,2) contains residues 33 to 190 of GP(1) and all residues of GP(2). The location of the receptor binding site was determined by a two-pronged approach. We identified a small receptor binding region (RBR), residues 90 to 149 of GP(1), by comparing the cell binding abilities of four RBR proteins produced in high yield. We characterized the binding properties of the optimal RBR (containing GP(1) residues 57 to 149) and then conducted a mutational analysis to identify critical binding residues. Substitutions at four lysines (K95, K114, K115, and K140) decreased binding and the ability of RBR proteins to inhibit GP(1,2)-mediated infection. K114, K115, and K140 lie in a small region modeled to be located on the top surface of the chalice following proteolytic priming; K95 lies deeper in the chalice bowl. Combined with those of Lee et al., our findings provide structural insight into how GP(1,2) is primed for fusion and define the core of the EBOV RBR (residues 90 to 149 of GP(1)) as a highly conserved region containing a two-stranded beta-sheet, the two intra-GP(1) disulfide bonds, and four critical Lys residues.

SUBMITTER: Dube D

PROVIDER: S-EPMC2655554 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The primed ebolavirus glycoprotein (19-kilodalton GP1,2): sequence and residues critical for host cell binding.

Dube Derek D Brecher Matthew B MB Delos Sue E SE Rose Sean C SC Park Edward W EW Schornberg Kathryn L KL Kuhn Jens H JH White Judith M JM

Journal of virology 20090114 7

Entry of ebolavirus (EBOV) into cells is mediated by its glycoprotein (GP(1,2)), a class I fusion protein whose structure was recently determined (J. E. Lee et al., Nature 454:177-182, 2008). Here we confirmed two major predictions of the structural analysis, namely, the residues in GP(1) and GP(2) that remain after GP(1,2) is proteolytically primed by endosomal cathepsins for fusion and residues in GP(1) that are critical for binding to host cells. Mass spectroscopic analysis indicated that pri ...[more]

PMID: 19144707

Similar Datasets

Project description:We have produced a new Ebola virus pseudotype, E-S-FLU, that can be handled in biosafety level 1/2 containment for laboratory analysis. The E-S-FLU virus is a single-cycle influenza virus coated with Ebolavirus glycoprotein, and it encodes enhanced green fluorescence protein as a reporter that replaces the influenza virus hemagglutinin. MDCK-SIAT1 cells were transduced to express Ebolavirus glycoprotein as a stable transmembrane protein for E-S-FLU virus production. Infection of cells with the E-S-FLU virus was dependent on the Niemann-Pick C1 protein, which is the well-characterized receptor for Ebola virus entry at the late endosome/lysosome membrane. The E-S-FLU virus was neutralized specifically by an anti-Ebolavirus glycoprotein antibody and a variety of small drug molecules that are known to inhibit the entry of wild-type Ebola virus. To demonstrate the application of this new Ebola virus pseudotype, we show that a single laboratory batch was sufficient to screen a library (LOPAC1280; Sigma) of 1,280 pharmacologically active compounds for inhibition of virus entry. A total of 215 compounds inhibited E-S-FLU virus infection, while only 22 inhibited the control H5-S-FLU virus coated in H5 hemagglutinin. These inhibitory compounds have very dispersed targets and mechanisms of action, e.g., calcium channel blockers, estrogen receptor antagonists, antihistamines, serotonin uptake inhibitors, etc., and this correlates with inhibitor screening results obtained with other pseudotypes or wild-type Ebola virus in the literature. The E-S-FLU virus is a new tool for Ebola virus cell entry studies and is easily applied to high-throughput screening assays for small-molecule inhibitors or antibodies.IMPORTANCE Ebola virus is in the Filoviridae family and is a biosafety level 4 pathogen. There are no FDA-approved therapeutics for Ebola virus. These characteristics warrant the development of surrogates for Ebola virus that can be handled in more convenient laboratory containment to study the biology of the virus and screen for inhibitors. Here we characterized a new surrogate, named E-S-FLU virus, that is based on a disabled influenza virus core coated with the Ebola virus surface protein but does not contain any genetic information from the Ebola virus itself. We show that E-S-FLU virus uses the same cell entry pathway as wild-type Ebola virus. As an example of the ease of use of E-S-FLU virus in biosafety level 1/2 containment, we showed that a single production batch could provide enough surrogate virus to screen a standard small-molecule library of 1,280 candidates for inhibitors of viral entry.

Dataset Information

The primed ebolavirus glycoprotein (19-kilodalton GP1,2): sequence and residues critical for host cell binding.

Publications

The primed ebolavirus glycoprotein (19-kilodalton GP1,2): sequence and residues critical for host cell binding.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets