Identification of two amino acid residues on Ebola virus glycoprotein 1 critical for cell entry.
ABSTRACT Using site-directed mutagenesis and retroviral vector pseudotyping of the wild type or mutated glycoprotein of Zaire ebolavirus (ZEBOV), we analyzed 15 conserved residues in the N-terminus of the filovirus glycoprotein 1 (GP1) in order to identify residues critical for cell entry. Results from infectivity assays and Western blot analyses identified two phenylalanine residues at positions 88 and 159 that appear to be critical for ZEBOV entry in vitro. We extended this observation by introduction of alanines at either position 88 or 159 of Ivory Coast Ebolavirus (CIEBOV) and observed the same phenotype. Further, we showed that introduction of each of the two mutations in a recombinant full-length clone of ZEBOV (Mayinga strain) that also carried the coding sequence for GFP could not be rescued, suggesting the mutants rendered the virus non-infectious. The two phenylalanines that are critical for both ZEBOV and CIEBOV entry are found in two linear domains of GP1 that are highly conserved among filoviruses, and thus could provide a target for rational development of broadly cross-protective vaccines or antiviral therapies.
Article: The primed ebolavirus glycoprotein (19-kilodalton GP1,2): sequence and residues critical for host cell binding.[show abstract] [hide abstract]
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.Journal of Virology 02/2009; 83(7):2883-91. · 5.40 Impact Factor
Article: Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus.[show abstract] [hide abstract]
ABSTRACT: Interferon-inducible transmembrane proteins 1, 2, and 3 (IFITM1, 2, and 3) are recently identified viral restriction factors that inhibit infection mediated by the influenza A virus (IAV) hemagglutinin (HA) protein. Here we show that IFITM proteins restricted infection mediated by the entry glycoproteins (GP(1,2)) of Marburg and Ebola filoviruses (MARV, EBOV). Consistent with these observations, interferon-β specifically restricted filovirus and IAV entry processes. IFITM proteins also inhibited replication of infectious MARV and EBOV. We observed distinct patterns of IFITM-mediated restriction: compared with IAV, the entry processes of MARV and EBOV were less restricted by IFITM3, but more restricted by IFITM1. Moreover, murine Ifitm5 and 6 did not restrict IAV, but efficiently inhibited filovirus entry. We further demonstrate that replication of infectious SARS coronavirus (SARS-CoV) and entry mediated by the SARS-CoV spike (S) protein are restricted by IFITM proteins. The profile of IFITM-mediated restriction of SARS-CoV was more similar to that of filoviruses than to IAV. Trypsin treatment of receptor-associated SARS-CoV pseudovirions, which bypasses their dependence on lysosomal cathepsin L, also bypassed IFITM-mediated restriction. However, IFITM proteins did not reduce cellular cathepsin activity or limit access of virions to acidic intracellular compartments. Our data indicate that IFITM-mediated restriction is localized to a late stage in the endocytic pathway. They further show that IFITM proteins differentially restrict the entry of a broad range of enveloped viruses, and modulate cellular tropism independently of viral receptor expression.PLoS Pathogens 01/2011; 7(1):e1001258. · 9.13 Impact Factor