The Tetherin Antagonism of the Ebola Virus Glycoprotein Requires an Intact Receptor-Binding Domain and Can Be Blocked by GP1-Specific Antibodies

Abstract

The glycoprotein of Ebola virus (EBOV GP), a member of the family Filoviridae, facilitates viral entry into target cells. In addition, EBOV GP antagonizes the antiviral activity of the host cell protein tetherin, which may otherwise restrict EBOV release from infected cells. However, it is unclear how EBOV GP antagonizes tetherin, and it is unknown whether the GP of Lloviu virus (LLOV), a filovirus found in dead bats in Northern Spain, also counteracts tetherin. Here, we show that LLOV GP antagonizes tetherin, indicating that tetherin may not impede LLOV spread in human cells. Moreover, we demonstrate that appropriate processing of N-glycans in tetherin/GP-coexpressing cells is required for tetherin counteraction by EBOV GP. Furthermore, we show that an intact receptor-binding domain (RBD) in the GP1 subunit of EBOV GP is a prerequisite for tetherin counteraction. In contrast, blockade of Niemann-Pick disease type C1 (NPC1), a cellular binding partner of the RBD, did not interfere with tetherin antagonism. Finally, we provide evidence that an antibody directed against GP1, which protects mice from a lethal EBOV challenge, may block GP-dependent tetherin antagonism. Our data, in conjunction with previous reports, indicate that tetherin antagonism is conserved among the GPs of all known filoviruses and demonstrate that the GP1 subunit of EBOV GP plays a central role in tetherin antagonism. IMPORTANCEFiloviruses are reemerging pathogens that constitute a public health threat. Understanding how Ebola virus (EBOV), a highly pathogenic filovirus responsible for the 2013-2016 Ebola virus disease epidemic in western Africa, counteracts antiviral effectors of the innate immune system might help to define novel targets for antiviral intervention. Similarly, determining whether Lloviu virus (LLOV), a filovirus detected in bats in northern Spain, is inhibited by innate antiviral effectors in human cells might help to determine whether the virus constitutes a threat to humans. The present study shows that LLOV, like EBOV, counteracts the antiviral effector protein tetherin via its glycoprotein (GP), suggesting that tetherin does not pose a defense against LLOV spread in humans. Moreover, our work identifies the GP1 subunit of EBOV GP, in particular an intact receptor-binding domain, as critical for tetherin counteraction and provides evidence that antibodies directed against GP1 can interfere with tetherin counteraction. Infection with Ebola virus (EBOV) (formerly Zaire ebolavirus), a member of the genus Ebolavirus within the family Filoviridae, causes severe and frequently fatal disease. The Ebola virus disease (EVD) epidemic in Western Africa in 2013 to 2016 was associated with 11,316 deaths and entailed secondary cases in the United States and Spain (1, 2), indicating that EVD constitutes a global public health threat. The interferon (IFN) system, an important component of innate immunity, is a first-line defense against infection by EBOV and other viruses (3, 4). Sensors of the IFN system detect viral invaders an...

Release of Immunomodulatory Ebola Virus Glycoprotein-Containing Microvesicles Is Suppressed by Tetherin in a Species-Specific Manner

“…We identified single amino acid residues in the RBD that are important for GP-virosome formation ( Figures 5A and 5B). Notably, the same residues have been previously demonstrated to be involved in GP-mediated entry and tetherin antagonism but were dispensable for GP virion incorporation and augmentation of VP40-driven VLP release (Brinkmann et al, 2016). A thus far unknown RBD-binding partner is supposed to be involved in GP-driven entry (Dube et al, 2008(Dube et al, , 2010 and tetherin counteraction (Brinkmann et al, 2016), and according to our results the same factor might also be involved in regulating GP-virosome release.…”

“…Fruit bat tetherins, Epo and Hyp tetherin, where cloned from fruit bat cell lines EpoNi/22.1 and HypNi/1.1 (Hoffmann et al, 2013), respectively, and sequences have been submitted to GenBank under accession numbers MG792836 and MG792837. The plasmids coding for the GP RBD mutants (F88A, L111A, L122A, W104A), GP LXXXL, LASV-GPC, ELE and LEL GP chimera, wild-type GP as well as the GP A82V mutant derived from the 2014 Ebola outbreak in West Africa are described elsewhere (Brinkmann et al, 2016;Gnirß et al, 2014;Hacke et al, 2015;Hoffmann et al, 2017). The GP-coding sequences of those mutants were amplified by PCR (forward 5 0 -CGTCTAGAATATGGGTGTTACAGGAATATTGC-3 0 and reverse 5 0 -TACGCGTTTCTAAAA GACAAATTTGCATATACAG-3 0 ) and subsequently cloned into the pCG-IRES-GFP vector using the restriction enzymes MluI and XbaI.…”

“…The RBD and TMD of EBOV-GP Are Determinants of GP-Virosome Release To elucidate which GP residues or domains are required for the release of GP-virosomes, we analyzed a set of previously characterized GP mutants containing single amino acid substitutions in the RBD (F88A, L111A, L122A, and W104A) (Brinkmann et al, 2016;Manicassamy et al, 2005;Martinez et al, 2013) or mutation of a conserved GXXXA motif in the TMD (LXXXL) (González-Herná ndez et al, 2018; Hacke et al, 2015). 293T cells were transfected to express the various GP variants and GP-virosome release in cell culture supernatants was analyzed using western (A) 293T cells were transfected to express EBOV-GP or an empty control plasmid (mock) and increasing amounts of tetherin.…”

Tetherin Inhibits Nipah Virus but Not Ebola Virus Replication in Fruit Bat Cells

“…Plasmids, mutagenesis, and transfection. Expression plasmids for HIV-1 p55-Gag (Gag), HIV-1 Vpu (Vpu), EBOV-GP, VSV glycoprotein (VSV-G), Nipah virus fusion (F), and attachment glycoprotein (G), EBOV-VP40 harboring an N-terminal cMYC tag, and DC-SIGN have been described elsewhere (35,44,(48)(49)(50). To generate expression plasmids for human, gorilla, African green monkey, pig, rat, mouse, and artificial tetherin, the respective ORFs were amplified from existing plasmids (32,51,52) and inserted into the pCAGGS expression vector.…”

Cooperation of the Ebola Virus Proteins VP40 and GP 1,2 with BST2 To Activate NF-κB Independently of Virus-Like Particle Trapping

“…As mentioned above, HIV-1 Vpu is the prototype antagonist of BST2 (2,3); Vpu mistraffics BST2 away from the plasma membrane, removes it from virion assembly sites, and targets it for degradation (13,(16)(17)(18)(19)(20)(21). On the other hand, the Ebola virus envelope glycoprotein (GP 1,2 ) antagonizes BST2 to enhance virion release through an unclear mechanism that requires neither the removal of BST2 from the cell surface nor its degradation but might instead involve dissociation of BST2 from the Ebola virus matrix protein, VP40; this activity requires proper N-linked glycosylation of the GP1 subunit and the interaction of BST2 with the GP2 subunit (12,(22)(23)(24)(25)(26)(27)(28).…”