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

The Ebola virus glycoprotein (EBOV-GP) forms GP-containing microvesicles, so-called virosomes, which are secreted from GP-expressing cells. However, determinants of GP-virosome release and their functionality are poorly understood. We characterized GP-mediated virosome formation and delineated the role of the antiviral factor tetherin (BST2, CD317) in this process. Residues in the EBOV-GP receptor-binding domain (RBD) promote GP-virosome secretion, while tetherin suppresses GP-virosomes by interactions involving the GP-transmembrane domain. Tetherin from multiple species interfered with GP-virosome release, and tetherin from the natural fruit bat reservoir showed the highest inhibitory activity. Moreover, analyses of GP from various ebolavirus strains, including the EBOV responsible for the West African epidemic, revealed the most efficient GP-virosome formation by highly pathogenic ebolaviruses. Finally, EBOV-GP-virosomes were immunomodulatory and acted as decoys for EBOV-neutralizing antibodies. Our results indicate that GP-virosome formation might be a determinant of EBOV immune evasion and pathogenicity, which is suppressed by tetherin.

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

Ebola virus (EBOV) and Nipah virus (NiV) infection of humans can cause fatal disease and constitutes a public health threat. In contrast, EBOV and NiV infection of fruit bats, the putative (EBOV) or proven (NiV) natural reservoir, is not associated with disease, and it is currently unknown how these animals control the virus. The human interferon (IFN)-stimulated antiviral effector protein tetherin (CD317, BST-2) blocks release of EBOV- and NiV-like particles from cells and is counteracted by the EBOV glycoprotein (GP). In contrast, it is unknown whether fruit bat tetherin restricts virus infection and is susceptible to GP-driven antagonism. Here, we report the sequence of fruit bat tetherin and show that its expression is IFN stimulated and associated with strong antiviral activity. Moreover, we demonstrate that EBOV-GP antagonizes tetherin orthologues of diverse species but fails to efficiently counteract fruit bat tetherin in virus-like particle (VLP) release assays. However, unexpectedly, tetherin was dispensable for robust IFN-mediated inhibition of EBOV spread in fruit bat cells. Thus, the VLP-based model systems mimicking tetherin-mediated inhibition of EBOV release and its counteraction by GP seem not to adequately reflect all aspects of EBOV release from IFN-stimulated fruit bat cells, potentially due to differences in tetherin expression levels that could not be resolved by the present study. In contrast, tetherin expression was essential for IFN-dependent inhibition of NiV infection, demonstrating that IFN-induced fruit bat tetherin exerts antiviral activity and may critically contribute to control of NiV and potentially other highly virulent viruses in infected animals.IMPORTANCE Ebola virus and Nipah virus (EBOV and NiV) can cause fatal disease in humans. In contrast, infected fruit bats do not develop symptoms but can transmit the virus to humans. Why fruit bats but not humans control infection is largely unknown. Tetherin is an antiviral host cell protein and is counteracted by the EBOV glycoprotein in human cells. Here, employing model systems, we show that tetherin of fruit bats displays higher antiviral activity than human tetherin and is largely resistant against counteraction by the Ebola virus glycoprotein. Moreover, we demonstrate that induction of tetherin expression is critical for interferon-mediated inhibition of NiV but, for at present unknown reasons, not EBOV spread in fruit bat cells. Collectively, our findings identify tetherin as an antiviral effector of innate immune responses in fruit bats, which might allow these animals to control infection with NiV and potentially other viruses that cause severe disease in humans.

A GXXXA Motif in the Transmembrane Domain of the Ebola Virus Glycoprotein Is Required for Tetherin Antagonism.

The interferon-induced antiviral host cell protein tetherin can inhibit the release of several enveloped viruses from infected cells. The Ebola virus (EBOV) glycoprotein (GP) antagonizes tetherin, but the domains and amino acids in GP that are required for tetherin antagonism have not been fully defined. A GXXXA motif within the transmembrane domain (TMD) of EBOV-GP was previously shown to be important for GP-mediated cellular detachment. Here, we investigated whether this motif also contributes to tetherin antagonism. Mutation of the GXXXA motif did not impact GP expression or particle incorporation and only modestly reduced EBOV-GP-driven entry. In contrast, the GXXXA motif was required for tetherin antagonism in transfected cells. Moreover, alteration of the GXXXA motif increased tetherin sensitivity of a replication-competent vesicular stomatitis virus (VSV) chimera encoding EBOV-GP. Although these results await confirmation with authentic EBOV, they indicate that a GXXXA motif in the TMD of EBOV-GP is important for tetherin antagonism. Moreover, they provide the first evidence that GP can antagonize tetherin in the context of an infectious EBOV surrogate.IMPORTANCE The glycoprotein (GP) of Ebola virus (EBOV) inhibits the antiviral host cell protein tetherin and may promote viral spread in tetherin-positive cells. However, tetherin antagonism by GP has so far been demonstrated only with virus-like particles, and it is unknown whether GP can block tetherin in infected cells. Moreover, a mutation in GP that selectively abrogates tetherin antagonism is unknown. Here, we show that a GXXXA motif in the transmembrane domain of EBOV-GP, which was previously reported to be required for GP-mediated cell rounding, is also important for tetherin counteraction. Moreover, analysis of this mutation in the context of vesicular stomatitis virus chimeras encoding EBOV-GP revealed that GP-mediated tetherin counteraction is operative in infected cells. To our knowledge, these findings demonstrate for the first time that GP can antagonize tetherin in infected cells and provide a tool to study the impact of GP-dependent tetherin counteraction on EBOV spread.

The glycoprotein of vesicular stomatitis virus promotes release of virus-like particles from tetherin-positive cells.

Vesicular stomatitis virus (VSV) release from infected cells is inhibited by the interferon (IFN)-inducible antiviral host cell factor tetherin (BST-2, CD317). However, several viruses encode tetherin antagonists and it is at present unknown whether residual VSV spread in tetherin-positive cells is also promoted by a virus-encoded tetherin antagonist. Here, we show that the viral glycoprotein (VSV-G) antagonizes tetherin in transfected cells, although with reduced efficiency as compared to the HIV-1 Vpu protein. Tetherin antagonism did not involve alteration of tetherin expression and was partially dependent on a GXXXG motif in the transmembrane domain of VSV-G. However, mutation of the GXXXG motif did not modulate tetherin sensitivity of infectious VSV. These results identify VSV-G as a tetherin antagonist in transfected cells but fail to provide evidence for a contribution of tetherin antagonism to viral spread.

Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin.

The cellular serine protease TMPRSS2, a member of the type II transmembrane serine protease (TTSP) family, cleaves and activates the hemagglutinin of influenza A viruses (FLUAV) in cell culture and is essential for spread of diverse FLUAV in mice. Non-human primates (NHP), in particular rhesus and cynomolgus macaques, serve as animal models for influenza and experimental FLUAV infection of common marmosets has recently also been reported. However, it is currently unknown whether the NHP orthologues of human TMPRSS2 cleave and activate FLUAV hemagglutinin and contribute to viral spread in respiratory tissue. Here, we cloned and functionally analyzed the macaque and marmoset orthologues of human TMPRSS2. In addition, we analyzed the macaque orthologues of human TMPRSS4 and HAT, which also belong to the TTSP family. We found that all NHP orthologues of human TMPRSS2, TMPRSS4 and HAT cleave and activate HA upon directed expression and provide evidence that endogenous TMPRSS2 is expressed in the respiratory epithelium of rhesus macaques. Finally, we demonstrate that a serine protease inhibitor active against TMPRSS2 suppresses FLUAV spread in precision-cut lung slices of human, macaque and marmoset origin. These results indicate that FLUAV depends on serine protease activity for spread in diverse NHP and in humans. Moreover, our findings suggest that macaques and marmosets may serve as models to study FLUAV activation by TMPRSS2 in human patients.

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

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. IMPORTANCE: Filoviruses 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.