Formation of cytoplasmic inclusion bodies (IBs) is a hallmark of infections with non-segmented negative-strand RNA viruses (order Mononegavirales ). We show here that Nipah virus (NiV), a bat-derived highly pathogenic member of the Paramyxoviridae family, differs from mononegaviruses of the Rhabdo- , Filo- and Pneumoviridae families by forming two types of IBs with distinct localizations, formation kinetics, and protein compositions. IBs in the perinuclear region form rapidly upon expression of the nucleocapsid proteins. These IB peri are highly mobile and associate with the aggresome marker y-tubulin. IB peri can recruit unrelated overexpressed cytosolic proteins but do not contain the viral matrix (M) protein. Additionally, NiV forms an as yet undescribed IB population at the plasma membrane (IB PM ) that is y-tubulin-negative but contains the M protein. Infection studies with recombinant NiV revealed that IB PM require the M protein for their formation, and most likely represent sites of NiV assembly and budding. The identification of this novel type of plasma membrane-associated IBs not only provides new insights into NiV biology and may open new avenues to develop novel antiviral approaches to treat these highly pathogenic viruses, it also provides a basis for a more detailed characterization of IBs and their role in virus assembly and replication in infections with other Mononegavirale s.

In recent years, novel henipavirus-related sequences have been identified in bats in Africa. To evaluate the potential of African bat henipaviruses to spread in non-bat mammalian cells, we compared the biological functions of the surface glycoproteins G and F of the prototype African henipavirus GH-M74a with those of the glycoproteins of Nipah virus (NiV), a well-characterized pathogenic member of the henipavirus genus. Glycoproteins are central determinants for virus tropism, as efficient binding of henipavirus G proteins to cellular ephrin receptors and functional expression of fusion-competent F proteins are indispensable prerequisites for virus entry and cellto-cell spread. In this study, we analysed the ability of the GH-M74a G and F proteins to cause cell-to-cell fusion in mammalian cell types readily permissive to NiV or Hendra virus infections. Except for limited syncytium formation in a bat cell line derived from Hypsignathus monstrosus, HypNi/1.1 cells, we did not observe any fusion. The highly restricted fusion activity was predominantly due to the F protein. Whilst GH-M74a G protein was found to interact with the main henipavirus receptor ephrin-B2 and induced syncytia upon co-expression with heterotypic NiV F protein, GH-M74a F protein did not cause evident fusion in the presence of heterotypic NiV G protein. Pulse-chase and surface biotinylation analyses revealed delayed F cleavage kinetics with a reduced expression of cleaved and fusion-active GH-M74a F protein on the cell surface. Thus, the F protein of GH-M74a showed a functional defect that is most likely caused by impaired trafficking leading to less efficient proteolytic activation and surface expression.

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.

Nipah virus (NiV) matrix protein (NiV M) plays a major role in virus assembly. It undergoes nuclear transit before accumulating at the plasma membrane and recruiting nucleocapsids to the budding sites. Because nuclear NiV M cannot be detected in all cell types, we wondered whether it can reach the cell surface by bypassing the nucleus. Using an M mutant with a defective nuclear export signal (MNESmut), however, we revealed that the nuclear import of M is ubiquitous, because MNESmut was retained in the nuclei of all cell types tested. Because a functional nuclear transit is a general prerequisite for M surface transport, we wanted to characterize the effect of nuclear-retained M protein in a full viral context and generated a recombinant NiV-MNESmut. Mutant NiV-MNESmut caused increased cell-cell fusion and produced lower virus titers. As expected for an assembly defective NiV, perinuclear inclusions (IBperi) were formed, but inclusions at the plasma membrane (IBPM), which probably represent the viral assembly platforms, were not found. It is interesting to note that the transport-defective MNESmut was recruited to IBperi. This probably prevents overaccumulation of nonfunctional M proteins in the cytoplasm and nuclei of NiV-infected cells and thus provides first evidence that IBperi are functionally relevant aggresome-like compartments.

Highly pathogenic Nipah virus (NiV) causes symptomatic infections in pigs and humans. The severity of respiratory symptoms is much more pronounced in pigs than in humans, suggesting species-specific differences of NiV replication in porcine and human airways. Here, we present a comparative study on productive NiV replication in primary airway epithelial cell cultures of the two species. We reveal that NiV growth substantially differs in primary cells between pigs and humans, with a more rapid spread of infection in human airway epithelia. Increased replication, correlated with higher endogenous expression levels of the main NiV entry receptor ephrin-B2, not only significantly differed between airway cells of the two species but also varied between cells from different human donors. To our knowledge, our study provides the first experimental evidence of species-specific and individual differences in NiV receptor expression and replication kinetics in primary airway epithelial cells. It remains to be determined whether and how these differences contribute to the viral host range and pathogenicity.