Protective CD8+ T Cell Response Induced by Modified Vaccinia Virus Ankara Delivering Ebola Virus Nucleoprotein.

The urgent need for vaccines against Ebola virus (EBOV) was underscored by the large outbreak in West Africa (2014-2016). Since then, several promising vaccine candidates have been tested in pre-clinical and clinical studies. As a result, two vaccines were approved for human use in 2019/2020, of which one includes a heterologous adenovirus/Modified Vaccinia virus Ankara (MVA) prime-boost regimen. Here, we tested new vaccine candidates based on the recombinant MVA vector, encoding the EBOV nucleoprotein (MVA-EBOV-NP) or glycoprotein (MVA-EBOV-GP) for their efficacy after homologous prime-boost immunization in mice. Our aim was to investigate the role of each antigen in terms of efficacy and correlates of protection. Sera of mice vaccinated with MVA-EBOV-GP were virus-neutralizing and MVA-EBOV-NP immunization readily elicited interferon-γ-producing NP-specific CD8+ T cells. While mock-vaccinated mice succumbed to EBOV infection, all vaccinated mice survived and showed drastically decreased viral loads in sera and organs. In addition, MVA-EBOV-NP vaccinated mice became susceptible to lethal EBOV infection after depletion of CD8+ T cells prior to challenge. This study highlights the potential of MVA-based vaccines to elicit humoral immune responses as well as a strong and protective CD8+ T cell response and contributes to understanding the possible underlying mechanisms.

The Ebola Virus Nucleoprotein Recruits the Host PP2A-B56 Phosphatase to Activate Transcriptional Support Activity of VP30.

Transcription of the Ebola virus genome depends on the viral transcription factor VP30 in its unphosphorylated form, but the underlying molecular mechanism of VP30 dephosphorylation is unknown. Here we show that the Ebola virus nucleoprotein (NP) recruits the host PP2A-B56 protein phosphatase through a B56-binding LxxIxE motif and that this motif is essential for VP30 dephosphorylation and viral transcription. The LxxIxE motif and the binding site of VP30 in NP are in close proximity, and both binding sites are required for the dephosphorylation of VP30. We generate a specific inhibitor of PP2A-B56 and show that it suppresses Ebola virus transcription and infection. This work dissects the molecular mechanism of VP30 dephosphorylation by PP2A-B56, and it pinpoints this phosphatase as a potential target for therapeutic intervention.

A Polymorphism within the Internal Fusion Loop of the Ebola Virus Glycoprotein Modulates Host Cell Entry.

The large scale of the Ebola virus disease (EVD) outbreak in West Africa in 2013-2016 raised the question whether the host cell interactions of the responsible Ebola virus (EBOV) strain differed from those of other ebolaviruses. We previously reported that the glycoprotein (GP) of the virus circulating in West Africa in 2014 (EBOV2014) exhibited reduced ability to mediate entry into two nonhuman primate (NHP)-derived cell lines relative to the GP of EBOV1976. Here, we investigated the molecular determinants underlying the differential entry efficiency. We found that EBOV2014-GP-driven entry into diverse NHP-derived cell lines, as well as human monocyte-derived macrophages and dendritic cells, was reduced compared to EBOV1976-GP, although entry into most human- and all bat-derived cell lines tested was comparable. Moreover, EBOV2014 replication in NHP but not human cells was diminished relative to EBOV1976, suggesting that reduced cell entry translated into reduced viral spread. Mutagenic analysis of EBOV2014-GP and EBOV1976-GP revealed that an amino acid polymorphism in the receptor-binding domain, A82V, modulated entry efficiency in a cell line-independent manner and did not account for the reduced EBOV2014-GP-driven entry into NHP cells. In contrast, polymorphism T544I, located in the internal fusion loop in the GP2 subunit, was found to be responsible for the entry phenotype. These results suggest that position 544 is an important determinant of EBOV infectivity for both NHP and certain human target cells.IMPORTANCE The Ebola virus disease outbreak in West Africa in 2013 entailed more than 10,000 deaths. The scale of the outbreak and its dramatic impact on human health raised the question whether the responsible virus was particularly adept at infecting human cells. Our study shows that an amino acid exchange, A82V, that was acquired during the epidemic and that was not observed in previously circulating viruses, increases viral entry into diverse target cells. In contrast, the epidemic virus showed a reduced ability to enter cells of nonhuman primates compared to the virus circulating in 1976, and a single amino acid exchange in the internal fusion loop of the viral glycoprotein was found to account for this phenotype.

Functional Characterization of Adaptive Mutations during the West African Ebola Virus Outbreak.

The Ebola virus (EBOV) outbreak in West Africa started in December 2013, claimed more than 11,000 lives, threatened to destabilize a whole region, and showed how easily health crises can turn into humanitarian disasters. EBOV genomic sequences of the West African outbreak revealed nonsynonymous mutations, which induced considerable public attention, but their role in virus spread and disease remains obscure. In this study, we investigated the functional significance of three nonsynonymous mutations that emerged early during the West African EBOV outbreak. Almost 90% of more than 1,000 EBOV genomes sequenced during the outbreak carried the signature of three mutations: a D759G substitution in the active center of the L polymerase, an A82V substitution in the receptor binding domain of surface glycoprotein GP, and an R111C substitution in the self-assembly domain of RNA-encapsidating nucleoprotein NP. Using a newly developed virus-like particle system and reverse genetics, we found that the mutations have an impact on the functions of the respective viral proteins and on the growth of recombinant EBOVs. The mutation in L increased viral transcription and replication, whereas the mutation in NP decreased viral transcription and replication. The mutation in the receptor binding domain of the glycoprotein GP improved the efficiency of GP-mediated viral entry into target cells. Recombinant EBOVs with combinations of the three mutations showed a growth advantage over the prototype isolate Makona C7 lacking the mutations. This study showed that virus variants with improved fitness emerged early during the West African EBOV outbreak. IMPORTANCE: The dimension of the Ebola virus outbreak in West Africa was unprecedented. Amino acid substitutions in the viral L polymerase, surface glycoprotein GP, and nucleocapsid protein NP emerged, were fixed early in the outbreak, and were found in almost 90% of the sequences. Here we showed that these mutations affected the functional activity of viral proteins and improved viral growth in cell culture. Our results demonstrate emergence of adaptive changes in the Ebola virus genome during virus circulation in humans and prompt further studies on the potential role of these changes in virus transmissibility and pathogenicity.

Characterization of African bat henipavirus GH-M74a glycoproteins.

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 cell-to-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.

Nipah virus entry and egress from polarized epithelial cells.

Highly pathogenic Nipah virus (NiV) infections are transmitted via airway secretions and urine, commonly via the respiratory route. Epithelial surfaces represent important replication sites in both primary and systemic infection phases. NiV entry and spread from polarized epithelial cells therefore determine virus entry and dissemination within a new host and influence virus shedding via mucosal surfaces in the respiratory and urinary tract. To date, there is no knowledge regarding the entry and exit sites of NiV in polarized epithelial cells. In this report, we show for the first time that NiV can infect polarized kidney epithelial cells (MDCK) from both cell surfaces, while virus release is primarily restricted to the apical plasma membrane. Substantial amounts of basolateral infectivity were detected only after infection with high virus doses, at time points when the integrity of the cell monolayer was largely disrupted as a result of cell-to-cell fusion. Confocal immunofluorescence analyses of envelope protein distribution at early and late infection stages suggested that apical virus budding is determined by the polarized sorting of the NiV matrix protein, M. Studies with stably M-expressing and with monensin-treated cells furthermore demonstrated that M protein transport is independent from the glycoproteins, implying that the M protein possesses an intrinsic apical targeting signal.

Canine distemper virus matrix protein influences particle infectivity, particle composition, and envelope distribution in polarized epithelial cells and modulates virulence.

In paramyxoviruses, the matrix (M) protein mediates the interaction between the envelope and internal proteins during particle assembly and egress. In measles virus (MeV), M mutations, such as those found in subacute sclerosing panencephalitis (SSPE) strains, and differences in vaccine and wild-type M proteins can affect the strength of interaction with the envelope glycoproteins, assembly efficiency, and spread. However, the contribution of the M protein to the replication and pathogenesis of the closely related canine distemper virus (CDV) has not been characterized. To this end this, we generated a recombinant wild-type CDV carrying a vaccine strain M protein. The recombinant virus retained the parental growth phenotype in VerodogSLAMtag cells, but displayed an increased particle-to-infectivity ratio very similar to that of the vaccine strain, likely due to inefficient H protein incorporation. Even though infectious virus was released only from the apical surface, consistent with the release polarity of the wild-type CDV strain, envelope protein distribution in polarized epithelial cells reproduced the bipolar pattern seen in vaccine strain-infected cells. Most notably, the chimeric virus was completely attenuated in ferrets and caused only a mild and transient leukopenia, indicating that the differences in particle infectivity and envelope protein sorting mediated by the vaccine M protein contribute importantly to vaccine strain attenuation.

Nipah virus fusion protein: influence of cleavage site mutations on the cleavability by cathepsin L, trypsin and furin.

Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae which originated from bats, encodes for a fusion (F) protein which is proteolytically processed within endosomes by cathepsin L. We show here that sequence requirements for NiV F activation differ markedly from other para- or orthomyxoviral fusion proteins. In contrast to other viral fusion proteins with monobasic cleavage sites, processing of NiV F proteins with one single basic amino acid in the cleavage peptide by exogenous trypsin is very inefficient, and introduction of a consensus sequence for furin does not result in cleavage by this ubiquitous protease. In contrast, a multibasic cleavage peptide in the NiV F protein completely impairs proteolytic processing and the generation of biological activity.

Sorting signals in the measles virus wild-type glycoproteins differently influence virus spread in polarized epithelia and lymphocytes.

The spread of virus infection within an organism is partially dictated by the receptor usage of the virus and can be influenced by sorting signals present in the viral glycoproteins expressed in infected cells. In previous studies, we have shown that the haemagglutinin (H) and fusion protein (F) of the measles virus (MV) vaccine strain MV(Edm) harbour tyrosine-dependent sorting signals which influence virus spread in both lymphocytes and epithelial cells to a similar degree. In contrast with the vaccine strain, MV wild-type virus does not use CD46 but CD150/SLAM and a not clearly identified molecule on epithelial cells as receptors. To determine differences in viral spread between vaccine and wild-type virus, we generated recombinant MV expressing glycoproteins of both the wild-type strain WTFb and the corresponding tyrosine mutants. In contrast with observations based on vaccine virus glycoproteins, mutations in wild-type virus H and F differently influenced cell-to-cell fusion and replication in polarized epithelia and lymphocytes. For wild-type H, our data suggest a key role of the cytoplasmic tyrosine signal for virus dissemination in vivo. It seems to be important for efficient virus spread between lymphocytes, while the tyrosine signal in the F protein gains importance in epithelial cells as both signals have to be intact to allow efficient spread of infection within epithelia.

Glycoprotein targeting signals influence the distribution of measles virus envelope proteins and virus spread in lymphocytes.

We previously demonstrated the presence of tyrosine-dependent motifs for specific sorting of two measles virus (MV) glycoproteins, H and F, to the basolateral surface in polarized epithelial cells. Targeted expression of the glycoproteins was found to be required for virus spread in epithelia via cell-to-cell fusion in vitro and in vivo. In the present study, recombinant MVs (rMVs) with substitutions of the critical tyrosines in the H and F cytoplasmic domains were used to determine whether the sorting signals also play a crucial role for MV replication and spread within lymphocytes, the main target cells of acute MV infection. Immunolocalization revealed that only standard glycoproteins are targeted specifically to the uropod of polarized lymphocytes and cluster on the surface of non-polarized lymphocytes. H and F proteins with tyrosine mutations did not accumulate in uropods, but were distributed homogeneously on the surface and did not colocalize markedly with the matrix (M) protein. Due to the defective interaction with the M protein, all mutant rMVs showed an enhanced fusion capacity, but only rMVs harbouring two mutated glycoproteins showed a marked decrease in virus release from infected lymphocytes. These results demonstrate clearly that the tyrosine-based targeting motifs in the MV glycoproteins are not only important in polarized epithelial cells, but are also active in lymphocytes, thus playing an important role in virus propagation in different key target cells during acute MV infection.