Martin Schwemmle

Mx GTPases: dynamin-like antiviral machines of innate immunity.

The Mx dynamin-like GTPases are key antiviral effector proteins of the type I and type III interferon (IFN) systems. They inhibit several different viruses by blocking early steps of the viral replication cycle. We focus on new structural and functional insights and discuss recent data revealing that human MxA (MX1) provides a safeguard against introduction of avian influenza A viruses (FLUAV) into the human population. The related human MxB (MX2) serves as restriction factor for HIV-1 and other primate lentiviruses.

Taxonomic reorganization of the family Bornaviridae

Knowledge of bornaviruses has expanded considerably during the last decade. A possible reservoir of mammalian Borna disease virus has been identified, divergent bornaviruses have been detected in birds and reptiles, and endogenous bornavirus-like elements have been discovered in the genomes of vertebrates of several species. Previous sequence comparisons and alignments have indicated that the members of the current family Bornaviridae are phylogenetically diverse and are not adequately classified in the existing bornavirus taxonomy supported by the International Committee on Taxonomy of Viruses (ICTV). We provide an update of these analyses and describe their implications for taxonomy. We propose retaining the family name Bornaviridae and the genus Bornavirus but reorganizing species classification. PAirwise Sequence Comparison (PASC) of bornavirus genomes and Basic Local Alignment Search Tool (BLAST) comparison of genomic and protein sequences, in combination with other already published phylogenetic analyses and known biological characteristics of bornaviruses, indicate that this genus should include at least five species: Mammalian 1 bornavirus (classical Borna disease virus and divergent Borna disease virus isolate No/98), Psittaciform 1 bornavirus (avian/psittacine bornaviruses 1, 2, 3, 4, 7), Passeriform 1 bornavirus (avian/canary bornaviruses C1, C2, C3, LS), Passeriform 2 bornavirus (estrildid finch bornavirus EF), and Waterbird 1 bornavirus (avian bornavirus 062CG). This classification is also in line with biological characteristics of these viruses and their vertebrate hosts. A snake bornavirus, proposed to be named Loveridge’s garter snake virus 1, should be classified as a member of an additional species (Elapid 1 bornavirus), unassigned to a genus, in the family Bornaviridae. Avian bornaviruses 5, 6, MALL, and another “reptile bornavirus” (“Gaboon viper virus”) should stay unclassified until further information becomes available. Finally, we propose new virus names and abbreviations when necessary to achieve clear differentiation and unique identification.

The Nuclear Export Protein of H5N1 Influenza A Viruses Recruits Matrix 1 (M1) Protein to the Viral Ribonucleoprotein to Mediate Nuclear Export

Background: NEP stimulates viral RNA synthesis and nuclear vRNP export. Results: NEP is required to stabilize M1-vRNP binding for nuclear export. Deletion of the last three amino acids of NEP abrogates nuclear export and the polymerase-enhancing function of NEP. Conclusion: The polymerase-enhancing function and the nuclear export function of NEP are linked functionally. Significance: This study provides new insights into the assembly of the nuclear export complex. In influenza A virus-infected cells, replication and transcription of the viral genome occurs in the nucleus. To be packaged into viral particles at the plasma membrane, encapsidated viral genomes must be exported from the nucleus. Intriguingly, the nuclear export protein (NEP) is involved in both processes. Although NEP stimulates viral RNA synthesis by binding to the viral polymerase, its function during nuclear export implicates interaction with viral ribonucleoprotein (vRNP)-associated M1. The observation that both interactions are mediated by the C-terminal moiety of NEP raised the question whether these two features of NEP are linked functionally. Here we provide evidence that the interaction between M1 and the vRNP depends on the NEP C terminus and its polymerase activity-enhancing property for the nuclear export of vRNPs. This suggests that these features of NEP are linked functionally. Furthermore, our data suggest that the N-terminal domain of NEP interferes with the stability of the vRNP-M1-NEP nuclear export complex, probably mediated by its highly flexible intramolecular interaction with the NEP C terminus. On the basis of our data, we propose a new model for the assembly of the nuclear export complex of Influenza A vRNPs.

The nucleoprotein of newly emerged H7N9 influenza A virus harbors a unique motif conferring resistance to antiviral human MxA.

ABSTRACT Interferon-induced Mx proteins show strong antiviral activity against influenza A viruses (IAVs). We recently demonstrated that the viral nucleoprotein (NP) determines resistance of seasonal and pandemic human influenza viruses to Mx, while avian isolates retain Mx sensitivity. We identified a surface-exposed cluster of amino acids in NP of pandemic A/BM/1/1918 (H1N1), comprising isoleucine-100, proline-283, and tyrosine-313, that is essential for reduced Mx sensitivity in cell culture and in vivo. This cluster has been maintained in all descendant seasonal strains, including A/PR/8/34 (PR/8). Accordingly, two substitutions in the NP of PR/8 [PR/8(mut)] to the Mx-sensitive amino acids (P283L and Y313F) led to attenuation in Mx1-positive mice. Serial lung passages of PR/8(mut) in Mx1 mice resulted in a single exchange of tyrosine to asparagine at position 52 in NP (in close proximity to the amino acid cluster at positions 100, 283, and 313), which partially compensates loss of Mx resistance in PR/8(mut). Intriguingly, the NP of the newly emerged avian-origin H7N9 virus also contains an asparagine at position 52 and shows reduced Mx sensitivity. N52Y substitution in NP results in increased sensitivity of the H7N9 virus to human Mx, indicating that this residue is a determinant of Mx resistance in mammals. Our data strengthen the hypothesis that the human Mx protein represents a potent barrier against zoonotic transmission of avian influenza viruses. However, the H7N9 viruses overcome this restriction by harboring an NP that is less sensitive to Mx-mediated host defense. This might contribute to zoonotic transmission of H7N9 and to the severe to fatal outcome of H7N9 infections in humans. IMPORTANCE The natural host of influenza A viruses (IAVs) are aquatic birds. Occasionally, these viruses cross the species barrier, as in early 2013 when an avian H7N9 virus infected humans in China. Since then, multiple transmissions of H7N9 viruses to humans have occurred, leaving experts puzzled about molecular causes for such efficient crossing of the species barrier compared to other avian influenza viruses. Mx proteins are known restriction factors preventing influenza virus replication. Unfortunately, some viruses (e.g., human IAV) have developed some resistance, which is associated with specific amino acids in their nucleoproteins, the target of Mx function. Here, we demonstrate that the novel H7N9 bird IAV already carries a nucleoprotein that overcomes the inhibition of viral replication by human MxA. This is the first example of an avian IAV that is naturally less sensitive to Mx-mediated inhibition and might explain why H7N9 viruses transmitted efficiently to humans.

Influenza A viruses escape from MxA restriction at the expense of efficient nuclear vRNP import

To establish a new lineage in the human population, avian influenza A viruses (AIV) must overcome the intracellular restriction factor MxA. Partial escape from MxA restriction can be achieved when the viral nucleoprotein (NP) acquires the critical human-adaptive amino acid residues 100I/V, 283P, and 313Y. Here, we show that introduction of these three residues into the NP of an avian H5N1 virus renders it genetically unstable, resulting in viruses harboring additional single mutations, including G16D. These substitutions restored genetic stability yet again yielded viruses with varying degrees of attenuation in mammalian and avian cells. Additionally, most of the mutant viruses lost the capacity to escape MxA restriction, with the exception of the G16D virus. We show that MxA escape is linked to attenuation by demonstrating that the three substitutions promoting MxA escape disturbed intracellular trafficking of incoming viral ribonucleoprotein complexes (vRNPs), thereby resulting in impaired nuclear import, and that the additional acquired mutations only partially compensate for this import block. We conclude that for adaptation to the human host, AIV must not only overcome MxA restriction but also an associated block in nuclear vRNP import. This inherent difficulty may partially explain the frequent failure of AIV to become pandemic.

Expected and Unexpected Features of the Newly Discovered Bat Influenza A-like Viruses.

Citation: Ma, W. J., Garcia-Sastre, A., & Schwemmle, M. (2015). Expected and Unexpected Features of the Newly Discovered Bat Influenza A-like Viruses. Plos Pathogens, 11(6), 6. doi:10.1371/journal.ppat.1004819

Influenza, a One Health paradigm—Novel therapeutic strategies to fight a zoonotic pathogen with pandemic potential

Influenza virus is a paradigm for a pathogen that frequently crosses the species barrier from animals to humans, causing severe disease in the human population. This ranges from frequent epidemics to occasional pandemic outbreaks with millions of death. All previous pandemics in humans were caused by animal viruses or virus reassortants carrying animal virus genes, underlining that the fight against influenza requires a One Health approach integrating human and veterinary medicine. Furthermore, the fundamental question of what enables a flu pathogen to jump from animals to humans can only be tackled in a transdisciplinary approach between virologists, immunologists and cell biologists. To address this need the German FluResearchNet was established as a first nationwide influenza research network that virtually integrates all national expertise in the field of influenza to unravel viral and host determinants of pathogenicity and species transmission and to explore novel avenues of antiviral intervention. Here we focus on the various novel anti-flu approaches that were developed as part of the FluResearchNet activities.

The Feat of Packaging Eight Unique Genome Segments.

Influenza A viruses (IAVs) harbor a segmented RNA genome that is organized into eight distinct viral ribonucleoprotein (vRNP) complexes. Although a segmented genome may be a major advantage to adapt to new host environments, it comes at the cost of a highly sophisticated genome packaging mechanism. Newly synthesized vRNPs conquer the cellular endosomal recycling machinery to access the viral budding site at the plasma membrane. Genome packaging sequences unique to each RNA genome segment are thought to be key determinants ensuring the assembly and incorporation of eight distinct vRNPs into progeny viral particles. Recent studies using advanced fluorescence microscopy techniques suggest the formation of vRNP sub-bundles (comprising less than eight vRNPs) during their transport on recycling endosomes. The formation of such sub-bundles might be required for efficient packaging of a bundle of eight different genomes segments at the budding site, further highlighting the complexity of IAV genome packaging.

Adaptive Mutations in the Nuclear Export Protein of Human-Derived H5N1 Strains Facilitate a Polymerase Activity-Enhancing Conformation

ABSTRACT The nuclear export protein (NEP) (NS2) of the highly pathogenic human-derived H5N1 strain A/Thailand/1(KAN-1)/2004 with the adaptive mutation M16I greatly enhances the polymerase activity in human cells in a concentration-dependent manner. While low NEP levels enhance the polymerase activity, high levels are inhibitory. To gain insights into the underlying mechanism, we analyzed the effect of NEP deletion mutants on polymerase activity after reconstitution in human cells. This revealed that the polymerase-enhancing function of NEP resides in the C-terminal moiety and that removal of the last three amino acids completely abrogates this activity. Moreover, compared to full-length NEP, the C-terminal moiety alone exhibited significantly higher activity and seemed to be deregulated, since even the highest concentration did not result in an inhibition of polymerase activity. To determine transient interactions between the N- and C-terminal domains in cis, we fused both ends of NEP to a split click beetle luciferase and performed fragment complementation assays. With decreasing temperature, increased luciferase activity was observed, suggesting that intramolecular binding between the C- and N-terminal domains is preferentially stabilized at low temperatures. This stabilizing effect was significantly reduced with the adaptive mutation M16I or a combination of adaptive mutations (M16I, Y41C, and E75G), which further increased polymerase activity also at 34°C. We therefore propose a model in which the N-terminal moiety of NEP exerts an inhibitory function by back-folding to the C-terminal domain. In this model, adaptive mutations in NEP decrease binding between the C- and N-terminal domains, thereby allowing the protein to “open up” and become active already at a low temperature.

Synthetically derived bat influenza A-like viruses reveal a cell type- but not species-specific tropism

Significance Since the discovery of bat influenza A-like genomic sequences (provisionally designated HL17NL10 and HL18NL11), it was uncertain whether these sequences encode infectious viruses and, if so, which cells might support propagation of these viruses. Using chimeric vesicular stomatitis virus (VSV) encoding HL18 or HL17, a particular cell line of canine origin was found to be highly susceptible to infection. Taking advantage of this cell line, we succeeded to generate recombinant HL17NL10 and HL18NL11. These viruses revealed marked differences to conventional influenza A viruses because they do not use the same receptor, in addition to initiating infection from the basolateral site of polarized epithelial cells. The established reverse genetic system will undoubtedly help characterizing these hitherto uncultivable viruses. Two novel influenza A-like viral genome sequences have recently been identified in Central and South American fruit bats and provisionally designated “HL17NL10” and “HL18NL11.” All efforts to isolate infectious virus from bats or to generate these viruses by reverse genetics have failed to date. Recombinant vesicular stomatitis virus (VSV) encoding the hemagglutinin-like envelope glycoproteins HL17 or HL18 in place of the VSV glycoprotein were generated to identify cell lines that are susceptible to bat influenza A-like virus entry. More than 30 cell lines derived from various species were screened but only a few cell lines were found to be susceptible, including Madin–Darby canine kidney type II (MDCK II) cells. The identification of cell lines susceptible to VSV chimeras allowed us to recover recombinant HL17NL10 and HL18NL11 viruses from synthetic DNA. Both influenza A-like viruses established a productive infection in MDCK II cells; however, HL18NL11 replicated more efficiently than HL17NL10 in this cell line. Unlike conventional influenza A viruses, bat influenza A-like viruses started the infection preferentially at the basolateral membrane of polarized MDCK II cells; however, similar to conventional influenza A viruses, bat influenza A-like viruses were released primarily from the apical site. The ability of HL18NL11 or HL17NL10 viruses to infect canine and human cells might reflect a zoonotic potential of these recently identified bat viruses.