Gülsah Gabriel

Interaction of Polymerase Subunit PB2 and NP with Importin α1 Is a Determinant of Host Range of Influenza A Virus

We have previously reported that mutations in the polymerase proteins PB1, PB2, PA, and the nucleocapsid protein NP resulting in enhanced transcription and replication activities in mammalian cells are responsible for the conversion of the avian influenza virus SC35 (H7N7) into the mouse-adapted variant SC35M. We show now that adaptive mutations D701N in PB2 and N319K in NP enhance binding of these proteins to importin α1 in mammalian cells. Enhanced binding was paralleled by transient nuclear accumulation and cytoplasmic depletion of importin α1 as well as increased transport of PB2 and NP into the nucleus of mammalian cells. In avian cells, enhancement of importin α1 binding and increased nuclear transport were not observed. These findings demonstrate that adaptation of the viral polymerase to the nuclear import machinery plays an important role in interspecies transmission of influenza virus.

Differential use of importin-α isoforms governs cell tropism and host adaptation of influenza virus

Influenza A viruses are a threat to humans due to their ability to cross species barriers, as illustrated by the 2009 H1N1v pandemic and sporadic H5N1 transmissions. Interspecies transmission requires adaptation of the viral polymerase to importin-α, a cellular protein that mediates transport into the nucleus where transcription and replication of the viral genome takes place. In this study, we analysed replication, host specificity and pathogenicity of avian and mammalian influenza viruses, in importin-α-silenced cells and importin-α-knockout mice, to understand the role of individual importin-α isoforms in adaptation. For efficient virus replication, the polymerase subunit PB2 and the nucleoprotein (NP) of avian viruses required importin-α3, whereas PB2 and NP of mammalian viruses showed importin-α7 specificity. H1N1v replication depended on both, importin-α3 and -α7, suggesting ongoing adaptation of this virus. Thus, differences in importin-α specificity are determinants of host range underlining the importance of the nuclear envelope in interspecies transmission.

Differential polymerase activity in avian and mammalian cells determines host range of influenza virus

ABSTRACT As recently shown, mutations in the polymerase genes causing increased polymerase activity in mammalian cells are responsible for the adaptation of the highly pathogenic avian influenza virus SC35 (H7N7) to mice (G. Gabriel et al., Proc. Natl. Acad. Sci. USA 102:18590-18595, 2005). We have now compared mRNA, cRNA, and viral RNA levels of SC35 and its mouse-adapted variant SC35M in avian and mammalian cells. The increase in levels of transcription and replication of SC35M in mammalian cells was linked to a decrease in avian cells. Thus, the efficiency of the viral polymerase is a determinant of both host specificity and pathogenicity.

Human-like PB2 627K Influenza Virus Polymerase Activity Is Regulated by Importin-α1 and -α7

Influenza A viruses may cross species barriers and transmit to humans with the potential to cause pandemics. Interplay of human- (PB2 627K) and avian-like (PB2 627E) influenza polymerase complexes with unknown host factors have been postulated to play a key role in interspecies transmission. Here, we have identified human importin-α isoforms (α1 and α7) as positive regulators of human- but not avian-like polymerase activity. Human-like polymerase activity correlated with efficient recruitment of α1 and α7 to viral ribonucleoprotein complexes (vRNPs) without affecting subcellular localization. We also observed that human-like influenza virus growth was impaired in α1 and α7 downregulated human lung cells. Mice lacking α7 were less susceptible to human- but not avian-like influenza virus infection. Thus, α1 and α7 are positive regulators of human-like polymerase activity and pathogenicity beyond their role in nuclear transport.

Adaptive mutations in the H5N1 polymerase complex.

Adaptation of the viral polymerase to host factors plays an important role in interspecies transmission of H5N1 viruses. Several adaptive mutations have been identified that, in general, determine not only host range, but also pathogenicity and transmissibility of the virus. The available evidence indicates that most of these mutations are found in the PB2 subunit of the polymerase. Particularly prominent mutations are located in the C-terminal domain of PB2 involving the amino acid exchanges E627K and D701N. Both mutations, that are also responsible for the adaptation of other avian viruses to mammalian hosts, have been described in human H5N1 isolates. In animal models, it could be demonstrated that they enhance pathogenicity in mice and induce contact transmission in guinea pigs. Mutation E627K has also been identified as a determinant of air-borne H5N1 transmission in ferrets. We are only beginning to understand the underlying mechanisms at the molecular level. Thus, mutation D701N promotes importin-α mediated nuclear transport in mammalian cells. Mutation E627K also enhances the replication rate in an importin-α dependent fashion in mammalian cells, yet without affecting nuclear entry of PB2. Numerous other adaptive mutations, some of which compensate for the lack of PB2 E627K, have been observed in PB2 as well as in the polymerase subunit PB1, the nucleoprotein NP, and the nuclear export protein NEP (NS2).

Sex, Immunity and Influenza

Sex-specific endocrine and immune responses are widely recognized to account for differential disease outcomes between females and males. Surprisingly, sex-specific risk assessments for influenza, a viral pathogen that affects human populations worldwide through seasonal epidemics and irregular occurring pandemics, are sparse and-if available-ambiguous. To date, this precludes proposing an unequivocal sex-dependent susceptibility to influenza. However, one undisputable observation recurrently confirmed during influenza seasons of the last decades is the significantly increased risk for pregnant women. This increased risk is likely attributable to the contradictory demands for the maternal immune system to adapt to pregnancy and to simultaneously mount an immune response to clear the influenza virus infection. Here, we review published evidence on the potential association between sex on influenza risk and propose that future epidemiologic studies should carefully dissect surveillance data for sex-specific effects. Moreover, we propose potential mechanisms involved in enhanced risk for severe influenza during pregnancy that could be studied to identify causal pathways.

The nuclear import machinery is a determinant of influenza virus host adaptation.

After viral entry into the cell, the nuclear envelope poses a major cellular barrier that needs to be overcome upon adaptation of highly pathogenic avian influenza viruses (HPAIV) to the new host. To ensure efficient viral transcription and replication in the nucleus of the host cell, the viral polymerase complex of avian influenza viruses needs to switch from recognition of avian to mammalian components of the nuclear import machinery. Recent evidence suggests that influenza viruses have evolved different mechanisms to utilize importin‐α isoforms as components of this machinery, bridging pre‐ and post‐nuclear import on both sides of the nuclear envelope.

Molecular Determinants of Pathogenicity in the Polymerase Complex

Viral pathogenesis involves numerous interactions between viral and cellular factors. In recent years, the influenza virus polymerase complex has emerged as a major determinant of interspecies transmission and pathogenicity. The viral RNA-dependent RNA polymerase, in concert with the nucleoprotein, mediates transcription and replication of the viral RNA genome in the nucleus of the infected cell. The activity by which the viral polymerase complex performs these processes in mammalian cells is considered to be a major contributor to viral pathogenicity in mammals. In this chapter, we summarise our current understanding on the pathogenicity determinants in the viral polymerase complex and highlight some of its cellular interaction partners. We particularly discuss the role of importin-α isoforms in host adaptation and pathogenesis as well as the role of the viral polymerase in regulating cellular responses to viral infection.

Spread of Infection and Lymphocyte Depletion in Mice Depends on Polymerase of Influenza Virus

SC35M is a mouse-adapted variant of the highly pathogenic avian influenza virus SC35. We have previously shown that interspecies adaptation is mediated by mutations in the viral polymerase and that it is paralleled by the acquisition of high pathogenicity for mice. In the present study, we have compared virus spread and organ tropism of SC35 and SC35M in mice. We show that SC35 virus causes mild bronchiolitis in these animals, whereas infection with the mouse-adapted SC35M virus leads to severe hemorrhagic pneumonia with dissemination to other organs, including the brain. In SC35M-infected animals, viral RNA and viral antigen were detected in monocytes and macrophages, and SC35M, unlike SC35, replicated in lymphocyte and macrophage cultures in vitro. SC35M did not induce an adequate cytokine response but, unlike SC35, caused severe lymphopenia in mice. These observations suggest that the high efficiency of the SC35M polymerase is responsible for infection and depletion of lymphocytes and other white blood cells, which results in immune suppression and systemic virus spread.

A new class of synthetic anti-lipopolysaccharide peptides inhibits influenza A virus replication by blocking cellular attachment.

Influenza A viruses are a continuous threat to human health as illustrated by the 2009 H1N1 pandemic. Since circulating influenza virus strains become increasingly resistant against currently available drugs, the development of novel antivirals is urgently needed. Here, we have evaluated a recently described new class of broad-spectrum antiviral peptides (synthetic anti-lipopolysaccharide peptides; SALPs) for their potential to inhibit influenza virus replication in vitro and in vivo. We found that particularly SALP PEP 19-2.5 shows high binding affinities for the influenza virus receptor molecule, N-Acetylneuraminic acid, leading to impaired viral attachment and cellular entry. As a result, replication of several influenza virus subtypes (H7N7, H3N2 and 2009 pandemic H1N1) was strongly reduced. Furthermore, mice co-treated with PEP 19-2.5 were protected against an otherwise 100% lethal H7N7 influenza virus infection. These findings show that SALPs exhibit antiviral activity against influenza viruses by blocking virus attachment and entry into host cells. Thus, SALPs present a new class of broad-spectrum antiviral peptides for further development for influenza virus therapy.