Identification and characterization of host specificity factors of a lethal human influenza H5N1 isolate

Influenza A viruses are major human and avian pathogens. Despite a species barrier, subtypes of influenza A can transmit from the avian reservoir to humans and widely spread in the population. Since H5N1 viruses circulate in the avian reservoir and cause high lethality rates when transmitted to humans, infections with the H5N1 subtype pose an ongoing threat. Although human-to-human transmission is a rare event, rapid evolution of the virus might result in a strain, which gains the ability to spread in the human population, leading to high morbidity and mortality. Advanced surveillance by understanding the mechanisms by which influenza viruses acquire the ability to cross the species barrier from birds to humans and new strategies to improve current vaccines are needed to control future pandemics. In this study, the fatal human case isolate A/Thailand/1(KAN-1)/2004 (H5N1) (KAN-1) was analyzed to examine mechanisms of H5N1 viruses to overcome host range restriction. • We were able to identify an adaptive mutation in KAN-1 hemagglutinin (HA). A polymorphism leading to an amino acid change in the HA that was previously reported to be positively selected during replication in humans altered the organ tropism of KAN- 1 in mice and ferrets. In the mouse model we found an increased replication of the selected variant in the lung. • In a genetic analysis of the KAN-1 virus, we identified further mutations crucial for adaptation to the mammalian host. Interestingly, the KAN-1 polymerase was poorly adapted to human cells, in contrast to other H5N1 viruses isolated from humans. We identified the NEP protein as a new pathogenicity factor of H5N1 viruses in humans, which is able to overcome this incomplete adaptation of the KAN-1 and avian H5N1 polymerases in human cells. Furthermore, functional studies revealed that the restriction of avian influenza polymerases in mammals is due to a general defect in RNA-replication and not transcription. • Since the human MxA GTPase is an important factor in the immune response against influenza viruses, we analyzed its antiviral activity against KAN-1. KAN-1 proved to be sensitive, while an isolate of the 2009 pandemic was relatively resistant to MxA. We were able to determine the viral nucleoprotein (NP) as the determinant for MxA sensitivity in vitro and in vivo. In addition, we identified mutations in NP responsible for resistance against MxA and could draw conclusions about the evolution of NP. • Based on our knowledge about protein-protein interactions in the polymerase complex, we developed a new strategy to create polymerase assembly mutants as a basis for live attenuated vaccines against H5N1. Vaccination of mice with these mutants showed protection against homologous and heterologous challenge with lethal doses of H5N1 viruses including KAN-1, therefore providing new options for live attenuated vaccine design

A 40-nm 256-Kb Half-Select Resilient 8T SRAM with Sequential Writing Technique

The interferon-induced dynamin-like MxA GTPase restricts the replication of influenza A viruses. We identified adaptive mutations in the nucleoprotein (NP) of pandemic strains A/Brevig Mission/1/1918 (1918) and A/Hamburg/4/2009 (pH1N1) that confer MxA resistance. These resistance-associated amino acids in NP differ between the two strains but form a similar discrete surface-exposed cluster in the body domain of NP, indicating that MxA resistance evolved independently. The 1918 cluster was conserved in all descendent strains of seasonal influenza viruses. Introduction of this cluster into the NP of the MxA-sensitive influenza virus A/Thailand/1(KAN-1)/04 (H5N1) resulted in a gain of MxA resistance coupled with a decrease in viral replication fitness. Conversely, introduction of MxA-sensitive amino acids into pH1N1 NP enhanced viral growth in Mx-negative cells. We conclude that human MxA represents a barrier against zoonotic introduction of avian influenza viruses and that adaptive mutations in the viral NP should be carefully monitored

Tumorigenic WAP-T Mouse Mammary Carcinoma Cells: A Model for a Self-Reproducing Homeostatic Cancer Cell System

BACKGROUND: In analogy to normal stem cell differentiation, the current cancer stem cell (CSC) model presumes a hierarchical organization and an irreversible differentiation in tumor tissue. Accordingly, CSCs should comprise only a small subset of the tumor cells, which feeds tumor growth. However, some recent findings raised doubts on the general applicability of the CSC model and asked for its refinement. METHODOLOGY/PRINCIPAL FINDINGS: In this study we analyzed the CSC properties of mammary carcinoma cells derived from transgenic (WAP-T) mice. We established a highly tumorigenic WAP-T cell line (G-2 cells) that displays stem-like traits. G-2 cells, as well as their clonal derivates, are closely related to primary tumors regarding histology and gene expression profiles, and reflect heterogeneity regarding their differentiation states. G-2 cultures comprise cell populations in distinct differentiation states identified by co-expression of cytoskeletal proteins (cytokeratins and vimentin), a combination of cell surface markers and a set of transcription factors. Cellular subsets sorted according to expression of CD24a, CD49f, CD61, Epcam, Sca1, and Thy1 cell surface proteins, or metabolic markers (e.g. ALDH activity) are competent to reconstitute the initial cellular composition. Repopulation efficiency greatly varies between individual subsets and is influenced by interactions with the respective complementary G-2 cellular subset. The balance between differentiation states is regulated in part by the transcription factor Sox10, as depletion of Sox10 led to up-regulation of Twist2 and increased the proportion of Thy1-expressing cells representing cells in a self-renewable, reversible, quasi-mesenchymal differentiation state. CONCLUSIONS/SIGNIFICANCE: G-2 cells constitute a self-reproducing cancer cell system, maintained by bi- and unidirectional conversion of complementary cellular subsets. Our work contributes to the current controversial discussion on the existence and nature of CSC and provides a basis for the incorporation of alternative hypotheses into the CSC model

One health, multiple challenges: The inter-species transmission of influenza A virus

Influenza A viruses are amongst the most challenging viruses that threaten both human and animal health. Influenza A viruses are unique in many ways. Firstly, they are unique in the diversity of host species that they infect. This includes waterfowl (the original reservoir), terrestrial and aquatic poultry, swine, humans, horses, dog, cats, whales, seals and several other mammalian species. Secondly, they are unique in their capacity to evolve and adapt, following crossing the species barrier, in order to replicate and spread to other individuals within the new species. Finally, they are unique in the frequency of inter-species transmission events that occur. Indeed, the consequences of novel influenza virus strain in an immunologically naïve population can be devastating. The problems that influenza A viruses present for human and animal health are numerous. For example, influenza A viruses in humans represent a major economic and disease burden, whilst the poultry industry has suffered colossal damage due to repeated outbreaks of highly pathogenic avian influenza viruses. This review aims to provide a comprehensive overview of influenza A viruses by shedding light on interspecies virus transmission and summarising the current knowledge regarding how influenza viruses can adapt to a new host

Identification of a PA-Binding Peptide with Inhibitory Activity against Influenza A and B Virus Replication

There is an urgent need for new drugs against influenza type A and B viruses due to incomplete protection by vaccines and the emergence of resistance to current antivirals. The influenza virus polymerase complex, consisting of the PB1, PB2 and PA subunits, represents a promising target for the development of new drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between the PB1 and PA subunits of the polymerase complex of influenza A virus using a small peptide derived from the PA-binding domain of PB1. However, this influenza A virus-derived peptide did not affect influenza B virus polymerase activity. Here we report that the PA-binding domain of the polymerase subunit PB1 of influenza A and B viruses is highly conserved and that mutual amino acid exchange shows that they cannot be functionally exchanged with each other. Based on phylogenetic analysis and a novel biochemical ELISA-based screening approach, we were able to identify an influenza A-derived peptide with a single influenza B-specific amino acid substitution which efficiently binds to PA of both virus types. This dual-binding peptide blocked the viral polymerase activity and growth of both virus types. Our findings provide proof of principle that protein-protein interaction inhibitors can be generated against influenza A and B viruses. Furthermore, this dual-binding peptide, combined with our novel screening method, is a promising platform to identify new antiviral lead compounds

Identification and characterization of host specificity factors of a lethal human influenza H5N1 isolate

Influenza A viruses are major human and avian pathogens. Despite a species barrier, subtypes of influenza A can transmit from the avian reservoir to humans and widely spread in the population. Since H5N1 viruses circulate in the avian reservoir and cause high lethality rates when transmitted to humans, infections with the H5N1 subtype pose an ongoing threat. Although human-to-human transmission is a rare event, rapid evolution of the virus might result in a strain, which gains the ability to spread in the human population, leading to high morbidity and mortality. Advanced surveillance by understanding the mechanisms by which influenza viruses acquire the ability to cross the species barrier from birds to humans and new strategies to improve current vaccines are needed to control future pandemics. In this study, the fatal human case isolate A/Thailand/1(KAN-1)/2004 (H5N1) (KAN-1) was analyzed to examine mechanisms of H5N1 viruses to overcome host range restriction. • We were able to identify an adaptive mutation in KAN-1 hemagglutinin (HA). A polymorphism leading to an amino acid change in the HA that was previously reported to be positively selected during replication in humans altered the organ tropism of KAN- 1 in mice and ferrets. In the mouse model we found an increased replication of the selected variant in the lung. • In a genetic analysis of the KAN-1 virus, we identified further mutations crucial for adaptation to the mammalian host. Interestingly, the KAN-1 polymerase was poorly adapted to human cells, in contrast to other H5N1 viruses isolated from humans. We identified the NEP protein as a new pathogenicity factor of H5N1 viruses in humans, which is able to overcome this incomplete adaptation of the KAN-1 and avian H5N1 polymerases in human cells. Furthermore, functional studies revealed that the restriction of avian influenza polymerases in mammals is due to a general defect in RNA-replication and not transcription. • Since the human MxA GTPase is an important factor in the immune response against influenza viruses, we analyzed its antiviral activity against KAN-1. KAN-1 proved to be sensitive, while an isolate of the 2009 pandemic was relatively resistant to MxA. We were able to determine the viral nucleoprotein (NP) as the determinant for MxA sensitivity in vitro and in vivo. In addition, we identified mutations in NP responsible for resistance against MxA and could draw conclusions about the evolution of NP. • Based on our knowledge about protein-protein interactions in the polymerase complex, we developed a new strategy to create polymerase assembly mutants as a basis for live attenuated vaccines against H5N1. Vaccination of mice with these mutants showed protection against homologous and heterologous challenge with lethal doses of H5N1 viruses including KAN-1, therefore providing new options for live attenuated vaccine design

Adaptation of avian influenza a virus polymerase in mammals to overcome the host species barrier

Avian influenza A viruses, such as the highly pathogenic avian H5N1 viruses, sporadically enter the human population but often do not transmit between individuals. In rare cases, however, they establish a new lineage in humans. In addition to well-characterized barriers to cell entry, one major hurdle which avian viruses must overcome is their poor polymerase activity in human cells. There is compelling evidence that these viruses overcome this obstacle by acquiring adaptive mutations in the polymerase subunits PB1, PB2, and PA and the nucleoprotein (NP) as well as in the novel polymerase cofactor nuclear export protein (NEP). Recent findings suggest that synthesis of the viral genome may represent the major defect of avian polymerases in human cells. While the precise mechanisms remain to be unveiled, it appears that a broad spectrum of polymerase adaptive mutations can act collectively to overcome this defect. Thus, identification and monitoring of emerging adaptive mutations that further increase polymerase activity in human cells are critical to estimate the pandemic potential of avian viruses

Multiple Natural Substitutions in Avian Influenza A Virus PB2 Facilitate Efficient Replication in Human Cells

A strong restriction of the avian influenza A virus polymerase in mammalian cells generally limits viral host-range switching. Although substitutions like E627K in the PB2 polymerase subunit can facilitate polymerase activity to allow replication in mammals, many human H5N1 and H7N9 viruses lack this adaptive substitution. Here, several previously unknown, naturally occurring, adaptive substitutions in PB2 were identified by bioinformatics, and their enhancing activity was verified using in vitro assays. Adaptive substitutions enhanced polymerase activity and virus replication in mammalian cells for avian H5N1 and H7N9 viruses but not for a partially human-adapted H5N1 virus. Adaptive substitutions toward basic amino acids were frequent and were mostly clustered in a putative RNA exit channel in a polymerase crystal structure. Phylogenetic analysis demonstrated divergent dependency of influenza viruses on adaptive substitutions. The novel adaptive substitutions found in this study increase basic understanding of influenza virus host adaptation and will help in surveillance efforts

Adaptive mutations in the nuclear export protein of human-derived H5N1 strains facilitate a polymerase activity-enhancing conformation

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