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Find video protocols related to scientific articles indexed in Pubmed.
Influenza, a One Health paradigm-Novel therapeutic strategies to fight a zoonotic pathogen with pandemic potential.
Int. J. Med. Microbiol.
PUBLISHED: 09-16-2014
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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.
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The nuclear export protein of H5N1 influenza A viruses recruits Matrix 1 (M1) protein to the viral ribonucleoprotein to mediate nuclear export.
J. Biol. Chem.
PUBLISHED: 06-02-2014
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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.
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Phosphorylation of highly conserved serine residues in the influenza A virus nuclear export protein NEP plays a minor role in viral growth in human cells and mice.
J. Virol.
PUBLISHED: 04-16-2014
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Phosphorylation at the highly conserved serine residues S23 to S25 in the nuclear export protein (NEP) of influenza A viruses was suspected to regulate its nuclear export activity or polymerase activity-enhancing function. Mutation of these phosphoacceptor sites to either alanine or aspartic acid showed only a minor effect on both activities but revealed the presence of other phosphoacceptor sites that might be involved in regulating NEP activity.
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An infectious bat-derived chimeric influenza virus harbouring the entry machinery of an influenza A virus.
Nat Commun
PUBLISHED: 03-07-2014
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In 2012, the complete genomic sequence of a new and potentially harmful influenza A-like virus from bats (H17N10) was identified. However, infectious influenza virus was neither isolated from infected bats nor reconstituted, impeding further characterization of this virus. Here we show the generation of an infectious chimeric virus containing six out of the eight bat virus genes, with the remaining two genes encoding the haemagglutinin and neuraminidase proteins of a prototypic influenza A virus. This engineered virus replicates well in a broad range of mammalian cell cultures, human primary airway epithelial cells and mice, but poorly in avian cells and chicken embryos without further adaptation. Importantly, the bat chimeric virus is unable to reassort with other influenza A viruses. Although our data do not exclude the possibility of zoonotic transmission of bat influenza viruses into the human population, they indicate that multiple barriers exist that makes this an unlikely event.
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Adaptive Mutations in the Nuclear Export Protein of Human-Derived H5N1 Strains Facilitate a Polymerase Activity-Enhancing Conformation.
J. Virol.
PUBLISHED: 10-23-2013
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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.
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Analysis of borna disease virus trafficking in live infected cells by using a virus encoding a tetracysteine-tagged p protein.
J. Virol.
PUBLISHED: 09-11-2013
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Borna disease virus (BDV) is a nonsegmented, negative-stranded RNA virus characterized by noncytolytic persistent infection and replication in the nuclei of infected cells. To gain further insight on the intracellular trafficking of BDV components during infection, we sought to generate recombinant BDV (rBDV) encoding fluorescent fusion viral proteins. We successfully rescued a virus bearing a tetracysteine tag fused to BDV-P protein, which allowed assessment of the intracellular distribution and dynamics of BDV using real-time live imaging. In persistently infected cells, viral nuclear inclusions, representing viral factories tethered to chromatin, appeared to be extremely static and stable, contrasting with a very rapid and active trafficking of BDV components in the cytoplasm. Photobleaching (fluorescence recovery after photobleaching [FRAP] and fluorescence loss in photobleaching [FLIP]) imaging approaches revealed that BDV components were permanently and actively exchanged between cellular compartments, including within viral inclusions, albeit with a fraction of BDV-P protein not mobile in these structures, presumably due to its association with viral and/or cellular proteins. We also obtained evidence for transfer of viral material between persistently infected cells, with routing of the transferred components toward the cell nucleus. Finally, coculture experiments with noninfected cells allowed visualization of cell-to-cell BDV transmission and movement of the incoming viral material toward the nucleus. Our data demonstrate the potential of tetracysteine-tagged recombinant BDV for virus tracking during infection, which may provide novel information on the BDV life cycle and on the modalities of its interaction with the nuclear environment during viral persistence.
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Adaptation of avian influenza A virus polymerase in mammals to overcome the host species barrier.
J. Virol.
PUBLISHED: 04-24-2013
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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.
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Absence of a robust innate immune response in rat neurons facilitates persistent infection of Borna disease virus in neuronal tissue.
Cell. Mol. Life Sci.
PUBLISHED: 02-27-2013
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Borna disease virus (BDV) persistently infects neurons of the central nervous system of various hosts, including rats. Since type I IFN-mediated antiviral response efficiently blocks BDV replication in primary rat embryo fibroblasts, it has been speculated that BDV is not effectively sensed by the host innate immune system in the nervous system. To test this assumption, organotypical rat hippocampal slice cultures were infected with BDV for up to 4 weeks. This resulted in the secretion of IFN and the up-regulation of IFN-stimulated genes. Using the rat Mx protein as a specific marker for IFN-induced gene expression, astrocytes and microglial cells were found to be Mx positive, whereas neurons, the major cell type in which BDV is replicating, lacked detectable levels of Mx protein. In uninfected cultures, neurons also remained Mx negative even after treatment with high concentrations of IFN-?. This non-responsiveness correlated with a lack of detectable nuclear translocation of both pSTAT1 and pSTAT2 in these cells. Consistently, neuronal dissemination of BDV was not prevented by treatment with IFN-?. These data suggest that the poor innate immune response in rat neurons renders this cell type highly susceptible to BDV infection even in the presence of exogenous IFN-?. Intriguingly, in contrast to rat neurons, IFN-? treatment of mouse neurons resulted in the up-regulation of Mx proteins and block of BDV replication, indicating species-specific differences in the type I IFN response of neurons between mice and rats.
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Pandemic influenza A viruses escape from restriction by human MxA through adaptive mutations in the nucleoprotein.
PLoS Pathog.
PUBLISHED: 02-13-2013
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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.
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Borna disease virus-induced neuronal degeneration dependent on host genetic background and prevented by soluble factors.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 01-14-2013
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Infection of newborn rats with Borne disease virus (BDV) results in selective degeneration of granule cell neurons of the dentate gyrus (DG). To study cellular countermechanisms that might prevent this pathology, we screened for rat strains resistant to this BDV-induced neuronal degeneration. To this end, we infected hippocampal slice cultures of different rat strains with BDV and analyzed for the preservation of the DG. Whereas infected cultures of five rat strains, including Lewis (LEW) rats, exhibited a disrupted DG cytoarchitecture, slices of three other rat strains, including Sprague-Dawley (SD), were unaffected. However, efficiency of viral replication was comparable in susceptible and resistant cultures. Moreover, these rat strain-dependent differences in vulnerability were replicated in vivo in neonatally infected LEW and SD rats. Intriguingly, conditioned media from uninfected cultures of both LEW and SD rats could prevent BDV-induced DG damage in infected LEW hippocampal cultures, whereas infection with BDV suppressed the availability of these factors from LEW but not in SD hippocampal cultures. To gain further insights into the genetic basis for this rat strain-dependent susceptibility, we analyzed DG granule cell survival in BDV-infected cultures of hippocampal neurons derived from the F1 and F2 offspring of the crossing of SD and LEW rats. Genome-wide association analysis revealed one resistance locus on chromosome (chr) 6q16 in SD rats and, surprisingly, a locus on chr3q21-23 that was associated with susceptibility. Thus, BDV-induced neuronal degeneration is dependent on the host genetic background and is prevented by soluble protective factors in the disease-resistant SD rat strain.
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Targeting of the influenza A virus polymerase PB1-PB2 interface indicates strain-specific assembly differences.
J. Virol.
PUBLISHED: 09-28-2011
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Assembly of the heterotrimeric influenza virus polymerase complex from the individual subunits PB1, PA, and PB2 is a prerequisite for viral replication. The conserved protein-protein interaction sites have been suggested as potential drug targets. To characterize the PB1-PB2 interface, we fused the PB1-binding domain of PB2 to green fluorescent protein (PB2(1-37)-GFP) and determined its competitive inhibitory effect on the polymerase activity of influenza A virus strains. Coexpression of PB2(1-37)-GFP in a polymerase reconstitution system led to substantial inhibition of the polymerase of A/WSN/33 (H1N1). Surprisingly, polymerases of other strains, including A/SC35M (H7N7), A/Puerto Rico/8/34 (H1N1), A/Hamburg/4/2009 (H1N1), and A/Thailand/1(KAN-1)/2004 (H5N1), showed various degrees of resistance. Individual exchange of polymerase subunits and the nucleoprotein between the sensitive WSN polymerase and the insensitive SC35M polymerase mapped the resistance to both PB1 and PA of SC35M polymerase. While PB2(1-37)-GFP bound equally well to the PB1 subunits of both virus strains, PB1-PA dimers of SC35M polymerase showed impaired binding compared to PB1-PA dimers of WSN polymerase. The use of PA(SC35M/WSN) chimeras revealed that the reduced affinity of the SC35M PB1-PA dimer was mediated by the N-terminal 277 amino acids of PA. Based on these observations, we speculate that the PB1-PA dimer formation of resistant polymerases shields the PB2(1-37) binding site, whereas sensitive polymerases allow this interaction, suggesting different assembly strategies of the trimeric polymerase complex between different influenza A virus strains.
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Reversion of PB2-627E to -627K during replication of an H5N1 Clade 2.2 virus in mammalian hosts depends on the origin of the nucleoprotein.
J. Virol.
PUBLISHED: 08-17-2011
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H5N1 highly pathogenic avian influenza viruses (HPAIV) of clade 2.2 spread from Southeast Asia to Europe. Intriguingly, in contrast to all common avian strains specifying glutamic acid at position 627 of the PB2 protein (PB2-627E), they carry a lysine at this position (PB2-627K), which is normally found only in human strains. To analyze the impact of this mutation on the host range of HPAIV H5N1, we altered PB2-627K to PB2-627E in the European isolate A/Swan/Germany/R65/2006 (R65). In contrast to the parental R65, multicycle growth and polymerase activity of the resulting mutant R65-PB2(K627E) were considerably impaired in mammalian but not in avian cells. Correspondingly, the 50% lethal dose (LD??) in mice was increased by three orders of magnitude, whereas virulence in chicken remained unchanged, resulting in 100% lethality, as was found for the parental R65. Strikingly, R65-PB2(K627E) reverted to PB2-627K after only one passage in mice but did not revert in chickens. To investigate whether additional R65 genes influence reversion, we passaged R65-PB2(K627E) reassortants containing genes from A/Hong Kong/156/97 (H5N1) (carrying PB2-627E), in avian and mammalian cells. Reversion to PB2-627K in mammalian cells required the presence of the R65 nucleoprotein (NP). This finding corresponds to results of others that during replication of avian strains in mammalian cells, PB2-627K restores an impaired PB2-NP association. Since this mutation is apparently not detrimental for virus prevalence in birds, it has not been eliminated. However, the prompt reversion to PB2-627K in MDCK cells and mice suggests that the clade 2.2 H5N1 HPAIV may have had a history of intermediate mammalian hosts.
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The viral nucleoprotein determines Mx sensitivity of influenza A viruses.
J. Virol.
PUBLISHED: 06-15-2011
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Host restriction factors play a crucial role in preventing trans-species transmission of viral pathogens. In mammals, the interferon-induced Mx GTPases are powerful antiviral proteins restricting orthomyxoviruses. Hence, the human MxA GTPase may function as an efficient barrier against zoonotic introduction of influenza A viruses into the human population. Successful viruses are likely to acquire adaptive mutations allowing them to evade MxA restriction. We compared the 2009 pandemic influenza A virus [strain A/Hamburg/4/09 (pH1N1)] with a highly pathogenic avian H5N1 isolate [strain A/Thailand/1(KAN-1)/04] for their relative sensitivities to human MxA and murine Mx1. The H5N1 virus was highly sensitive to both Mx GTPases, whereas the pandemic H1N1 virus was almost insensitive. Substitutions of the viral polymerase subunits or the nucleoprotein (NP) in a polymerase reconstitution assay demonstrated that NP was the main determinant of Mx sensitivity. The NP of H5N1 conferred Mx sensitivity to the pandemic H1N1 polymerase, whereas the NP of pandemic H1N1 rendered the H5N1 polymerase insensitive. Reassortant viruses which expressed the NP of H5N1 in a pH1N1 genetic background and vice versa were generated. Congenic Mx1-positive mice survived intranasal infection with these reassortants if the challenge virus contained the avian NP. In contrast, they succumbed to infection if the NP of pH1N1 origin was present. These findings clearly indicate that the origin of NP determines Mx sensitivity and that human influenza viruses acquired adaptive mutations to evade MxA restriction. This also explains our previous observations that human and avian influenza A viruses differ in their sensitivities to Mx.
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Identification of influenza virus inhibitors which disrupt of viral polymerase protein-protein interactions.
Methods
PUBLISHED: 06-09-2011
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Due to their ability to rapidly mutate, influenza viruses quickly develop resistance against many antiviral substances, leading to an urgent need for new compounds. The trimeric viral polymerase complex, a major target for the development of new inhibitors, must be assembled from the PB1, PB2, and PA subunits for successful infection. Here, we describe ELISA-based assays which allow the identification of peptides which impair polymerase complex formation. Since the protein-protein interaction domains of the viral polymerase are highly conserved, these inhibitors are also predicted to be active against a broad range of influenza strains. Using this method, identification of small molecules and lead compounds against influenza A and B viruses should be feasible.
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Influenza virus ribonucleoprotein complexes gain preferential access to cellular export machinery through chromatin targeting.
PLoS Pathog.
PUBLISHED: 04-21-2011
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In contrast to most RNA viruses, influenza viruses replicate their genome in the nucleus of infected cells. As a result, newly-synthesized vRNA genomes, in the form of viral ribonucleoprotein complexes (vRNPs), must be exported to the cytoplasm for productive infection. To characterize the composition of vRNP export complexes and their interplay with the nucleus of infected cells, we affinity-purified tagged vRNPs from biochemically fractionated infected nuclei. After treatment of infected cells with leptomycin B, a potent inhibitor of Crm1-mediated export, we isolated vRNP export complexes which, unexpectedly, were tethered to the host-cell chromatin with very high affinity. At late time points of infection, the cellular export receptor Crm1 also accumulated at the same regions of the chromatin as vRNPs, which led to a decrease in the export of other nuclear Crm1 substrates from the nucleus. Interestingly, chromatin targeting of vRNP export complexes brought them into association with Rcc1, the Ran guanine exchange factor responsible for generating RanGTP and driving Crm1-dependent nuclear export. Thus, influenza viruses gain preferential access to newly-generated host cell export machinery by targeting vRNP export complexes at the sites of Ran regeneration.
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The influenza A virus NS1 protein interacts with the nucleoprotein of viral ribonucleoprotein complexes.
J. Virol.
PUBLISHED: 03-16-2011
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The influenza A virus genome consists of eight RNA segments that associate with the viral polymerase proteins (PB1, PB2, and PA) and nucleoprotein (NP) to form ribonucleoprotein complexes (RNPs). The viral NS1 protein was previously shown to associate with these complexes, although it was not clear which RNP component mediated the interaction. Using individual TAP (tandem affinity purification)-tagged PB1, PB2, PA, and NP, we demonstrated that the NS1 protein interacts specifically with NP and not the polymerase subunits. The region of NS1 that binds NP was mapped to the RNA-binding domain.
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Influenza virus infection induces the nuclear relocalization of the Hsp90 co-chaperone p23 and inhibits the glucocorticoid receptor response.
PLoS ONE
PUBLISHED: 02-12-2011
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The genomic RNAs of influenza A viruses are associated with the viral polymerase subunits (PB1, PB2, PA) and nucleoprotein (NP), forming ribonucleoprotein complexes (RNPs). Transcription/replication of the viral genome occurs in the nucleus of infected cells. A role for Hsp90 in nuclear import and assembly of newly synthetized RNA-polymerase subunits has been proposed. Here we report that the p23 cochaperone of Hsp90, which plays a major role in glucocorticoid receptor folding and function, associates with influenza virus polymerase. We show that p23 is not essential for viral multiplication in cultured cells but relocalizes to the nucleus in influenza virus-infected cells, which may alter some functions of p23 and Hsp90. Moreover, we show that influenza virus infection inhibits glucocorticoid receptor-mediated gene transactivation, and that this negative effect can occur through a p23-independent pathway. Viral-induced inhibition of the glucocorticoid receptor response might be of significant importance regarding the physiopathology of influenza infections in vivo.
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Viral interference with neuronal integrity: what can we learn from the Borna disease virus?
Cell Tissue Res.
PUBLISHED: 01-27-2011
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The neurotropic Borna disease virus (BDV) is unusual in that it can persistently infect neurons of the central nervous system (CNS) without causing general cell death, reflecting its favourable adaptation to the brain. The activity-dependent enhancement of neuronal network activity is however disturbed after BDV infection, possibly by its effect on the protein kinase C signalling pathway. The best model for studying BDV, which has a non-cytolytic replication strategy in primary neurons, is the rat. Infection of adult rats results in a fatal immune-mediated disease, whereas BDV establishes persistent infection of the brain in newborn rats resulting in progressive neuronal cell loss in defined regions of the CNS. Our recently developed system of BDV-infected hippocampal slice cultures has clearly shown that the onset of granule cell loss begins after the formation of the mossy fibre projection. Quantitative analysis has revealed a significant increase in synaptic density on identified remaining granule cell dendrites at 6 weeks after infection, followed by a decline. Granule cells are the major target of entorhinal afferents. However, despite an almost complete loss of dentate granule cells during BDV infection, entorhinal axons persist in their correct layer, both in vivo and in slice cultures, possibly exploiting rewiring capabilities and thereby allowing new synapse formation with available targets. These morphological observations, together with electrophysiological and biochemical data, indicate that BDV is a suitable model virus for studying virus-induced morphological and functional changes of neurons and connectivity patterns.
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Host- and strain-specific regulation of influenza virus polymerase activity by interacting cellular proteins.
MBio
PUBLISHED: 01-01-2011
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Highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtype have recently emerged from avian zoonotic reservoirs to cause fatal human disease. Adaptation of HPAI virus RNA-dependent RNA polymerase (PB1, PB2, and PA proteins) and nucleoprotein (NP) to interactions with mammalian host proteins is thought to contribute to the efficiency of viral RNA synthesis and to disease severity. While proteomics experiments have identified a number of human proteins that associate with H1N1 polymerases and/or viral ribonucleoprotein (vRNP), how these host interactions might regulate influenza virus polymerase functions and host adaptation has been largely unexplored. We took a functional genomics (RNA interference [RNAi]) approach to assess the roles of a network of human proteins interacting with influenza virus polymerase proteins in viral polymerase activity from prototype H1N1 and H5N1 viruses. A majority (18 of 31) of the cellular proteins tested, including RNA-binding (DDX17, DDX5, NPM1, and hnRNPM), stress (PARP1, DDB1, and Ku70/86), and intracellular transport proteins, were required for efficient activity of both H1N1 and H5N1 polymerases. NXP2 and NF90 antagonized both polymerases, and six more RNA-associated proteins exhibited strain-specific phenotypes. Remarkably, 12 proteins differentially regulated H5N1 polymerase according to PB2 genotype at mammalian-adaptive residue 627. Among these, DEAD box RNA helicase DDX17/p72 facilitated efficient human-adapted (627K) H5N1 virus mRNA and viral RNA (vRNA) synthesis in human cells. Likewise, the chicken DDX17 homologue was required for efficient avian (627E) H5N1 infection in chicken DF-1 fibroblasts, suggesting that this conserved virus-host interaction contributes to PB2-dependent host species specificity of influenza virus and ultimately to the outcome of human HPAI infections.
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Disruption of the viral polymerase complex assembly as a novel approach to attenuate influenza A virus.
J. Biol. Chem.
PUBLISHED: 12-23-2010
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To develop a novel attenuation strategy applicable to all influenza A viruses, we targeted the highly conserved protein-protein interaction of the viral polymerase subunits PA and PB1. We postulated that impaired binding between PA and PB1 would negatively affect trimeric polymerase complex formation, leading to reduced viral replication efficiency in vivo. As proof of concept, we introduced single or multiple amino acid substitutions into the protein-protein-binding domains of either PB1 or PA, or both, to decrease binding affinity and polymerase activity substantially. As expected, upon generation of recombinant influenza A viruses (SC35M strain) containing these mutations, many pseudo-revertants appeared that partially restored PA-PB1 binding and polymerase activity. These polymerase assembly mutants displayed drastic attenuation in cell culture and mice. The attenuation of the polymerase assembly mutants was maintained in IFN?/? receptor knock-out mice. As exemplified using a H5N1 polymerase assembly mutant, this attenuation strategy can be also applied to other highly pathogenic influenza A virus strains. Thus, we provide proof of principle that targeted mutation of the highly conserved interaction domains of PA and PB1 represents a novel strategy to attenuate influenza A viruses.
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Identification of high-affinity PB1-derived peptides with enhanced affinity to the PA protein of influenza A virus polymerase.
Antimicrob. Agents Chemother.
PUBLISHED: 12-06-2010
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The influenza A virus polymerase complex, consisting of the subunits PB1, PB2, and PA, represents a promising target for the development of new antiviral drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between PA and PB1 using peptides derived from the extreme N terminus of PB1 (amino acids [aa] 1 to 15), comprising the PA-binding domain of PB1. To increase the binding affinity of these peptides, we performed a systematic structure-affinity relationship analysis. Alanine and aspartic acid scans revealed that almost all amino acids in the core binding region (aa 5 to 11) are indispensable for PA binding. Using a library of immobilized peptides representing all possible single amino acid substitutions, we were able to identify amino acid positions outside the core PA-binding region (aa 1, 3, 12, 14, and 15) that are variable and can be replaced by affinity-enhancing residues. Surface plasmon resonance binding studies revealed that combination of several affinity-enhancing mutations led to an additive effect. Thus, the feasibility to enhance the PA-binding affinity presents an intriguing possibility to increase antiviral activity of the PB1-derived peptide and one step forward in the development of an antiviral drug against influenza A viruses.
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Fusion-active glycoprotein G mediates the cytotoxicity of vesicular stomatitis virus M mutants lacking host shut-off activity.
J. Gen. Virol.
PUBLISHED: 07-14-2010
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The cytopathogenicity of vesicular stomatitis virus (VSV) has been attributed mainly to the host shut-off activity of the viral matrix (M) protein, which inhibits both nuclear transcription and nucleocytoplasmic RNA transport, thereby effectively suppressing the synthesis of type I interferon (IFN). The M protein from persistently VSV-infected cells was shown to harbour characteristic amino acid substitutions (M51R, V221F and S226R) implicated in IFN induction. This study demonstrates that infection of human fibroblasts with recombinant VSV containing the M51R substitution resulted in IFN induction, whereas neither the V221F nor the S226R substitution effected an IFN-inducing phenotype. Only when V221F was combined with S226R were the host shut-off activity of the M protein abolished and IFN induced, independently of M51R. The M33A substitution, previously implicated in VSV cytotoxicity, did not affect host shut-off activity. M-mutant VSV containing all four amino acid substitutions retained cytotoxic properties in both Vero cells and IFN-competent primary fibroblasts. Infected-cell death was associated with the formation of giant polynucleated cells, suggesting that the fusion activity of the VSV G protein was involved. Accordingly, M-mutant VSV expressing a fusion-defective G protein or with a deletion of the G gene showed significantly reduced cytotoxic properties and caused long-lasting infections in Vero cells and mouse hippocampal slice cultures. In contrast, a G-deleted VSV expressing wild-type M protein remained cytotoxic. These findings indicate that the host shut-off activity of the M protein dominates VSV cytotoxicty, whilst the fusion-active G protein is mainly responsible for the cytotoxicity remaining with M-mutant VSV.
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A polymorphism in the hemagglutinin of the human isolate of a highly pathogenic H5N1 influenza virus determines organ tropism in mice.
J. Virol.
PUBLISHED: 06-02-2010
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We characterized a human H5N1 virus isolate (KAN-1) encoding a hemagglutinin (HA) with a K-to-E substitution at amino acid position 222 that was previously described to be selected in the lung of the infected patient. In mice, the growth of the HA(222E)-encoding virus was mainly confined to the lung, but reversion to 222K allowed virus to spread to the brain. The HA(222E) variant showed an overall reduced binding affinity compared to that of HA(222K) for synthetic Neu5Ac2-3Gal-terminated receptor analogues, except for one analogue [Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)(6-HSO(3))GlcNAcbeta, Su-SLe(x)]. Our results suggest that human-derived mutations in HA of H5N1 viruses can affect viral replication efficiency and organ tropism.
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Limited compatibility of polymerase subunit interactions in influenza A and B viruses.
J. Biol. Chem.
PUBLISHED: 04-02-2010
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Despite their close phylogenetic relationship, natural intertypic reassortants between influenza A (FluA) and B (FluB) viruses have not been described. Inefficient polymerase assembly of the three polymerase subunits may contribute to this incompatibility, especially because the known protein-protein interaction domains, including the PA-binding domain of PB1, are highly conserved for each virus type. Here we show that substitution of the FluA PA-binding domain (PB1-A(1-25)) with that of FluB (PB1-B(1-25)) is accompanied by reduced polymerase activity and viral growth of FluA. Consistent with these findings, surface plasmon resonance spectroscopy measurements revealed that PA of FluA exhibits impaired affinity to biotinylated PB1-B(1-25) peptides. PA of FluB showed no detectable affinity to biotinylated PB1-A(1-25) peptides. Consequently, FluB PB1 harboring the PA-binding domain of FluA (PB1-AB) failed to assemble with PA and PB2 into an active polymerase complex. To regain functionality, we used a single amino acid substitution (T6Y) known to confer binding to PA of both virus types, which restored polymerase complex formation but surprisingly not polymerase activity for FluB. Taken together, our results demonstrate that the conserved virus type-specific PA-binding domains differ in their affinity to PA and thus might contribute to intertypic exclusion of reassortants between FluA and FluB viruses.
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Lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections.
J. Virol.
PUBLISHED: 03-24-2010
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Virus-infected cells secrete a broad range of interferons (IFN) which confer resistance to yet uninfected cells by triggering the synthesis of antiviral factors. The relative contributions of the various IFN subtypes to innate immunity against virus infections remain elusive. IFN-alpha, IFN-beta, and other type I IFN molecules signal through a common, universally expressed cell surface receptor, whereas type III IFN (IFN-lambda) uses a distinct cell-type-specific receptor complex for signaling. Using mice lacking functional receptors for type I IFN, type III IFN, or both, we found that IFN-lambda plays an important role in the defense against several human pathogens that infect the respiratory tract, such as influenza A virus, influenza B virus, respiratory syncytial virus, human metapneumovirus, and severe acute respiratory syndrome (SARS) coronavirus. These viruses were more pathogenic and replicated to higher titers in the lungs of mice lacking both IFN receptors than in mice with single IFN receptor defects. In contrast, Lassa fever virus, which infects via the respiratory tract but primarily replicates in the liver, was not influenced by the IFN-lambda receptor defect. Careful analysis revealed that expression of functional IFN-lambda receptor complexes in the lung and intestinal tract is restricted to epithelial cells and a few other, undefined cell types. Interestingly, we found that SARS coronavirus was present in feces from infected mice lacking receptors for both type I and type III IFN but not in those from mice lacking single receptors, supporting the view that IFN-lambda contributes to the control of viral infections in epithelial cells of both respiratory and gastrointestinal tracts.
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Protein kinase C-dependent phosphorylation of Borna disease virus P protein is required for efficient viral spread.
Arch. Virol.
PUBLISHED: 01-15-2010
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Mutational analysis of the phosphate acceptor sites of the Borna disease virus (BDV) phosphoprotein (P) has suggested a role of phosphorylation for viral spread. However, the studied mutant viruses also had two amino acid exchanges in the X protein, because the reading frames of P and X overlap. To determine the relative contribution of P and X to viral attenuation, we studied a P variant with serine-to-leucine substitutions (P(S26L,S28L)) in which the wild-type X sequence was conserved. Viral spread of rBDV-P(S26L,S28L) was impaired in human oligodendroglioma cells and in adult rats. Thus, BDV-P phosphorylation contributes to efficient viral dissemination.
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Identification of a PA-binding peptide with inhibitory activity against influenza A and B virus replication.
PLoS ONE
PUBLISHED: 07-31-2009
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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.
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Borna disease virus infection alters synaptic input of neurons in rat dentate gyrus.
Cell Tissue Res.
PUBLISHED: 06-29-2009
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Granule cells are major targets of entorhinal afferents terminating in a laminar fashion in the outer molecular layer of the dentate gyrus. Since Borna disease virus (BDV) infection of newborn rats causes a progressive loss of granule cells in the dentate gyrus, entorhinal fibres become disjoined from their main targets. We have investigated the extent to which entorhinal axons react to this loss of granule cells. Unexpectedly, anterograde DiI tracing has shown a prominent layered termination of the entorhinal projection, despite an almost complete loss of granule cells at 9 weeks after infection. Combined light- and electron-microscopic analysis of dendrites at the outer molecular layer of the dentate gyrus at 6 and 9 weeks post-infection has revealed a transient increase in the synaptic density of calbindin-positive granule cells and parvalbuminergic neurons after 6 weeks. In contrast, synaptic density reaches values similar to those of uninfected controls 9 weeks post-infection. These findings indicate that, after BDV infection, synaptic reorganization processes occur at peripheral dendrites of the remaining granule cells and parvalbuminergic neurons, including the unexpected persistence of entorhinal axons in the absence of their main targets.
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Mutation of the protein kinase C site in borna disease virus phosphoprotein abrogates viral interference with neuronal signaling and restores normal synaptic activity.
PLoS Pathog.
PUBLISHED: 04-13-2009
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Understanding the pathogenesis of infection by neurotropic viruses represents a major challenge and may improve our knowledge of many human neurological diseases for which viruses are thought to play a role. Borna disease virus (BDV) represents an attractive model system to analyze the molecular mechanisms whereby a virus can persist in the central nervous system (CNS) and lead to altered brain function, in the absence of overt cytolysis or inflammation. Recently, we showed that BDV selectively impairs neuronal plasticity through interfering with protein kinase C (PKC)-dependent signaling in neurons. Here, we tested the hypothesis that BDV phosphoprotein (P) may serve as a PKC decoy substrate when expressed in neurons, resulting in an interference with PKC-dependent signaling and impaired neuronal activity. By using a recombinant BDV with mutated PKC phosphorylation site on P, we demonstrate the central role of this protein in BDV pathogenesis. We first showed that the kinetics of dissemination of this recombinant virus was strongly delayed, suggesting that phosphorylation of P by PKC is required for optimal viral spread in neurons. Moreover, neurons infected with this mutant virus exhibited a normal pattern of phosphorylation of the PKC endogenous substrates MARCKS and SNAP-25. Finally, activity-dependent modulation of synaptic activity was restored, as assessed by measuring calcium dynamics in response to depolarization and the electrical properties of neuronal networks grown on microelectrode arrays. Therefore, preventing P phosphorylation by PKC abolishes viral interference with neuronal activity in response to stimulation. Our findings illustrate a novel example of viral interference with a differentiated neuronal function, mainly through competition with the PKC signaling pathway. In addition, we provide the first evidence that a viral protein can specifically interfere with stimulus-induced synaptic plasticity in neurons.
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Attenuation of rabies virus replication and virulence by picornavirus internal ribosome entry site elements.
J. Virol.
PUBLISHED: 02-14-2009
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Gene expression of nonsegmented negative-strand RNA viruses is regulated at the transcriptional level and relies on the canonical 5-end-dependent translation of capped viral mRNAs. Here, we have used internal ribosome entry sites (IRES) from picornaviruses to control the expression level of the phosphoprotein P of the neurotropic rabies virus (RV; Rhabdoviridae), which is critically required for both viral replication and escape from the host interferon response. In a dual luciferase reporter RV, the IRES elements of poliovirus (PV) and human rhinovirus type 2 (HRV2) were active in a variety of cell lines from different host species. While a generally lower activity of the HRV2 IRES was apparent compared to the PV IRES, specific deficits of the HRV2 IRES in neuronal cell lines were not observed. Recombinant RVs expressing P exclusively from a bicistronic nucleoprotein (N)-IRES-P mRNA showed IRES-specific reduction of replication in cell culture and in neurons of organotypic brain slice cultures, an increased activation of the beta interferon (IFN-beta) promoter, and increased sensitivity to IFN. Intracerebral infection revealed a complete loss of virulence of both PV- and HRV2 IRES-controlled RV for wild-type mice and for transgenic mice lacking a functional IFN-alpha receptor (IFNAR(-/-)). The virulence of HRV2 IRES-controlled RV was most severely attenuated and could be demonstrated only in newborn IFNAR(-/-) mice. Translational control of individual genes is a promising strategy to attenuate replication and virulence of live nonsegmented negative-strand RNA viruses and vectors and to study the function of IRES elements in detail.
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Affinity purification of influenza virus ribonucleoprotein complexes from the chromatin of infected cells.
J Vis Exp
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Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized. Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix. After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500).
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Bornavirus closely associates and segregates with host chromosomes to ensure persistent intranuclear infection.
Cell Host Microbe
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Bornaviruses are nonsegmented negative-strand RNA viruses that establish a persistent infection in the nucleus and occasionally integrate a DNA genome copy into the host chromosomal DNA. However, how these viruses achieve intranuclear infection remains unclear. We show that Borna disease virus (BDV), a mammalian bornavirus, closely associates with the cellular chromosome to ensure intranuclear infection. BDV generates viral factories within the nucleus using host chromatin as a scaffold. In addition, the viral ribonucleoprotein (RNP) interacts directly with the host chromosome throughout the cell cycle, using core histones as a docking platform. HMGB1, a host chromatin-remodeling DNA architectural protein, is required to stabilize RNP on chromosomes and for efficient BDV RNA transcription in the nucleus. During metaphase, the association of RNP with mitotic chromosomes allows the viral RNA to segregate into daughter cells and ensure persistent infection. Thus, bornaviruses likely evolved a chromosome-dependent life cycle to achieve stable intranuclear infection.
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Adaptive mutations in NEP compensate for defective H5N1 RNA replication in cultured human cells.
Nat Commun
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Infection of mammals by avian influenza viruses requires adaptive mutations to achieve high-level replication in the new host. However, the basic mechanism underlying this adaptation process is still unknown. Here we show that avian polymerases, lacking the human signature PB2-E627K, are incapable of generating usable complementary RNA templates in cultured human cells and therefore require adaptation. Characterization of the highly pathogenic human H5N1 isolate A/Thailand/1(KAN-1)/2004 that retained the avian PB2-E627 reveals that the defect in RNA replication is only partially compensated by mutations in the polymerase. Instead, mutations in the nuclear export protein are required for efficient polymerase activity. We demonstrate that adaptive mutations in nuclear export proteins of several human isolates enhance the polymerase activity of avian polymerases in human cultured cells. In conclusion, when crossing the species barrier, avian influenza viruses acquire adaptive mutations in nuclear export protein to escape restricted viral genome replication in mammalian cells.
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