RESUMO
Recently, A/H5N1 influenza viruses were shown to acquire airborne transmissibility between ferrets upon targeted mutagenesis and virus passage. The critical genetic changes in airborne A/Indonesia/5/05 were not yet identified. Here, five substitutions proved to be sufficient to determine this airborne transmission phenotype. Substitutions in PB1 and PB2 collectively caused enhanced transcription and virus replication. One substitution increased HA thermostability and lowered the pH of membrane fusion. Two substitutions independently changed HA binding preference from α2,3-linked to α2,6-linked sialic acid receptors. The loss of a glycosylation site in HA enhanced overall binding to receptors. The acquired substitutions emerged early during ferret passage as minor variants and became dominant rapidly. Identification of substitutions that are essential for airborne transmission of avian influenza viruses between ferrets and their associated phenotypes advances our fundamental understanding of virus transmission and will increase the value of future surveillance programs and public health risk assessments.
Assuntos
Virus da Influenza A Subtipo H5N1/fisiologia , Influenza Humana/transmissão , Influenza Humana/virologia , Substituição de Aminoácidos , Animais , Furões , Genoma Viral , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Humanos , Virus da Influenza A Subtipo H5N1/genética , Mutação , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Receptores Virais/metabolismo , Seleção GenéticaRESUMO
UNLABELLED: 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. IMPORTANCE: Influenza viruses from birds jump the species barrier into humans relatively frequently. Such influenza virus zoonoses may pose public health risks if the virus adapts to humans and becomes a pandemic threat. Relatively few amino acid substitutions-most notably in the receptor binding site of hemagglutinin and at positions 591 and 627 in the polymerase protein PB2-have been identified in pandemic influenza virus strains as determinants of host adaptation, to facilitate efficient virus replication and transmission in humans. Here, we show that substantial numbers of amino acid substitutions are functionally compensating for the lack of the above-mentioned mutations in PB2 and could facilitate influenza virus emergence in humans.
Assuntos
Substituição de Aminoácidos , Virus da Influenza A Subtipo H5N1/fisiologia , Subtipo H7N9 do Vírus da Influenza A/fisiologia , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação Viral , Adaptação Fisiológica , Animais , Aves , Linhagem Celular , Biologia Computacional , Células HEK293 , Especificidade de Hospedeiro , Humanos , Vírus da Influenza A Subtipo H1N1 , Virus da Influenza A Subtipo H5N1/enzimologia , Virus da Influenza A Subtipo H5N1/genética , Subtipo H7N9 do Vírus da Influenza A/enzimologia , Subtipo H7N9 do Vírus da Influenza A/genética , Influenza Aviária/virologia , Influenza Humana/virologia , Filogenia , RNA Polimerase Dependente de RNA/química , Proteínas Virais/químicaRESUMO
UNLABELLED: 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.
Assuntos
Evasão da Resposta Imune , Subtipo H7N9 do Vírus da Influenza A/imunologia , Influenza Aviária/virologia , Influenza Humana/imunologia , Proteínas de Resistência a Myxovirus/imunologia , Proteínas de Ligação a RNA/imunologia , Proteínas do Core Viral/imunologia , Animais , Aves , Linhagem Celular , China , Humanos , Subtipo H7N9 do Vírus da Influenza A/crescimento & desenvolvimento , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo , RNA Viral/genética , Proteínas de Ligação a RNA/genética , Análise de Sequência de DNA , Proteínas do Core Viral/genética , Zoonoses/transmissão , Zoonoses/virologiaRESUMO
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.
Assuntos
Núcleo Celular/metabolismo , Virus da Influenza A Subtipo H5N1/metabolismo , Mutação , Proteínas não Estruturais Virais/genética , Células HEK293 , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/fisiologia , Conformação Proteica , Dobramento de Proteína , Transporte Proteico , Temperatura , Proteínas não Estruturais Virais/química , Replicação ViralRESUMO
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.
Assuntos
Proteínas de Ligação ao GTP/genética , Evasão da Resposta Imune/genética , Vírus da Influenza A Subtipo H1N1/genética , Mutação , Nucleoproteínas/genética , Infecções por Orthomyxoviridae/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Resistência à Doença/genética , Evolução Molecular , Proteínas de Ligação ao GTP/química , Humanos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Resistência a Myxovirus , Infecções por Orthomyxoviridae/imunologia , Pandemias , Estrutura Secundária de Proteína , Seleção Genética , Zoonoses/virologiaRESUMO
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.
Assuntos
Adaptação Biológica/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Interações Hospedeiro-Patógeno/genética , Vírus da Influenza A/enzimologia , Influenza Aviária/virologia , Nucleoproteínas/genética , Proteínas Virais/metabolismo , Animais , Aves , RNA Polimerases Dirigidas por DNA/genética , Humanos , Modelos Biológicos , Mutação/genética , Especificidade da Espécie , Proteínas Virais/genética , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
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 IFNα/ß 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.
Assuntos
Virus da Influenza A Subtipo H5N1/enzimologia , Vírus da Influenza A Subtipo H7N7/enzimologia , Influenza Humana/enzimologia , Mutação , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/metabolismo , Animais , Cães , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/imunologia , Vírus da Influenza A Subtipo H7N7/genética , Vírus da Influenza A Subtipo H7N7/imunologia , Vacinas contra Influenza/biossíntese , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Influenza Humana/genética , Influenza Humana/imunologia , Interferon-alfa/genética , Interferon-alfa/imunologia , Interferon-alfa/metabolismo , Interferon beta/genética , Interferon beta/imunologia , Interferon beta/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/imunologia , Vacinas Atenuadas/biossíntese , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Replicação Viral/genética , Replicação Viral/imunologiaRESUMO
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.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Vírus da Influenza A Subtipo H1N1/fisiologia , Virus da Influenza A Subtipo H5N1/fisiologia , Proteínas de Ligação a RNA/metabolismo , Proteínas do Core Viral/metabolismo , Animais , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Células HEK293 , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Virus da Influenza A Subtipo H5N1/genética , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Proteínas de Resistência a Myxovirus , Células NIH 3T3 , Proteínas do Nucleocapsídeo , Proteínas de Ligação a RNA/genética , Vírus Reordenados/genética , Vírus Reordenados/fisiologia , Proteínas do Core Viral/genética , Proteínas Virais/metabolismoRESUMO
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.
Assuntos
Vírus da Influenza A/fisiologia , Multimerização Proteica , Proteínas Virais/metabolismo , Replicação Viral , Fusão Gênica Artificial , Sítios de Ligação , Linhagem Celular , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Ligação Proteica , Mapeamento de Interação de Proteínas , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Coloração e RotulagemRESUMO
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 (LD50) 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.
Assuntos
Substituição de Aminoácidos/genética , Especificidade de Hospedeiro , Virus da Influenza A Subtipo H5N1/crescimento & desenvolvimento , Virus da Influenza A Subtipo H5N1/patogenicidade , Mutação de Sentido Incorreto , RNA Polimerase Dependente de RNA/genética , Proteínas Virais/genética , Animais , Aves , Linhagem Celular , Galinhas , Modelos Animais de Doenças , Feminino , Humanos , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Dose Letal Mediana , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/virologia , Doenças das Aves Domésticas/mortalidade , Doenças das Aves Domésticas/virologia , Doenças dos Roedores/mortalidade , Doenças dos Roedores/virologia , Análise de SobrevidaRESUMO
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.
Assuntos
Vírus da Influenza A/metabolismo , Vírus da Influenza B/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Cães , Humanos , Cinética , Dados de Sequência Molecular , Nucleotídeos/química , Peptídeos/química , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Ressonância de Plasmônio de SuperfícieRESUMO
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.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Virus da Influenza A Subtipo H5N1/genética , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Humana/virologia , Polimorfismo Genético , Tropismo Viral , Substituição de Aminoácidos/genética , Animais , Encéfalo/virologia , Humanos , Pulmão/virologia , Camundongos , Mutação de Sentido Incorreto , Infecções por Orthomyxoviridae/virologia , Receptores Virais/metabolismo , Doenças dos Roedores/virologia , Supressão Genética , Ligação ViralRESUMO
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.
RESUMO
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.
Assuntos
Virus da Influenza A Subtipo H5N1/genética , Influenza Humana/virologia , Mutação de Sentido Incorreto , Proteínas não Estruturais Virais/genética , Replicação Viral , Substituição de Aminoácidos , Animais , Linhagem Celular , Feminino , Humanos , Virus da Influenza A Subtipo H5N1/enzimologia , Virus da Influenza A Subtipo H5N1/patogenicidade , Virus da Influenza A Subtipo H5N1/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , RNA Polimerase Dependente de RNA/química , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Alinhamento de Sequência , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo , VirulênciaRESUMO
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.