Characterization of the molecular mechanisms that prevent successful adaptation of avian influenza virus to the human host: the nuclear import of incoming vRNPs

为了在人类中建立新的病毒谱系，甲型禽流感病毒必须适应新的宿主及其先天免疫系统。我们最近可以确定病毒核蛋白（NP）中的适应性突变，这些突变是实现对人类限制性因子MxA的抗性所必需的。这些适应性突变在流行的人类毒株中高度保守，而在禽毒株中几乎不存在。有趣的是，将这些适应性突变引入A/Thailand/1(KAN-1)/04 （H5N1）的NP后，在缺乏MxA的情况下会产生强烈的衰减。事实上，在其他病毒中，获得这些适应性突变仍然伴随着部分衰减。这种衰减的原因尚不清楚。初步证据表明，即使在mx阴性细胞中，携带mxa适应性突变的vRNPs的核输入也受到干扰。该项目的目标是揭示导致核vRNP进口缺陷的分子机制。具体来说，我们希望通过生化和遗传方法确定介导NP中含有这些适应性突变的vRNPs核输入的细胞因子。此外，我们希望确定季节性流感病毒中发生的代偿性变化，以克服这种衰减。最后，我们想分析人源性H7N9 （a /Anhui/1/2013）的一种可能的新策略，即允许核vRNP不受阻碍地输入和病毒生长，尽管NP中存在Mx抗性氨基酸。通过这些研究，我们希望挑战我们的假设，即vRNP的核输入是甲型禽流感病毒在人类适应过程中的主要障碍。这些研究还可能有助于估计人类H7N9分离株的大流行潜力。

To establish a new virus lineage in humans, avian influenza A viruses have to adapt to the new host and its innate immune system. We could recently identify adaptive mutations in the viral nucleoprotein (NP) that are required to achieve resistance against the human restriction factor MxA. These adaptive mutations are highly conserved in circulating human strains and virtually absent in avian strains. Interestingly, introduction of these adaptive mutations into the NP of A/Thailand/1(KAN-1)/04 (H5N1) results in a strong attenuation in the absence of MxA. Indeed, acquisition of these adaptive mutations is still accompanied by partial attenuation in other viruses. The reason for this attenuation is unknown. Preliminary evidence indicates that the nuclear import of vRNPs harboring MxA-adaptive mutations is disturbed even in Mx-negative cells. The goal of this project is to unravel the molecular mechanisms that are responsible for this defect in nuclear vRNP import. Specifically, we want to identify the cellular factors that mediate nuclear import of vRNPs harboring these adaptive mutations in NP by biochemical and genetic approaches. Furthermore, we want to identify the compensatory changes that had occurred in seasonal influenza viruses to overcome this attenuation. Finally, we want to analyze a presumably new strategy of the human derived H7N9 (A/Anhui/1/2013) to allow unhindered nuclear vRNP import and viral growth despite the presence of Mx resistance amino acids in NP. With these studies we want to challenge our hypothesis that nuclear import of vRNP represent a major barrier for avian influenza A viruses during the adaptation process to humans. These studies may also contribute to estimate the pandemic potential of the human H7N9 isolates.