The role of NA in virus receptor interaction and HA:NA functional balance

 描述（由申请方提供）：甲型流感病毒（IAV）的年度季节性爆发引起严重的公共卫生和经济问题，并且可能导致疾病严重程度、发病率和死亡率增加的大流行，因此有必要研究使IAV能够在未感染宿主中建立感染的因素。通常情况下，具有大流行潜力的病毒是那些在禽类或猪宿主中正常复制的病毒，并且已经获得了感染和在人类之间传播的能力，而之前没有暴露于病原体。适应新宿主和在新宿主之间传播的过程是多因素的，然而，在进入过程中负责受体结合和病毒与宿主膜融合的血凝素（HA）蛋白起着重要作用。已知HA的底物唾液酸的构象是病毒种属嗜性的决定因素。禽病毒通过HA识别并结合以α 2，3连接的唾液酸终止的受体。人类病毒识别并结合终止于α-2，6连接的末端唾液酸的受体。A的唾液酸特异性被认为是限制禽流感病毒直接传播给人类的一个因素。的 IAV的神经氨酸酶（NA）蛋白是一种唾液酸酶，并且被认为从气道衬里的粘蛋白和已经感染的细胞的表面清除唾液酸，使得病毒可以发芽并传播以感染其它附近的细胞。然而，NA在感染前的作用尚未得到广泛的表征。HA和NA共享一个底物，因此有人提出，两种蛋白质之间必须存在功能平衡，以使病毒维持生产性感染。我们推测，不仅NA的唾液酸酶与HA的结合亲和力相匹配，而且NA的唾液酸切割特异性与HA的唾液酸结合特异性相平衡。可能的是，NA在持续的HA结合之前作用于未感染宿主细胞的细胞表面聚糖，并去除某些结构，因此限制了HA的潜在受体的范围。我们建议使用聚糖微阵列技术来确定一系列NA的特异性和NA活性对HA结合谱的影响。我们还将通过在不同的细胞培养系统（包括鸡胚、猪和人原代上皮细胞以及Madin-达比犬肾细胞）中传代具有相同内部基因的重组病毒（表达一系列亚型的HA和NA蛋白），研究适应新宿主物种对HA和NA的影响。我们希望看到这两种蛋白质的变化，反映了识别不同唾液酸键构象的必要性。我们将扩展我们的研究，以检查HA和NA的共同进化，以建立一个功能平衡，通过连续传代重组病毒含有非同源HA：NA对在相同的细胞培养系统。拟议的研究结果将扩大我们的知识NA的作用之前，受体结合和进入和适应新的主机的过程中，HA和NA的作用和它们的功能平衡的跨物种传播的影响。

 DESCRIPTION (provided by applicant): Annual seasonal outbreaks of Influenza A virus (IAV) cause serious public health and economic concerns, and the potential for pandemics resulting in disease of increased severity, morbidity and mortality necessitates the study of factors that enable IAV to establish an infection in a naαve host. Frequently, viruses with pandemic potential are those that replicate in an avian or swine host normally and have acquired to ability to infect and transmit between humans with no prior exposure to the pathogen. The process of adaptation to and transmitting between new hosts is multifactorial, however the hemagglutinin (HA) protein, responsible for receptor binding and fusion of the viral and host membranes during entry, plays a significant role. It is known that the conformation of sialic acid, the substrate fo HA, is a determinant of species tropism for the virus. Avian viruses recognize and bind, via the HA, receptors that terminate in α2,3 linked sialic acid. Human viruses recognize and bind receptors that terminate in α-2,6 linked terminal sialic acid. The sialic acid specificity of the A is considered to be a factor in the restriction of direct transmission of avian viruses to humans. The neuraminidase (NA) protein of IAV is a sialidase and is thought to clear the sialic acids, from mucins lining the airway and from the surface of already infected cells, so that the virus may bud out and disseminate to infect other nearby cells. However, the role of NA prior to infection has not been as extensively characterized. The HA and NA share a substrate, so it has been proposed that a functional balance between the two proteins must exist in order for the virus to maintain a productive infection. We postulate that not only does the sialidase of the NA match the binding affinity of the HA as suggested by others, but also that the sialic acid cleavage specificity of NA balances the sialic acid binding specificity of HA. It is possible that the NA acs on the cell surface glycans of an uninfected host cell prior to sustained HA binding and removes certain structures, therefore limiting the range of potential receptors for HA. We propose to use glycan microarray technology to determine the specificity of a range of NAs and the effect of NA activity on the binding profiles of HA. We will also examine the affects of adaptation to a new host species on the HA and NA, by passaging recombinant viruses with the same internal genes expressing the HA and NA proteins of a range of subtypes in different cell culture systems, including embryonated chicken eggs, swine and human primary epithelial cells, and Madin- Darby canine kidney cells. We expect to see changes in both proteins reflecting the necessity of recognition of a different sialic acid linkage conformation. We will extend our studies to examine the co-evolution of the HA and NA to establish a functional balance by serially passaging recombinant viruses containing non-cognate HA:NA pairs in the same cell culture systems. The findings of the proposed studies will expand our knowledge of the role of NA prior to receptor binding and entry and of the process of adaptation to a new host, with implications for the role of HA and NA and their functional balance in the event of cross-species transmission.