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

 描述(由申请人提供)：甲型流感病毒每年的季节性暴发引起严重的公共卫生和经济问题，而大流行可能导致更严重的疾病、发病率和死亡率，因此有必要研究使甲型流感病毒在NAVαVE宿主中确定感染的因素。通常，具有大流行潜力的病毒是那些在禽类或猪的宿主中正常复制并已获得感染和在人与人之间传播的能力，而不事先接触病原体的病毒。新宿主的适应和传递过程是多因素的，但血凝素(HA)蛋白在进入宿主过程中负责病毒和宿主膜的受体结合和融合，发挥着重要作用。众所周知，唾液酸的构象是血凝素的底物，是病毒物种趋向性的决定因素。禽流感病毒通过HA识别和结合终止于α2，3连接的唾液酸的受体。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。这个 IAV的神经氨酸酶(NA)蛋白是一种唾液酸酶，被认为可以清除呼吸道内粘蛋白和已经感染的细胞表面的唾液酸，从而使病毒可以萌芽并传播到感染附近的其他细胞。然而，NA在感染前的作用还没有得到广泛的表征。HA和NA共用一个底物，因此有人提出，为了维持病毒的有效感染，这两种蛋白之间必须存在功能平衡。我们推测，NA的唾液酸酶不仅与其他人提出的HA的结合亲和力相匹配，而且NA的唾液酸裂解专一性平衡了HA的唾液酸结合专一性。在HA持续结合之前，未感染宿主细胞表面的NA-AC可能会糖化，并移除某些结构，从而限制了HA的潜在受体范围。我们建议使用葡聚糖微阵列技术来确定一系列NA的特异性以及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中传递具有相同内部基因的重组病毒来研究适应新宿主物种对HA和NA的影响，这些重组病毒表达一系列亚型的HA和NA蛋白，包括胚胎鸡蛋、猪和人类原代上皮细胞以及Madin-Darby犬肾细胞。我们希望看到这两种蛋白质的变化，反映出识别不同唾液酸链构象的必要性。我们将继续研究HA和NA的协同进化，通过在相同的细胞培养系统中连续传代含有非同源HA：NA对的重组病毒来建立功能平衡。这些研究结果将扩大我们对NA在受体结合和进入之前的作用以及适应新宿主的过程的认识，并对HA和NA的作用以及它们在跨物种传播中的功能平衡产生影响。

 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.