Hanson Anthony, Imai Masaki, Hatta Masato, McBride Ryan, Imai Hirotaka, Taft Andrew, Zhong Gongxun, Watanabe Tokiko, Suzuki Yasuo, Neumann Gabriele, Paulson James C, Kawaoka Yoshihiro
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Departments of Cell and Molecular Biology and Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA.
J Virol. 2015 Dec 30;90(6):2981-92. doi: 10.1128/JVI.02790-15.
Highly pathogenic avian influenza viruses of the H5N1 subtype continue to circulate in poultry in Asia, Africa, and the Middle East. Recently, outbreaks of novel reassortant H5 viruses have also occurred in North America. Although the number of human infections with highly pathogenic H5N1 influenza viruses continues to rise, these viruses remain unable to efficiently transmit between humans. However, we and others have identified H5 viruses capable of respiratory droplet transmission in ferrets. Two experimentally introduced mutations in the viral hemagglutinin (HA) receptor-binding domain conferred binding to human-type receptors but reduced HA stability. Compensatory mutations in HA (acquired during virus replication in ferrets) were essential to restore HA stability. These stabilizing mutations in HA also affected the pH at which HA undergoes an irreversible switch to its fusogenic form in host endosomes, a crucial step for virus infectivity. To identify additional stabilizing mutations in an H5 HA, we subjected a virus library possessing random mutations in the ectodomain of an H5 HA (altered to bind human-type receptors) to three rounds of treatment at 50°C. We isolated several mutants that maintained their human-type receptor-binding preference but acquired an appreciable increase in heat stability and underwent membrane fusion at a lower pH; collectively, these properties may aid H5 virus respiratory droplet transmission in mammals.
We have identified mutations in HA that increase its heat stability and affect the pH that triggers an irreversible conformational change (a prerequisite for virus infectivity). These mutations were identified in the genetic background of an H5 HA protein that was mutated to bind to human cells. The ability to bind to human-type receptors, together with physical stability and an altered pH threshold for HA conformational change, may facilitate avian influenza virus transmission via respiratory droplets in mammals.
H5N1亚型高致病性禽流感病毒继续在亚洲、非洲和中东的家禽中传播。最近，新型重配H5病毒也在北美爆发。尽管感染高致病性H5N1流感病毒的人类数量持续上升，但这些病毒仍无法在人与人之间有效传播。然而，我们和其他人已经鉴定出能够在雪貂中通过呼吸道飞沫传播的H5病毒。病毒血凝素（HA）受体结合域中的两个实验性引入突变赋予了与人型受体的结合能力，但降低了HA的稳定性。HA中的补偿性突变（在雪貂体内病毒复制过程中获得）对于恢复HA稳定性至关重要。HA中的这些稳定突变还影响了HA在宿主内体中不可逆地转变为其融合形式的pH值，这是病毒感染性的关键步骤。为了鉴定H5 HA中的其他稳定突变，我们对一个在H5 HA胞外域具有随机突变（改变后可结合人型受体）的病毒文库在50°C下进行了三轮处理。我们分离出了几个突变体，它们保持了与人型受体结合的偏好，但热稳定性显著提高，并且在较低的pH值下发生膜融合；总体而言，这些特性可能有助于H5病毒在哺乳动物中通过呼吸道飞沫传播。
我们已经鉴定出HA中的突变，这些突变增加了其热稳定性并影响了触发不可逆构象变化（病毒感染性的先决条件）的pH值。这些突变是在一个被突变以结合人类细胞的H5 HA蛋白的遗传背景中鉴定出来的。与人型受体结合的能力，连同物理稳定性和HA构象变化的pH阈值改变，可能促进禽流感病毒在哺乳动物中通过呼吸道飞沫传播。
