Compensatory Evolution of Net-Charge in Influenza A Virus Hemagglutinin

The propagation of influenza A virus depends on the balance between the activities of hemagglutinin (HA) for binding to host cells and neuraminidase (NA) for releasing from infected cells (HA-NA balance). Since the host cell membrane and the sialic acid receptor are negatively charged, the amino acid substitutions increasing (charge+) and decreasing (charge−) the positive charge of HA subunit 1 (HA1) enhance and reduce, respectively, the binding avidity and affinity. The positive charge of HA1 in human influenza A virus bearing subtype H3N2 (A/H3N2 virus) was observed to have increased during evolution, but the evolutionary mechanism for this observation was unclear because this may disrupt the HA-NA balance. Here we show, from the phylogenetic analysis of HA for human A/H3N2 and A/H1N1 viruses, that the relative frequencies of charge+ and charge− substitutions were elevated on the branches where the number of N-glycosylation sites (NGS) increased and decreased, respectively, compared to those where the number of NGS did not change. On the latter branches, the net-charge of HA1 appeared to have been largely maintained to preserve its structure and function. Since the charge+ and charge− substitutions in HA1 have opposite effects to the gain and loss of NGS on the binding and release of the virus, the net-charge of HA1 may have evolved to compensate for the effect of the gain and loss of NGS, probably through changing the avidity. Apparently, the relative frequency of charge− substitutions in HA1 of A/H3N2 virus was elevated after the introduction of oseltamivir, and that of charge+ substitutions in HA1 of A/H1N1 virus was elevated after the spread of oseltamivir resistance. These observations may also be explained by the compensatory effect of the net-charge in HA1 on the NA activity for keeping the HA-NA balance.甲型流感病毒的传播依赖于血凝素（hemagglutinin, HA）结合宿主细胞与神经氨酸酶（neuraminidase, NA）从感染细胞释放的活性平衡（HA-NA平衡）。由于宿主细胞膜与唾液酸受体均带负电荷，可增加HA1亚基正电荷的氨基酸替换（charge+）会增强病毒的结合亲合力与亲和力，而降低该正电荷的替换（charge−）则会相应减弱结合活性。此前研究观察到，携带H3N2亚型的人类甲型流感病毒（A/H3N2病毒）的HA1正电荷在进化过程中有所升高，但该现象的进化机制尚不明确——因为这一变化可能打破HA-NA平衡。本文通过对人类A/H3N2与A/H1N1病毒的HA蛋白开展系统发育分析，发现相较于N-糖基化位点（NGS）数量未发生改变的进化分支，charge+与charge−替换的相对频率分别在NGS数量增加和减少的分支中显著升高。在NGS数量未改变的分支上，HA1的净电荷基本保持稳定，以维持其结构与功能。由于HA1中的charge+与charge−替换，对病毒结合与释放的影响恰好与NGS的获得与丢失相反，因此HA1的净电荷可能通过改变病毒结合的亲合力，进化出补偿NGS获得与丢失所带来影响的能力。此外，A/H3N2病毒HA1的charge−替换相对频率在奥司他韦引入后显著升高，而A/H1N1病毒HA1的charge+替换相对频率在奥司他韦耐药毒株传播后有所上升。上述观测结果也可通过HA1净电荷对NA活性的补偿效应来解释，以维持HA-NA平衡。