HA-Dependent Tropism of H5N1 and H7N9 Influenza Viruses to Human Endothelial Cells Is Determined by Reduced Stability of the HA, Which Allows the Virus To Cope with Inefficient Endosomal Acidification and Constitutively Expressed IFITM3

Abstract

Previous studies revealed that certain avian influenza A viruses (IAVs), including zoonotic H5N1 and H7N9 IAVs, infect cultured human lung microvascular endothelial cells (HULEC) more efficiently than other IAVs and that tropism to HULEC is determined by viral hemagglutinin (HA). To characterize mechanisms of HA-mediated endotheliotropism, we used 2:6 recombinant IAVs harboring HAs from distinctive avian and human viruses and found that efficient infection of HULEC correlated with low conformational stability of the HA. We next studied effects on viral infectivity of single-point amino acid substitutions in the HA of 2:6 recombinant virus A/Vietnam/1203/2004-PR8 (H5N1). Substitutions H8Q, H103Y, T315I, and K582I (K58I in the HA2 subunit), which increased stability of the HA, markedly reduced viral infectivity for HULEC, whereas substitutions K189N and K218Q, which altered typical H5N1 virus-like receptor specificity and reduced binding avidity of the HA, led to only marginal reduction of infectivity. None of these substitutions affected virus infection in MDCK cells. We confirmed the previous observation of elevated basal expression of IFITM3 protein in HULEC and found that endosomal acidification is less efficient in HULEC than in MDCK cells. In accord with these findings, counteraction of IFITM3-mediated restriction by amphotericin B and reduction of endosomal pH by moderate acidification of the extracellular medium enhanced infectivity of viruses with stable HA for HULEC without significant effect on infectivity for MDCK cells. Collectively, our results indicate that relatively high pH optimum of fusion of the HA of zoonotic H5N1 and H7N9 IAVs allows them to overcome antiviral effects of inefficient endosomal acidification and IFITM3 in human endothelial cells. IMPORTANCE Receptor specificity of the HA of IAVs is known to be a critical determinant of viral cell tropism. Here, we show that fusion properties of the HA may also play a key role in the tropism. Thus, we demonstrate that IAVs having a relatively low pH optimum of fusion cannot efficiently infect human endothelial cells owing to their relatively high endosomal pH and increased expression of fusion-inhibiting IFITM3 protein. These restrictions can be overcome by IAVs with elevated pH of fusion, such as zoonotic H5N1 and H7N9. Our results illustrate that the infectivity of IAVs depends on an interplay between HA conformational stability, endosomal acidification and IFITM3 expression in target cells, and the extracellular pH. Given significant variation of levels of HA stability among animal, human, and zoonotic IAVs, our findings prompt further studies on the fusion-dependent tropism of IAVs to different cell types in humans and its role in viral host range and pathogenicity.

Topology, Antiviral Functional Residues and Mechanism of IFITM1

“…In addition, our work provided new insights into the antiviral mechanism of IFITM1 related to inhibiting organelles acidification. The previous research also showed that the endosomes in HULEC cells with a high basal level of IFITM3 were less acidic than in MDCKs [35]. The mechanism may be applicable to explain how IFITM1 restricts other viruses entering cells by clathrin-mediated endocytosis.…”

Current Progress on Host Antiviral Factor IFITMs

“…Embryonic fibroblasts (MEFs) from ifitmDel -/mice are more sensitive to IAV than MEFs from wild-type mice, and the anti-IAV effect of type I and II IFN is weakened in ifitmDel -/mice (26). Moreover, the infection of pseudo-retroviruses with different hemagglutinin proteins (H1, H3, H5, and H7) as outer membranes can be effectively inhibited by IFITM1, 2, and 3 (7,24,(27)(28)(29). Studies have shown that arboviruses, including DENV and WNV, have similar sensitivity to IFITM-mediated restriction (30).…”

Endothelial Cells in Emerging Viral Infections

“…Influenzavirus HA belongs to the category of fusion proteins triggered by exposure to low pH alone. Interestingly, human lung microvascular endothelial cells show less efficient endosomal acidification than human epithelial cells ( 159 ), and this renders endothelial cells less permissive to infection by seasonal influenzavirus and LPAIV strains. In addition, endothelial cells express high constitutive levels of antiviral interferon-induced transmembrane protein (IFITM) 3 in endosomal and lysosomal compartments.…”

“…This protein arrests endosomal fusion of seasonal human influenzavirus in endothelial cells at the hemifusion stage ( 160 ). Overcoming these obstacles, HPAIV strains have a less stable conformation of the HA molecule that allows fusion to take place at a higher pH ( 159 ) and allows infection in early endosomal compartments. The ability to efficiently fuse at a higher pH probably also mediates the relative resistance of HPAIV to the antiviral action of IFITM3 ( 161 ).…”