RESUMEN
Influenza A virus (IAV) and influenza B virus (IBV) cause yearly epidemics with significant morbidity and mortality. When zoonotic IAVs enter the human population, the viral hemagglutinin (HA) requires adaptation to achieve sustained virus transmission. In contrast, IBV has been circulating in humans, its only host, for a long period of time. Whether this entailed adaptation of IBV HA to the human airways is unknown. To address this question, we compared two seasonal IAVs (A/H1N1 and A/H3N2) and two IBVs (B/Victoria and B/Yamagata lineages) with regard to host-dependent activity of HA as the mediator of membrane fusion during viral entry. We first investigated proteolytic activation of HA by covering all type II transmembrane serine protease (TTSP) and kallikrein enzymes, many of which proved to be present in human respiratory epithelium. The IBV HA0 precursor is cleaved by a broader panel of TTSPs and activated with much higher efficiency than IAV HA0. Accordingly, knockdown of a single protease, TMPRSS2, abrogated spread of IAV but not IBV in human respiratory epithelial cells. Second, the HA fusion pH values proved similar for IBV and human-adapted IAVs (with one exception being the HA of 1918 IAV). Third, IBV HA exhibited higher expression at 33°C, a temperature required for membrane fusion by B/Victoria HA. This indicates pronounced adaptation of IBV HA to the mildly acidic pH and cooler temperature of human upper airways. These distinct and intrinsic features of IBV HA are compatible with extensive host adaptation during prolonged circulation of this respiratory virus in the human population.IMPORTANCE Influenza epidemics are caused by influenza A and influenza B viruses (IAV and IBV, respectively). IBV causes substantial disease; however, it is far less studied than IAV. While IAV originates from animal reservoirs, IBV circulates in humans only. Virus spread requires that the viral hemagglutinin (HA) is active and sufficiently stable in human airways. We resolve here how these mechanisms differ between IBV and IAV. Whereas human IAVs rely on one particular protease for HA activation, this is not the case for IBV. Superior activation of IBV by several proteases should enhance shedding of infectious particles. IBV HA exhibits acid stability and a preference for 33°C, indicating pronounced adaptation to the human upper airways, where the pH is mildly acidic and a cooler temperature exists. These adaptive features are rationalized by the long existence of IBV in humans and may have broader relevance for understanding the biology and evolution of respiratory viruses.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Virus de la Influenza B/genética , Gripe Humana/virología , Pulmón/virología , Replicación Viral/genética , Línea Celular , Células Epiteliales/patología , Células Epiteliales/virología , Regulación de la Expresión Génica , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Interacciones Huésped-Patógeno/genética , Humanos , Concentración de Iones de Hidrógeno , Subtipo H1N1 del Virus de la Influenza A/metabolismo , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H3N2 del Virus de la Influenza A/metabolismo , Subtipo H3N2 del Virus de la Influenza A/patogenicidad , Virus de la Influenza B/metabolismo , Virus de la Influenza B/patogenicidad , Gripe Humana/patología , Calicreínas/clasificación , Calicreínas/genética , Calicreínas/metabolismo , Pulmón/patología , Fusión de Membrana , Proteínas de la Membrana/clasificación , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteolisis , Mucosa Respiratoria/patología , Mucosa Respiratoria/virología , Serina Endopeptidasas/deficiencia , Serina Endopeptidasas/genética , Serina Proteasas/clasificación , Serina Proteasas/genética , Serina Proteasas/metabolismo , Especificidad de la Especie , Temperatura , Internalización del VirusRESUMEN
The possible resistance of influenza virus against existing antiviral drugs calls for new therapeutic concepts. One appealing strategy is to inhibit virus entry, in particular at the stage of internalization. This requires a better understanding of virus-host interactions during the entry process, including the role of receptor tyrosine kinases (RTKs). To search for cellular targets, we evaluated a panel of 276 protein kinase inhibitors in a multicycle antiviral assay in Madin-Darby canine kidney cells. The RTK inhibitor Ki8751 displayed robust anti-influenza A and B virus activity and was selected for mechanistic investigations. Ki8751 efficiently disrupted the endocytic process of influenza virus in different cell lines carrying platelet-derived growth factor receptor ß (PDGFRß), an RTK that is known to act at GM3 ganglioside-positive lipid rafts. The more efficient virus entry in CHO-K1 cells compared to the wild-type ancestor (CHO-wt) cells indicated a positive effect of GM3, which is abundant in CHO-K1 but not in CHO-wt cells. Entering virus localized to GM3-positive lipid rafts and the PDGFRß-containing endosomal compartment. PDGFRß/GM3-dependent virus internalization involved PDGFRß phosphorylation, which was potently inhibited by Ki8751, and desialylation of activated PDGFRß by the viral neuraminidase. Virus uptake coincided with strong activation of the Raf/MEK/Erk cascade, but not of PI3K/Akt or phospholipase C-γ. We conclude that influenza virus efficiently hijacks the GM3-enhanced PDGFRß signalling pathway for cell penetration, providing an opportunity for host cell-targeting antiviral intervention.