Hemagglutinin Receptor Specificity and Structural Analyses of Respiratory Droplet-Transmissible H5N1 Viruses

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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 humantype 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 humantype receptors) to three rounds of treatment at 50°C. We isolated several mutants that maintained their human-type receptorbinding 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. IMPORTANCEWe 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.

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Identification of Stabilizing Mutations in an H5 Hemagglutinin Influenza Virus Protein

Hanson

Imai

Hatta

et al. 2016

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“…We demonstrated that compensatory, HA-stabilizing mutations may therefore be critical for the selection of avian influenza viruses with human-type receptor specificity that are transmitted among mammals (58,59). Increased HA stability may also affect the pH at which HA undergoes an irreversible conformational change that is critical for HA-mediated fusion of the viral and endosomal membranes.…”

Section: Figmentioning

confidence: 99%

Identification of Amino Acid Changes That May Have Been Critical for the Genesis of A(H7N9) Influenza Viruses

Macken

Kawaoka

2014

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“…In addition, since the G219S substitution increased overall binding to avian and human receptors at the expense of HA stability, putative stability substitutions that would compensate for the decrease in HA stability caused by the G219S substitution were tested. Two substitutions-H103Y and T315I (H5 numbering)-were previously shown to increase the stability of A/H5N1 viruses via different mechanisms (10,11,(18)(19)(20). Using computational modeling (21), a N94K substitution located at the trimer interface of AN1 HA which could potentially have an impact similar to that seen with the H103Y substitution in HA of A/H5N1 was identified.…”

Section: R Ecent Human Infections With Influenza A/h7n9 Viruses Inmentioning

confidence: 99%

Amino Acid Substitutions That Affect Receptor Binding and Stability of the Hemagglutinin of Influenza A/H7N9 Virus

Schrauwen

Richard

Burke

et al. 2016

bReceptor-binding preference and stability of hemagglutinin have been implicated as crucial determinants of airborne transmission of influenza viruses. Here, amino acid substitutions previously identified to affect these traits were tested in the context of an A/H7N9 virus. Some combinations of substitutions, most notably G219S and K58I, resulted in relatively high affinity for ␣2,6-linked sialic acid receptor and acid and temperature stability. Thus, the hemagglutinin of the A/H7N9 virus may adopt traits associated with airborne transmission. R ecent human infections with influenza A/H7N9 viruses in China have raised concern of a pandemic threat emerging from avian reservoirs. As of November 2015, 681 laboratory-confirmed human cases of infection with A/H7N9 have been reported, including 275 fatalities (1). To date, no sustained humanto-human transmission of A/H7N9 viruses has been detected, although some of these viruses possess known mammalian adaption markers in the hemagglutinin (HA) and in the polymerase genes (2). Most A/H7N9 virus isolates can bind both avian and human influenza virus receptors (3), as the result of a leucine at amino acid position 217 (H7 numbering, corresponding to position 226 in H3 numbering [4]). Studies in ferrets have demonstrated that the airborne transmissibility of A/H7N9 viruses was more efficient than that of other avian influenza viruses but less efficient than that of pandemic and seasonal human influenza viruses (2, 5-9). Phenotypic traits of HA such as human receptor binding preference and increased acid or temperature stability have been shown to be critical for airborne transmission of avian A/H5N1 viruses between ferrets (10, 11). Here, we describe several amino acid substitutions that affect both of these properties of the HA of A/H7N9 A/Anhui/1/13 virus (AN1).The impact of two substitutions in the receptor-binding site (RBS), L217Q and G219S, on binding affinity and HA stability was investigated first. The L217Q substitution was previously detected in a minor virus population that emerged in ferrets upon inoculation with the AN1 virus isolate (6). The G219S substitution has been previously shown to increase the binding of A/H7N9 viruses to human trachea, alveolar epithelium, and synthetic glycans (12, 13). Recombinant viruses containing 7 gene segments of the mouse-adapted virus A/Puerto-Rico/8/34 and HA of AN1 with or without substitutions of interest were produced in 293T cells and propagated in Madin-Darby canine kidney (MDCK) cells by reverse genetics according to standard procedures (14). Properties of binding of recombinant viruses to ␣2,3-and ␣2,6-linked sialic acids (SA), the avian and human receptors, respectively, were assessed using a solid-phase binding assay (adjusted from the method described in reference 11). The latter analysis confirmed that the wild-type AN1 (AN1 WT ) virus binds to both avian and human influenza virus receptors (Fig. 1A and F), as shown previously (6, 15). AN1 L217Q virus predominantly bound to avian receptors but showed residual b...