Identification of Stabilizing Mutations in an H5 Hemagglutinin Influenza Virus Protein

Abstract

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.

Conformational Stability of the Hemagglutinin of H5N1 Influenza A Viruses Influences Susceptibility to Broadly Neutralizing Stem Antibodies

“…Previously, we found that an L415I (418 in H3 numbering) change in seasonal H1N1 strains accounted for the resistance of H1 HA pseudoviruses to cross-neutralizing human sera ( 35 ). The same mutation was also reported to increase the stability of H5 HA ( 36 ), though 419I is not naturally present in the H5 sequences that we studied.…”

“…Levels of HA stability differ among different strains. D94N (101 in H3 HA numbering), S217P (221 in H3 HA numbering), M226V (230 in H3 HA numbering), and L419I (418 in H3 HA numbering) have been reported to increase H5 HA stability ( 36 ). Among the H5 HAs that we investigated here, we noted that (i) the HAs of AH/1, TK/1, and OS/102 contain 94N, (ii) the HA of ID/5 contains 94S, (iii) the HA of GX/13 contains 94I, (iv) the HA of HK/156 contains 94N and 217P, and (v) the HA of HK/3088 contains 94N and 217I.…”

Viral factors in influenza pandemic risk assessment

“…Such research could use all of the approaches described above and higher-throughput assays that could be developed with improved technology, for example as described in Box 3. This will include generating mutations not found in known strains in nature to probe for those that could be involved in human adaptation in the future (Hanson et al, 2015; Thyagarajan and Bloom, 2014). The goal would be to identify classes of functionally equivalent substitutions, sufficient individually or in defined combinations to confer a trait of interest when introduced into a defined, avian-adapted genetic background.…”

Hydrogen Bond Variations of Influenza A Viruses During Adaptation in Human

“…2g ). It has been well proved that Q226I/L in the RBP increased the ability to bind a2–6 sialic acid 26 – 30 , which implies its role in inter-molecular interactions is more important than that in intra-molecular interactions in HA. Besides, dual mutations E190D and G225D of receptor binding sites had no effect on H-bonds in H1 HA according to our assessment (Fig.…”