scholarly journals Amino acid substitutions in the H5N1 avian influenza haemagglutinin alter pH of fusion and receptor binding to promote a highly pathogenic phenotype in chickens

2021 ◽  
Vol 102 (11) ◽  
Author(s):  
Joshua E. Sealy ◽  
Wendy A. Howard ◽  
Eleonora Molesti ◽  
Munir Iqbal ◽  
Nigel J. Temperton ◽  
...  
Keyword(s):  
Amino Acid ◽  
Avian Influenza ◽  
Binding Site ◽  
Receptor Binding ◽  
Viral Fitness ◽  
Amino Acid Substitutions ◽  
Receptor Binding Site ◽  
Highly Pathogenic ◽  
Ha Gene ◽  
H5n1 Avian Influenza

Highly pathogenic H5N1 avian influenza viruses cause devastating outbreaks in farmed poultry with serious consequences for animal welfare and economic losses. Zoonotic infection of humans through close contact with H5N1 infected birds is often severe and fatal. England experienced an outbreak of H5N1 in turkeys in 1991 that led to thousands of farmed bird mortalities. Isolation of clonal populations of one such virus from this outbreak uncovered amino acid differences in the virus haemagglutinin (HA) gene whereby the different genotypes could be associated with distinct pathogenic outcomes in chickens; both low pathogenic (LP) and high pathogenic (HP) phenotypes could be observed despite all containing a multi-basic cleavage site (MBCS) in the HA gene. Using reverse genetics, three amino acid substitutions in HA were examined for their ability to affect pathogenesis in the chicken. Restoration of amino acid polymorphisms close to the receptor binding site that are commonly found in H5 viruses only partially improved viral fitness in vitro and in vivo. A third novel substitution in the fusion peptide, HA2G4R, enabled the HP phenotype. HA2G4R decreased the pH stability of HA and increased the pH of HA fusion. The substitutions close to the receptor binding site optimised receptor binding while modulating the pH of HA fusion. Importantly, this study revealed pathogenic determinants beyond the MBCS.

scholarly journals Phenotypic Effects of Substitutions within the Receptor Binding Site of Highly Pathogenic Avian Influenza H5N1 Virus Observed during Human Infection

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
Dirk Eggink ◽  
Monique Spronken ◽  
Roosmarijn van der Woude ◽  
Jocynthe Buzink ◽  
Frederik Broszeit ◽  
...  
Keyword(s):  
Amino Acid ◽  
Avian Influenza ◽  
Binding Site ◽  
Receptor Binding ◽  
Human Infection ◽  
Influenza Viruses ◽  
Receptor Specificity ◽  
Receptor Binding Site ◽  
Human Type ◽  
Human Infections

ABSTRACT Highly pathogenic avian influenza (HPAI) viruses are enzootic in wild birds and poultry and continue to cause human infections with high mortality. To date, more than 850 confirmed human cases of H5N1 virus infection have been reported, of which ∼60% were fatal. Global concern persists that these or similar avian influenza viruses will evolve into viruses that can transmit efficiently between humans, causing a severe influenza pandemic. It was shown previously that a change in receptor specificity is a hallmark for adaptation to humans and evolution toward a transmittable virus. Substantial genetic diversity was detected within the receptor binding site of hemagglutinin of HPAI A/H5N1 viruses, evolved during human infection, as detected by next-generation sequencing. Here, we investigated the functional impact of substitutions that were detected during these human infections. Upon rescue of 21 mutant viruses, most substitutions in the receptor binding site (RBS) resulted in viable virus, but virus replication, entry, and stability were often impeded. None of the tested substitutions individually resulted in a clear switch in receptor preference as measured with modified red blood cells and glycan arrays. Although several combinations of the substitutions can lead to human-type receptor specificity, accumulation of multiple amino acid substitutions within a single hemagglutinin during human infection is rare, thus reducing the risk of virus adaptation to humans. IMPORTANCE H5 viruses continue to be a threat for public health. Because these viruses are immunologically novel to humans, they could spark a pandemic when adapted to transmit between humans. Avian influenza viruses need several adaptive mutations to bind to human-type receptors, increase hemagglutinin (HA) stability, and replicate in human cells. However, knowledge on adaptive mutations during human infections is limited. A previous study showed substantial diversity within the receptor binding site of H5N1 during human infection. We therefore analyzed the observed amino acid changes phenotypically in a diverse set of assays, including virus replication, stability, and receptor specificity. None of the tested substitutions resulted in a clear step toward a human-adapted virus capable of aerosol transmission. It is notable that acquiring human-type receptor specificity needs multiple amino acid mutations, and that variability at key position 226 is not tolerated, reducing the risk of them being acquired naturally.

scholarly journals Correction for Koel et al., Antigenic Variation of Clade 2.1 H5N1 Virus Is Determined by a Few Amino Acid Substitutions Immediately Adjacent to the Receptor Binding Site

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Björn F. Koel ◽  
Stefan van der Vliet ◽  
David F. Burke ◽  
Theo M. Bestebroer ◽  
Eny E. Bharoto ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Receptor Binding ◽  
H5n1 Virus ◽  
Antigenic Variation ◽  
Amino Acid Substitutions ◽  
Receptor Binding Site

scholarly journals Substitution Arg140Gly in Hemagglutinin Reduced the Virulence of Highly Pathogenic Avian Influenza Virus H7N1

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1584
Author(s):  
Anastasia Treshchalina ◽  
Yulia Postnikova ◽  
Elizaveta Boravleva ◽  
Alexandra Gambaryan ◽  
Alla Belyakova ◽  
...  
Keyword(s):  
Amino Acid ◽  
Avian Influenza ◽  
Receptor Binding ◽  
Genome Stability ◽  
Laboratory Strain ◽  
Influenza Viruses ◽  
Single Amino Acid ◽  
Head Part ◽  
Receptor Binding Site ◽  
Highly Pathogenic

The H7 subtype of avian influenza viruses (AIV) stands out among other AIV. The H7 viruses circulate in ducks, poultry and equines and have repeatedly caused outbreaks of disease in humans. The laboratory strain A/chicken/Rostock/R0p/1934 (H7N1) (R0p), which was previously derived from the highly pathogenic strain A/FPV/Rostock/1934 (H7N1), was studied in this work to ascertain its biological property, genome stability and virulent changing mechanism. Several virus variants were obtained by serial passages in the chicken lungs. After 10 passages of this virus through the chicken lungs we obtained a much more pathogenic variant than the starting R0p. The study of intermediate passages showed a sharp increase in pathogenicity between the fifth and sixth passage. By cloning these variants, a pair of strains (R5p and R6p) was obtained, and the complete genomes of these strains were sequenced. Single amino acid substitution was revealed, namely reversion Gly140Arg in HA1. This amino acid is located at the head part of the hemagglutinin, adjacent to the receptor-binding site. In addition to the increased pathogenicity in chicken and mice, R6p differs from R5p in the shape of foci in cell culture and an increased affinity for a negatively charged receptor analogue, while maintaining a pattern of receptor-binding specificity and the pH of conformational change of HA.

scholarly journals Single-Point Mutations within the Coxsackie B Virus Receptor-Binding Site Promote Resistance against Soluble Virus Receptor Traps

2020 ◽  
Vol 94 (19) ◽  
Author(s):  
Sandra Pinkert ◽  
Anja Kopp ◽  
Vanessa Brückner ◽  
Henry Fechner ◽  
Antje Beling
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Receptor Binding ◽  
Hela Cells ◽  
Viral Fitness ◽  
Single Amino Acid ◽  
Receptor Binding Site ◽  
Virus Receptor ◽  
Development Of Resistance ◽  
Coxsackie B

ABSTRACT Coxsackie B viruses (CVB) cause a wide spectrum of diseases, ranging from mild respiratory syndromes and hand, foot, and mouth disease to life-threatening conditions, such as pancreatitis, myocarditis, and encephalitis. Previously, we and others found that the soluble virus receptor trap sCAR-Fc strongly attenuates CVB3 infection in mice. In this study, we investigated whether treatment with sCAR-Fc results in development of resistance by CVB3. Two CVB3 strains (CVB3-H3 and CVB3 Nancy) were passaged in HeLa cells in the presence of sCAR-Fc. The CVB3-H3 strain did not develop resistance, whereas two populations of CVB3 Nancy mutants emerged, one with complete (CVB3M) and one with partial (CVB3K) resistance. DNA sequence alignment of the resistant virus variant CVB3M with CVB3 Nancy revealed an amino acid exchange from Asn(N) to Ser(S) at position 139 of the CVB3 capsid protein VP2 (N2139S), an amino acid predicted to be involved in the virus’s interaction with its cognate receptor CAR. Insertion of the N2139S mutation into CVB3-H3 by site-directed mutagenesis promoted resistance of the engineered CVB3-H3N2139S to sCAR-Fc. Interestingly, development of resistance by CVB3-H3N2139S and the exemplarily investigated CVB3M-clone 2 (CVB3M2) against soluble CAR did not compromise the use of cellular CAR for viral infection. Infection of HeLa cells showed that sCAR-Fc resistance, however, negatively affected both virus stability and viral replication compared to that of the parental strains. These data demonstrate that during sCAR-Fc exposure, CVB3 can develop resistance against sCAR-Fc by single-amino-acid exchanges within the virus-receptor binding site, which, however, come at the expense of viral fitness. IMPORTANCE The emergence of resistant viruses is one of the most frequent obstacles preventing successful therapy of viral infections, representing a significant threat to human health. We investigated the emergence of resistant viruses during treatment with sCAR-Fc, a well-studied, highly effective antiviral molecule against CVB infections. Our data show the molecular aspects of resistant CVB3 mutants that arise during repetitive sCAR-Fc usage. However, drug resistance comes at the price of lower viral fitness. These results extend our knowledge of the development of resistance by coxsackieviruses and indicate potential limitations of antiviral therapy using soluble receptor molecules.

Nicotinic receptor binding site probed with unnatural amino acid incorporation in intact cells

Science ◽  
1995 ◽  
Vol 268 (5209) ◽  
pp. 439-442 ◽  
Author(s):  
M. Nowak ◽  
P. Kearney ◽  
Sampson ◽  
M. Saks ◽  
C. Labarca ◽  
...  
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Receptor Binding ◽  
Nicotinic Receptor ◽  
Unnatural Amino Acid ◽  
Amino Acid Incorporation ◽  
Receptor Binding Site ◽  
Intact Cells ◽  
Acid Incorporation

scholarly journals Amino acid substitutions in human growth hormone affect coiled-coil content and receptor binding

2021 ◽  
Author(s):  
Andrei Rajkovic ◽  
Sandesh Kanchugal ◽  
Eldar Abdurakhmanov ◽  
Rebecca Howard ◽  
Astrid Gräslund ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Amino Acid ◽  
Binding Site ◽  
Human Growth Hormone ◽  
Receptor Binding ◽  
Coiled Coil ◽  
Human Growth ◽  
Zinc Binding ◽  
Amino Acid Substitutions ◽  
Great Relevance

The interaction between human Growth Hormone (hGH) and hGH Receptor (hGHR) has great relevance to human diseases such as acromegaly and cancer. HGH has been extensively engineered by other workers to improve binding and other properties. We used a computational screen to select substitutions at single hGH positions within the hGHR-binding site. We find that, while many successfully slow down dissociation of the hGH-hGHR complex once bound, they also slow down the association of hGH to hGHR. We are particularly interested in E174 which belongs to the hGH zinc-binding triad, and which spans coiled-coil helices and obeys the coiled-coil heptad pattern. Surprisingly, substituting E174 with A leads to substantial increase in an experimental measure of coiled-coil content. E174A is known to increase affinity of hGH against hGHR; here we show that this is simply because the off-rate is slowed down more than the on-rate, in line with what has been found for other affinity-improving mutations. For E174Y (and mutations at other sites) the slowdown in on-rate was greater, leading to decreased affinity. The results point to a link between coiled-coiling, zinc binding, and hGHR-binding affinity in hGH, and also suggest rules for choosing affinity-increasing substitutions.

scholarly journals Novel Mutations Evading Avian Immunity around the Receptor Binding Site of the Clade 2.3.2.1c Hemagglutinin Gene Reduce Viral Thermostability and Mammalian Pathogenicity

Viruses ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 923 ◽  
Author(s):  
An ◽  
Lee ◽  
Hong ◽  
Song ◽  
Kim ◽  
...  
Keyword(s):  
Binding Site ◽  
Receptor Binding ◽  
Mammalian Cells ◽  
Influenza A ◽  
Receptor Binding Site ◽  
Receptor Affinity ◽  
Replication Efficiency ◽  
Adaptive Mutations ◽  
H5n1 Avian Influenza ◽  
Clade 2.3.2.1C

Abstract: Since 2007, highly pathogenic clade 2.3.2 H5N1 avian influenza A (A(H5N1)) viruses have evolved to clade 2.3.2.1a, b, and c; currently only 2.3.2.1c A(H5N1) viruses circulate in wild birds and poultry. During antigenic evolution, clade 2.3.2.1a and c A(H5N1) viruses acquired both S144N and V223I mutations around the receptor binding site of hemagglutinin (HA), with S144N generating an N-glycosylation sequon. We introduced single or combined reverse mutations, N144S and/or I223V, into the HA gene of the clade 2.3.2.1c A(H5N1) virus and generated PR8-derived, 2 + 6 recombinant A(H5N1) viruses. When we compared replication efficiency in embryonated chicken eggs, mammalian cells, and mice, the recombinant virus containing both N144S and I223V mutations showed increased replication efficiency in avian and mammalian hosts and pathogenicity in mice. The N144S mutation significantly decreased avian receptor affinity and egg white inhibition, but not all mutations increased mammalian receptor affinity. Interestingly, the combined reverse mutations dramatically increased the thermostability of HA. Therefore, the adaptive mutations possibly acquired to evade avian immunity may decrease viral thermostability as well as mammalian pathogenicity.

scholarly journals Avian Influenza A Viruses Differ from Human Viruses by Recognition of Sialyloligosaccharides and Gangliosides and by a Higher Conservation of the HA Receptor-Binding Site

Virology ◽  
1997 ◽  
Vol 233 (1) ◽  
pp. 224-234 ◽  
Author(s):  
M.N. Matrosovich ◽  
A.S. Gambaryan ◽  
S. Teneberg ◽  
V.E. Piskarev ◽  
S.S. Yamnikova ◽  
...  
Keyword(s):  
Avian Influenza ◽  
Binding Site ◽  
Receptor Binding ◽  
Influenza A ◽  
Influenza A Viruses ◽  
Receptor Binding Site ◽  
Human Viruses

scholarly journals Genetic diversification of H5N1 highly pathogenic avian influenza A virus during replication in wild ducks

2011 ◽  
Vol 92 (9) ◽  
pp. 2105-2110 ◽  
Author(s):  
Yohei Watanabe ◽  
Madiha S. Ibrahim ◽  
Hany F. Ellakany ◽  
Hatem S. Abd El-Hamid ◽  
Kazuyoshi Ikuta
Keyword(s):  
Amino Acid ◽  
Avian Influenza ◽  
Influenza A Virus ◽  
Influenza A ◽  
Highly Pathogenic Avian Influenza ◽  
Amino Acid Substitutions ◽  
Highly Pathogenic ◽  
Pathogenic Avian Influenza ◽  
Novel Variants ◽  
Avian Influenza A Virus

Highly pathogenic avian influenza A virus subtype H5N1 can potentially generate novel variants during replication of infected hosts. To determine which H5N1 variants predominate in wild birds, we determined the sequences of RT-PCR amplified viral genes from several organs of infected chickens and ducks from Egypt, where H5N1 outbreaks in birds are endemic. Comparison of the sequences in viruses from trachea, lung, brain and liver revealed diversification with different amino acid substitutions in different ducks, but no diversification in chickens. These specific amino acid substitutions were rare among viruses currently circulating in Egypt. In addition, the H5N1 variants showed distinct growth kinetics in duck, canine and human cells. Our findings suggested that ducks can generate H5N1 variants with novel amino acid substitutions that might serve as aetiological agents for new influenza virus outbreaks and epidemics.

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