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

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.

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

Journal of General Virology ◽

10.1099/jgv.0.001672 ◽

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.

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

Science ◽

10.1126/science.7716551 ◽

1995 ◽

Vol 268 (5209) ◽

pp. 439-442 ◽

Cited By ~ 157

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

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

Journal of Virology ◽

10.1128/jvi.00195-20 ◽

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.

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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 ◽

10.1128/mbio.01645-14 ◽

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

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A novel amino acid substitution at the receptor-binding site on the hemagglutinin of H3N2 influenza A viruses isolated from 6 cases with acute encephalopathy during the 1997–1998 season in Tokyo

Archives of Virology ◽

10.1007/s007050050491 ◽

1999 ◽

Vol 144 (1) ◽

pp. 147-155 ◽

Cited By ~ 16

Author(s):

S.-I. Mori ◽

M. Nagashima ◽

Y. Sasaki ◽

K. Mori ◽

Y. Tabei ◽

...

Keyword(s):

Amino Acid ◽

Binding Site ◽

Receptor Binding ◽

Amino Acid Substitution ◽

Influenza A ◽

Acute Encephalopathy ◽

Influenza A Viruses ◽

Receptor Binding Site ◽

H3n2 Influenza

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

Viruses ◽

10.3390/v13081584 ◽

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.

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CryoEM Structure of an Influenza Virus Receptor-Binding Site Antibody–Antigen Interface

Journal of Molecular Biology ◽

10.1016/j.jmb.2017.05.011 ◽

2017 ◽

Vol 429 (12) ◽

pp. 1829-1839 ◽

Cited By ~ 15

Author(s):

Yuhang Liu ◽

Junhua Pan ◽

Simon Jenni ◽

Donald D. Raymond ◽

Tim Caradonna ◽

...

Keyword(s):

Influenza Virus ◽

Binding Site ◽

Receptor Binding ◽

Receptor Binding Site ◽

Virus Receptor

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Nicotinic Receptor Binding Site Probed with Unnatural Amino Acid Incorporation in Intact Cells.

10.21236/ada299991 ◽

1995 ◽

Author(s):

Henry A. Lester

Keyword(s):

Amino Acid ◽

Binding Site ◽

Receptor Binding ◽

Nicotinic Receptor ◽

Unnatural Amino Acid ◽

Amino Acid Incorporation ◽

Receptor Binding Site ◽

Intact Cells ◽

Acid Incorporation

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Mutation at Residue 523 Creates a Second Receptor Binding Site on Human Parainfluenza Virus Type 1 Hemagglutinin-Neuraminidase Protein

Journal of Virology ◽

10.1128/jvi.00969-06 ◽

2006 ◽

Vol 80 (18) ◽

pp. 9009-9016 ◽

Cited By ~ 18

Author(s):

Tatiana Bousse ◽

Toru Takimoto

Keyword(s):

Amino Acid ◽

Binding Site ◽

Receptor Binding ◽

Reverse Genetics ◽

Sendai Virus ◽

Parainfluenza Virus ◽

Amino Acid Difference ◽

Receptor Binding Site ◽

Human Parainfluenza Virus

ABSTRACT The paramyxovirus hemagglutinin-neuraminidase (HN) is a multifunctional protein mediating hemagglutination (HA), neuraminidase (NA), and fusion promotion activities. It has been a matter of debate whether HN contains combined or separate sites for HA and NA activities. To clear the issue, we determined the presence of the second binding site on human parainfluenza virus (hPIV) type 1, 2, and 3 and Sendai virus (SeV) HN proteins. Results of virus elution from erythrocytes at an elevated temperature and HA inhibition by NA inhibitor BCX-2798 suggest that all hPIVs bind to the receptor only through the NA catalytic site, while SeV HN has an additional receptor binding site. Comparison of SeV and hPIV1 HN sequences revealed two amino acid differences at residues 521 and 523 in the region close to the second binding site identified in Newcastle disease virus HN. We mutated hPIV1 HN at position 523 from Asn to the residue of SeV HN, Asp, and rescued a recombinant SeV that carries the mutated hPIV1 HN by a reverse genetics system. The hPIV1 HN with Asp at position 523 hemagglutinated in the presence of BCX-2798, suggesting that the amino acid difference at position 523 is critical for the formation of a second binding site. Creation of the second binding site on hPIV1 HN, however, did not significantly affect the growth or fusion activity of the recombinant virus. Our study indicates that the presence and requirement of a second binding site vary among paramyxoviruses.

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