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

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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N-Linked Glycan at Residue 523 of Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase Masks a Second Receptor-Binding Site

Journal of Virology ◽

10.1128/jvi.02331-09 ◽

2010 ◽

Vol 84 (6) ◽

pp. 3094-3100 ◽

Cited By ~ 16

Author(s):

Vasiliy P. Mishin ◽

Makiko Watanabe ◽

Garry Taylor ◽

John DeVincenzo ◽

Michael Bose ◽

...

Keyword(s):

Binding Site ◽

Virus Type ◽

Receptor Binding ◽

Critical Role ◽

Glycosylation Site ◽

Parainfluenza Virus ◽

Receptor Binding Site ◽

Human Parainfluenza Virus ◽

Type 3

ABSTRACT The hemagglutinin-neuraminidase (HN) glycoprotein plays a critical role in parainfluenza virus replication. We recently found that in addition to the catalytic binding site, HN of human parainfluenza virus type 1 (hPIV-1) may have a second receptor-binding site covered by an N-linked glycan at residue 173, which is near the region of the second receptor-binding site identified in Newcastle disease virus (NDV) HN (I. A. Alymova, G. Taylor, V. P. Mishin, M. Watanabe, K. G. Murti, K. Boyd, P. Chand, Y. S. Babu, and A. Portner, J. Virol. 82:8400-8410, 2008). Sequence analysis and superposition of the NDV and hPIV-3 HN dimer structures revealed that, similar to what was seen in hPIV-1, the N-linked glycan at residue 523 on hPIV-3 HN may cover a second receptor-binding site. Removal of this N-linked glycosylation site by an Asn-to-Asp substitution at residue 523 (N523D) changed the spectrum of the mutant virus's receptor specificity, delayed its elution from both turkey and chicken red blood cells, reduced mutant sensitivity (by about half) to the selective HN inhibitor BCX 2855 in hemagglutination inhibition tests, and slowed its growth in LLC-MK2 cells. The neuraminidase activity of the mutant and its sensitivity to BCX 2855 in neuraminidase inhibition assays did not change, indicating that the mutation did not affect the virus's catalytic-binding site and that all observed effects were caused by the exposure of the purported second receptor-binding site. Our data are consistent with the idea that, similar to the case for hPIV-1, the N-linked glycan shields a second receptor-binding site on hPIV-3 HN.

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The nucleoproteins of human parainfluenza virus type 1 and Sendai virus share amino acid sequences and antigenic and structural determinants

Journal of General Virology ◽

10.1099/0022-1317-72-4-983 ◽

1991 ◽

Vol 72 (4) ◽

pp. 983-987 ◽

Cited By ~ 34

Author(s):

D. Lyn ◽

D. S. Gill ◽

R. A. Scroggs ◽

A. Portner

Keyword(s):

Amino Acid ◽

Virus Type ◽

Sendai Virus ◽

Amino Acid Sequences ◽

Parainfluenza Virus ◽

Structural Determinants ◽

Human Parainfluenza Virus

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Loss of the N-Linked Glycan at Residue 173 of Human Parainfluenza Virus Type 1 Hemagglutinin-Neuraminidase Exposes a Second Receptor-Binding Site

Journal of Virology ◽

10.1128/jvi.00474-08 ◽

2008 ◽

Vol 82 (17) ◽

pp. 8400-8410 ◽

Cited By ~ 15

Author(s):

Irina V. Alymova ◽

Garry Taylor ◽

Vasiliy P. Mishin ◽

Makiko Watanabe ◽

K. Gopal Murti ◽

...

Keyword(s):

Binding Site ◽

Receptor Binding ◽

Sendai Virus ◽

Receptor Binding Site ◽

Parainfluenza Viruses ◽

Mutant Sequence ◽

Lethal Infection ◽

Human Parainfluenza Virus

ABSTRACT BCX 2798 (4-azido-5-isobutyrylamino-2,3-didehydro-2,3,4,5-tetradeoxy-d-glycero-d-galacto-2-nonulopyranosic acid) effectively inhibited the activities of the hemagglutinin-neuraminidase (HN) of human parainfluenza viruses (hPIV) in vitro and protected mice from lethal infection with a recombinant Sendai virus whose HN was replaced with that of hPIV-1 (rSeV[hPIV-1HN]) (I. V. Alymova, G. Taylor, T. Takimoto, T. H. Lin., P. Chand, Y. S. Babu, C. Li, X. Xiong, and A. Portner, Antimicrob. Agents Chemother. 48:1495-1502, 2004). The ability of BCX 2798 to select drug-resistant variants in vivo was examined. A variant with an Asn-to-Ser mutation at residue 173 (N173S) in HN was recovered from mice after a second passage of rSeV(hPIV-1HN) in the presence of BCX 2798 (10 mg/kg of body weight daily). The N173S mutant remained sensitive to BCX 2798 in neuraminidase inhibition assays but was more than 10,000-fold less sensitive to the compound in hemagglutination inhibition tests than rSeV(hPIV-1HN). Its susceptibility to BCX 2798 in plaque reduction assays was reduced fivefold and did not differ from that of rSeV(hPIV-1HN) in mice. The N173S mutant failed to be efficiently eluted from erythrocytes and released from cells. It demonstrated reduced growth in cell culture and superior growth in mice. The results for gel electrophoresis analysis were consistent with the loss of the N-linked glycan at residue 173 in the mutant. Sequence and structural comparisons revealed that residue 173 on hPIV-1 HN is located close to the region of the second receptor-binding site identified in Newcastle disease virus HN. Our study suggests that the N-linked glycan at residue 173 masks a second receptor-binding site on hPIV-1 HN.

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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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Recombinant Sendai Virus Expressing the G Glycoprotein of Respiratory Syncytial Virus (RSV) Elicits Immune Protection against RSV

Journal of Virology ◽

10.1128/jvi.78.11.6043-6047.2004 ◽

2004 ◽

Vol 78 (11) ◽

pp. 6043-6047 ◽

Cited By ~ 60

Author(s):

Toru Takimoto ◽

Julia L. Hurwitz ◽

Chris Coleclough ◽

Cecilia Prouser ◽

Sateesh Krishnamurthy ◽

...

Keyword(s):

Virus Type ◽

Virus Vaccine ◽

Reverse Genetics ◽

Sendai Virus ◽

Parainfluenza Virus ◽

Effective Vaccine ◽

Live Virus ◽

Live Virus Vaccine ◽

Syncytial Virus

ABSTRACT Although RSV causes serious pediatric respiratory disease, an effective vaccine does not exist. To capture the strengths of a live virus vaccine, we have used the murine parainfluenza virus type 1 (Sendai virus [SV]) as a xenogeneic vector to deliver the G glycoprotein of RSV. It was previously shown (J. L. Hurwitz, K. F. Soike, M. Y. Sangster, A. Portner, R. E. Sealy, D. H. Dawson, and C. Coleclough, Vaccine 15:533-540, 1997) that intranasal SV protected African green monkeys from challenge with the related human parainfluenza virus type 1 (hPIV1), and SV has advanced to clinical trials as a vaccine for hPIV1 (K. S. Slobod, J. L. Shenep, J. Lujan-Zilbermann, K. Allison, B. Brown, R. A. Scroggs, A. Portner, C. Coleclough, and J. L. Hurwitz, Vaccine, in press). Recombinant SV expressing RSV G glycoprotein was prepared by using reverse genetics, and intranasal inoculation of cotton rats elicited RSV-specific antibody and elicited protection from RSV challenge. RSV G-recombinant SV is thus a promising live virus vaccine candidate for RSV.

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