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

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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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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Efficacy of Novel Hemagglutinin-Neuraminidase Inhibitors BCX 2798 and BCX 2855 against Human Parainfluenza Viruses In Vitro and In Vivo

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.5.1495-1502.2004 ◽

2004 ◽

Vol 48 (5) ◽

pp. 1495-1502 ◽

Cited By ~ 56

Author(s):

Irina V. Alymova ◽

Garry Taylor ◽

Toru Takimoto ◽

Tsu-Hsing Lin ◽

Pooran Chand ◽

...

Keyword(s):

Sendai Virus ◽

Three Dimensional ◽

Parainfluenza Virus ◽

Dimensional Structure ◽

The Novel ◽

Virus Infections ◽

Parainfluenza Viruses ◽

Human Parainfluenza Viruses

ABSTRACT Human parainfluenza viruses are important respiratory tract pathogens, especially of children. However, no vaccines or specific therapies for infections caused by these viruses are currently available. In the present study we characterized the efficacy of the novel parainfluenza virus inhibitors BCX 2798 and BCX 2855, which were designed based on the three-dimensional structure of the hemagglutinin-neuraminidase (HN) protein. The compounds were highly effective in inhibiting hemagglutinin (HA) and neuraminidase (NA) activities and the growth of hPIV-1, hPIV-2, and hPIV-3 in LLC-MK2 cells. The concentrations required to reduce the activity to 50% of that of a control ranged from 0.1 to 6.0 μM in HA inhibition assays and from 0.02 to 20 μM in NA inhibition assays. The concentrations required to inhibit virus replication to 50% of the level of the control ranged from 0.7 to 11.5 μM. BCX 2798 and BCX 2855 were inactive against influenza virus HA and NA and bacterial NA. In mice infected with a recombinant Sendai virus whose HN gene was replaced with that of hPIV-1 [rSV(hHN)], intranasal administration of BCX 2798 (10 mg/kg per day) and of BCX 2855 (50 mg/kg per day) 4 h before the start of infection resulted in a significant reduction in titers of virus in the lungs and protection from death. Treatment beginning 24 h after the start of infection did not prevent death. Together, our results indicate that BCX 2798 and BCX 2855 are effective inhibitors of parainfluenza virus HN and may limit parainfluenza virus infections in humans.

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Novel Monoclonal Antibody Directed at the Receptor Binding Site on the Avian Sarcoma and Leukosis Virus Env Complex

Journal of Virology ◽

10.1128/jvi.76.15.7518-7527.2002 ◽

2002 ◽

Vol 76 (15) ◽

pp. 7518-7527 ◽

Cited By ~ 13

Author(s):

Christina Ochsenbauer-Jambor ◽

Sue E. Delos ◽

Mary Ann Accavitti ◽

Judith M. White ◽

Eric Hunter

Keyword(s):

Monoclonal Antibody ◽

Binding Site ◽

Receptor Binding ◽

Mammalian Cells ◽

Leukemia Virus ◽

Glycosylation Site ◽

Enzyme Linked Immunosorbent Assay ◽

Receptor Binding Site ◽

Avian Sarcoma

ABSTRACT We report here on the generation of a mouse monoclonal antibody directed against Rous sarcoma virus (RSV) subgroup A Env that will be useful in functional and structural analysis of RSV Env, as well as in approaches employing the RCAS/Tva system for gene targeting. BALB/c mice were primed and given boosters twice with EnvA-expressing NIH 3T3 cells. Resulting hybridomas were tested by enzyme-linked immunosorbent assay against RCANBP virions and SU-A-immunoglobulin G immunoadhesin. One highly reactive hybridoma clone, mc8C5, was subcloned and tested in immunofluorescence, immunoprecipitation (IP), and Western blotting assays. In all three assays, mc8C5-4 subgroup-specifically recognizes SR-A Env, through the SU domain, expressed from different vectors in both avian and mammalian cells. This multifunctionality is notable for a mouse monoclonal. We furthermore observed a preference for binding to terminally glycosylated Env over core-glycosylated Env precursor in IPs, suggesting that the epitope is at least partially conformational and dependent on glycosylation. Most importantly, we found mc8C5-4 inhibited Env function: in vitro, the monoclonal not only interferes with binding of the EnvA receptor, Tva, but it also blocks the Tva-induced conformational change required for activation of the fusion peptide, without inducing that change itself. Infection of Tva-expressing avian or mammalian cells by avian sarcoma and leukosis virus (ASLV) or EnvA-pseudotyped murine leukemia virus, respectively, is efficiently inhibited by mc8C5-4. The apparent interference of the monoclonal with the EnvA-Tva complex formation suggests that the epitope seen by mc8C5 overlaps with the receptor binding site. This is supported by the observation that mutations of basic residues in hr2 or of the downstream glycosylation site, which both impair Tva-binding to EnvA, have similar effects on the binding of mc8C5. Thus, anti-ASLV-SU-A mc8C5-4 proves to be a unique new immunoreagent that targets the receptor-binding site on a prototypical retroviral envelope.

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A Second Receptor Binding Site on Human Parainfluenza Virus Type 3 Hemagglutinin-Neuraminidase Contributes to Activation of the FusionMechanism

Journal of Virology ◽

10.1128/jvi.02617-06 ◽

2007 ◽

Vol 81 (7) ◽

pp. 3216-3228 ◽

Cited By ~ 67

Author(s):

Matteo Porotto ◽

Micaela Fornabaio ◽

Glen E. Kellogg ◽

Anne Moscona

Keyword(s):

Binding Site ◽

Receptor Binding ◽

Cell Interaction ◽

Binding Activity ◽

Viral Fusion ◽

Receptor Binding Site ◽

Computational Data ◽

Human Parainfluenza Virus ◽

Type 3 ◽

Entry Receptor

ABSTRACT The hemagglutinin-neuraminidase (HN) protein of paramyxoviruses carries out three discrete activities that each affect the ability of HN to promote viral fusion and entry: receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein. The interrelationship between the receptor binding and fusion-triggering functions of HN has not been clear. For human parainfluenza type 3 (HPIV3), one bifunctional site on HN can carry out both receptor binding and neuraminidase activities, and this site's receptor binding can be inhibited by the small receptor analog zanamivir. We now report experimental evidence, complemented by computational data, for a second receptor binding site near the HPIV3 HN dimer interface. This second binding site can mediate receptor binding even in the presence of zanamivir, and it differs from the second receptor binding site of the paramyxovirus Newcastle disease virus in its function and its relationship to the primary binding site. This second binding site of HPIV3 HN is involved in triggering F. We suggest that the two receptor binding sites on HPIV3 HN each contribute in distinct ways to virus-cell interaction; one is the multifunctional site that contains both binding and neuraminidase activities, and the other contains binding activity and also is involved in fusion promotion.

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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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Structural Insight into a Human Neutralizing Antibody against Influenza Virus H7N9

Journal of Virology ◽

10.1128/jvi.01850-17 ◽

2017 ◽

Vol 92 (5) ◽

Cited By ~ 7

Author(s):

Cong Chen ◽

Liguo Liu ◽

Yan Xiao ◽

Sheng Cui ◽

Jianmin Wang ◽

...

Keyword(s):

Crystal Structure ◽

Receptor Binding ◽

Influenza A ◽

Human Monoclonal Antibody ◽

H7n9 Virus ◽

Receptor Binding Site ◽

Fragment Antigen Binding ◽

Insight Into

ABSTRACT Since its first emergence in East China in early 2013, many cases of avian influenza A H7N9 have been reported. The disease has extended to 22 provinces in mainland China and some surrounding areas. Strategies to combat viral infection are urgently needed. We previously isolated a human monoclonal antibody, HNIgGA6, that neutralized the H7N9 virus both in vitro and in vivo . In this study, we determined the crystal structure of viral hemagglutinin (HA) globular head bound to the fragment antigen-binding region (Fab) of HNIgGA6. The crystal structure shows that the tip of the HNIgGA6 heavy-chain complementarity-determining region 3 (HCDR3) directly interposes into the receptor binding site (RBS) and mimics, in many respects, the interaction of the sialic acid receptor. Three residues at Y98, H183, and E190, which are critical to human cellular receptor binding, are also essential for HNIgGA6 recognition. Meanwhile, dual mutations at V186G and L226Q in RBS were able to disrupt viral HA1 binding with the antibody. Our study provides a better understanding of the mechanism for protective antibody recognition and a sound foundation for the design of therapeutic drugs and vaccines against H7N9 influenza. IMPORTANCE Neutralization by antibody is one of the most important mechanisms for a host to defend against viral infections. Human-originated antibody HNIgGA6 was generated in response to the natural infectious H7N9 virus and showed potential for use in suppression of H7N9 infection, with possible therapeutic implications. The crystal structure of the HNIgGA6/HA1 complex provided new insight into the protective immune response to H7N9 virus in humans, as well as possibilities for the development of effective H7N9 pandemic vaccines and antiviral molecules.

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Divergent Requirement of Fc-Fcγ Receptor Interactions for In Vivo Protection against Influenza Viruses by Two Pan-H5 Hemagglutinin Antibodies

Journal of Virology ◽

10.1128/jvi.02065-16 ◽

2017 ◽

Vol 91 (11) ◽

Cited By ~ 4

Author(s):

Shuangshuang Wang ◽

Huanhuan Ren ◽

Wenbo Jiang ◽

Honglin Chen ◽

Hongxing Hu ◽

...

Keyword(s):

Binding Site ◽

Receptor Binding ◽

Virus Strain ◽

Influenza Virus Infection ◽

Influenza Viruses ◽

Target Cells ◽

Fcγ Receptor ◽

Receptor Binding Site ◽

Virus Strains

ABSTRACT Recent studies have shown that Fc-Fcγ receptor (FcγR) interactions are required for in vivo protection against influenza viruses by broadly reactive anti-hemagglutinin (HA) stem, but not virus strain-specific, anti-receptor binding site (RBS), antibodies (Abs). Since only a few Abs recognizing epitopes in the head region but outside the RBS have been tested against single-challenge virus strains, it remains unknown whether Fc-FcγR interactions are required for in vivo protection by Abs recognizing epitopes outside the RBS and whether the requirement is virus strain specific or epitope specific. In the present study, we therefore investigated the requirements for in vivo protection using two pan-H5 Abs, 65C6 and 100F4. We generated chimeric Abs, 65C6/IgG2a and 100F4/IgG2a, which preferentially engage activating FcγRs, and isogenic forms, 65C6/D265A and 100F4/D265A, which do not bind FcγR. Virus neutralizing activity, binding, antibody-dependent cellular cytotoxicity (ADCC), and in vivo protection of these Abs were compared using three H5 strains, A/Shenzhen/406H/2006 (SZ06), A/chicken/Shanxi/2/2006 (SX06), and A/chicken/Netherlands/14015526/2014 (NE14). We found that all four chimeric Abs bound and neutralized the SZ06 and NE14 strains but poorly inhibited the SX06 strain. 65C6/IgG2a and 100F4/IgG2a, but not 65C6/D265A and 100F4/D265A, mediated ADCC against target cells expressing HA derived from all three virus strains. Interestingly, both 65C6/IgG2a and 65C6/D265A demonstrated comparable protection against all three virus strains in vivo; however, 100F4/IgG2a, but not 100F4/D265A, showed in vivo protection. Thus, we conclude that Fc-FcγR interactions are required for in vivo protection by 100F4, but not by 65C6, and therefore, protection is not virus strain specific but epitope specific. IMPORTANCE Abs play an important role in immune protection against influenza virus infection. Fc-FcγR interactions are required for in vivo protection by broadly neutralizing antistem, but not by virus strain-specific, anti-receptor binding site (RBS), Abs. Whether such interactions are necessary for protection by Abs that recognize epitopes outside RBS is not fully understood. In the present study, we investigated in vivo protection mechanisms against three H5 strains by two pan-H5 Abs, 65C6 and 100F4. We show that although these two Abs have similar neutralizing, binding, and ADCC activities against all three H5 strains in vitro, they have divergent requirements for Fc-FcγR interactions to protect against the three H5 strains in vivo. The Fc-FcγR interactions are required for in vivo protection by 100F4, but not by 65C6. Thus, we conclude that Fc-FcγR interactions for in vivo protection by pan-H5 Abs is not strain specific, but epitope specific.

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Evidence of a Potential Receptor-Binding Site on the Nipah Virus G Protein (NiV-G): Identification of Globular Head Residues with a Role in Fusion Promotion and Their Localization on an NiV-G Structural Model

Journal of Virology ◽

10.1128/jvi.00190-06 ◽

2006 ◽

Vol 80 (15) ◽

pp. 7546-7554 ◽

Cited By ~ 30

Author(s):

Vanessa Guillaume ◽

Hamide Aslan ◽

Michelle Ainouze ◽

Mathilde Guerbois ◽

T. Fabian Wild ◽

...

Keyword(s):

Binding Site ◽

G Protein ◽

Receptor Binding ◽

Measles Virus ◽

Structural Model ◽

Nipah Virus ◽

Hendra Virus ◽

Receptor Binding Site ◽

Globular Head

ABSTRACT As a preliminary to the localization of the receptor-binding site(s) on the Nipah virus (NiV) glycoprotein (NiV-G), we have undertaken the identification of NiV-G residues that play a role in fusion promotion. To achieve this, we have used two strategies. First, as NiV and Hendra virus (HeV) share a common receptor and their cellular tropism is similar, we hypothesized that residues functioning in receptor attachment could be conserved between their respective G proteins. Our initial strategy was to target charged residues (which can be expected to be at the surface of the protein) conserved between the NiV-G and HeV-G globular heads. Second, we generated NiV variants that escaped neutralization by anti-NiV-G monoclonal antibodies (MAbs) that neutralize NiV both in vitro and in vivo, likely by blocking receptor attachment. The sequencing of such “escape mutants” identified NiV-G residues present in the epitopes to which the neutralizing MAbs are directed. Residues identified via these two strategies whose mutation had an effect on fusion promotion were localized on a new structural model for the NiV-G protein. Our results suggest that seven NiV-G residues, including one (E533) that was identified using both strategies, form a contiguous site on the top of the globular head that is implicated in ephrinB2 binding. This site commences near the shallow depression in the center of the top surface of the globular head and extends to the rim of the barrel-like structure on the top loops of β-sheet 5. The topology of this site is strikingly similar to that proposed to form the SLAM receptor site on another paramyxovirus attachment protein, that of the measles virus hemagglutinin.

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