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A Computational Assay that Explores the Hemagglutinin/Neuraminidase Functional Balance Reveals the Neuraminidase Secondary Site as a Novel Anti-Influenza Target.

ABSTRACT: Studies of pathogen-host specificity, virulence, and transmissibility are critical for basic research as well as for assessing the pandemic potential of emerging infectious diseases. This is especially true for viruses such as influenza, which continue to affect millions of people annually through both seasonal and occasional pandemic events. Although the influenza virus has been fairly well studied for decades, our understanding of host-cell binding and its relation to viral transmissibility and infection is still incomplete. Assessing the binding mechanisms of complex biological systems with atomic-scale detail is challenging given current experimental limitations. Much remains to be learned, for example, about how the terminal residue of influenza-binding host-cell receptors (sialic acid) interacts with the viral surface. Here, we present an integrative structural-modeling and physics-based computational assay that reveals the sialic acid association rate constants (k on) to three influenza sites: the hemagglutinin (HA), neuraminidase (NA) active, and NA secondary binding sites. We developed a series of highly detailed (atomic-resolution) structural models of fully intact influenza viral envelopes. Brownian dynamics simulations of these systems showed how structural properties, such as stalk height and secondary-site binding, affect sialic acid k on values. Comparing the k on values of the three sialic acid binding sites across different viral strains suggests a detailed model of encounter-complex formation and indicates that the secondary NA binding site may play a compensatory role in host-cell receptor binding. Our method elucidates the competition among these sites, all present on the same virion, and provides a new technology for directly studying the functional balance between HA and NA.

SUBMITTER: Amaro RE

PROVIDER: S-EPMC6276040 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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A Computational Assay that Explores the Hemagglutinin/Neuraminidase Functional Balance Reveals the Neuraminidase Secondary Site as a Novel Anti-Influenza Target.

Amaro Rommie E RE Ieong Pek U PU Huber Gary G Dommer Abigail A Steven Alasdair C AC Bush Robin M RM Durrant Jacob D JD Votapka Lane W LW

ACS central science 20181109 11

Studies of pathogen-host specificity, virulence, and transmissibility are critical for basic research as well as for assessing the pandemic potential of emerging infectious diseases. This is especially true for viruses such as influenza, which continue to affect millions of people annually through both seasonal and occasional pandemic events. Although the influenza virus has been fairly well studied for decades, our understanding of host-cell binding and its relation to viral transmissibility an ...[more]

PMID: 30555910

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Project description:Influenza A virus (IAV) neuraminidase (NA) receptor-destroying activity and hemagglutinin (HA) receptor-binding affinity need to be balanced with the host receptor repertoire for optimal viral fitness. NAs of avian, but not human viruses, contain a functional 2nd sialic acid (SIA)-binding site (2SBS) adjacent to the catalytic site, which contributes to sialidase activity against multivalent substrates. The receptor-binding specificity and potentially crucial contribution of the 2SBS to the HA-NA balance of virus particles is, however, poorly characterized. Here, we elucidated the receptor-binding specificity of the 2SBS of N2 NA and established an important role for this site in the virion HA-NA-receptor balance. NAs of H2N2/1957 pandemic virus with or without a functional 2SBS and viruses containing this NA were analysed. Avian-like N2, with a restored 2SBS due to an amino acid substitution at position 367, was more active than human N2 on multivalent substrates containing α2,3-linked SIAs, corresponding with the pronounced binding-specificity of avian-like N2 for these receptors. When introduced into human viruses, avian-like N2 gave rise to altered plaque morphology and decreased replication compared to human N2. An opposite replication phenotype was observed when N2 was combined with avian-like HA. Specific bio-layer interferometry assays revealed a clear effect of the 2SBS on the dynamic interaction of virus particles with receptors. The absence or presence of a functional 2SBS affected virion-receptor binding and receptor cleavage required for particle movement on a receptor-coated surface and subsequent NA-dependent self-elution. The contribution of the 2SBS to virus-receptor interactions depended on the receptor-binding properties of HA and the identity of the receptors used. We conclude that the 2SBS is an important and underappreciated determinant of the HA-NA-receptor balance. The rapid loss of a functional 2SBS in pandemic viruses may have served to balance the novel host receptor-repertoire and altered receptor-binding properties of the corresponding HA protein.

Project description:Although a polybasic HA0 cleavage site is considered the dominant virulence determinant for highly pathogenic avian influenza (HPAI) H5 and H7 viruses, naturally occurring virus isolates possessing a polybasic HA0 cleavage site have been identified that are low pathogenic in chickens. In this study, we generated a reassortant H5N3 virus that possessed the hemagglutinin (HA) gene from H5N1 HPAI A/swan/Germany/R65/2006 and the remaining gene segments from low pathogenic A/chicken/British Columbia/CN0006/2004 (H7N3). Despite possessing the HA0 cleavage site GERRRKKR/GLF, this rH5N3 virus exhibited a low pathogenic phenotype in chickens. Although rH5N3-inoculated birds replicated and shed virus and seroconverted, transmission to naive contacts did not occur. To determine whether this virus could evolve into a HPAI form, it underwent six serial passages in chickens. A progressive increase in virulence was observed with the virus from passage number six being highly transmissible. Whole-genome sequencing demonstrated the fixation of 12 nonsynonymous mutations involving all eight gene segments during passaging. One of these involved the catalytic site of the neuraminidase (NA; R293K) and is associated with decreased neuraminidase activity and resistance to oseltamivir. Although introducing the R293K mutation into the original low-pathogenicity rH5N3 increased its virulence, transmission to naive contact birds was inefficient, suggesting that one or more of the remaining changes that had accumulated in the passage number six virus also play an important role in transmissibility. Our findings show that the functional linkage and balance between HA and NA proteins contributes to expression of the HPAI phenotype.To date, the contribution that hemagglutinin-neuraminidase balance can have on the expression of a highly pathogenic avian influenza virus phenotype has not been thoroughly examined. Reassortment, which can result in new hemagglutinin-neuraminidase combinations, may have unpredictable effects on virulence and transmission characteristics of a virus. Our data show the importance of the neuraminidase in complementing a polybasic HA0 cleavage site. Furthermore, it demonstrates that adaptive changes selected for during the course of virus evolution can result in unexpected traits such as antiviral drug resistance.

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Project description:Some avian influenza (AI) viruses have a deletion of up to 20 to 30 amino acids in their neuraminidase (NA) stalk. This has been associated with changes in virus replication and host range. Currently prevalent H9N2 AI viruses have only a 2- or 3-amino-acid deletion, and such deletions were detected in G1 and Y280 lineage viruses, respectively. The effect of an NA deletion on the H9N2 phenotype has not been fully elucidated. In this study, we isolated G1 mutants that carried an 8-amino-acid deletion in their NA stalk. To systematically analyze the effect of NA stalk length and concomitant (de)glycosylation on G1 replication and host range, we generated G1 viruses that had various NA stalk lengths and that were either glycosylated or not glycosylated. The stalk length was correlated with NA sialidase activity, using low-molecular-weight substrates, and with virus elution efficacy from erythrocytes. G1 virus replication in avian cells and eggs was positively correlated with the NA stalk length but was negatively correlated in human cells and mice. NA stalk length modulated G1 virus entry into host cells, with shorter stalks enabling more efficient G1 entry into human cells. However, with a hemagglutinin (HA) with a higher α2,6-linked sialylglycan affinity, the effect of NA stalk length on G1 virus infection was reversed, with shorter NA stalks reducing virus entry into human cells. These results indicate that a balance between HA binding affinity and NA sialidase activity, modulated by NA stalk length, is required for optimal G1 virus entry into human airway cells.IMPORTANCE H9N2 avian influenza (AI) virus, one of the most prevalent AI viruses, has caused repeated poultry and human infections, posing a huge public health risk. The H9N2 virus has diversified into multiple lineages, with the G1 lineage being the most prevalent worldwide. In this study, we isolated G1 variants carrying an 8-amino-acid deletion in their NA stalk, which is, to our knowledge, the longest deletion found in H9N2 viruses in the field. The NA stalk length was found to modulate G1 virus entry into host cells, with the effects being species specific and dependent on the corresponding HA binding affinity. Our results suggest that, in nature, H9N2 G1 viruses balance their HA and NA functions by the NA stalk length, leading to the possible association of host range and virulence in poultry and mammals during the evolution of G1 lineage viruses.

Dataset Information

A Computational Assay that Explores the Hemagglutinin/Neuraminidase Functional Balance Reveals the Neuraminidase Secondary Site as a Novel Anti-Influenza Target.

Publications

A Computational Assay that Explores the Hemagglutinin/Neuraminidase Functional Balance Reveals the Neuraminidase Secondary Site as a Novel Anti-Influenza Target.

Similar Datasets

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