Virus Hemagglutinin-neuraminidase Research Articles

Different Origins of Newcastle Disease Virus Hemagglutinin-Neuraminidase Protein Modulate the Replication Efficiency and Pathogenicity of the Virus.

To investigate the exact effects of different origins of Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein to the biological characteristics of the virus, we systematically studied the correlation between the HN protein and NDV virulence by exchanging the HN of velogenic or lentogenic NDV strains with the HN from other strains of different virulence. The results revealed that the rSG10 or rLaSota derivatives bearing the HN gene of other viruses exhibited decreased or increased hemadsorption (HAd), neuraminidase and fusion promotion activities. In vitro and in vivo tests further showed that changes in replication level, tissue tropism and virulence of the chimeric viruses were also consistent with these biological activities. These findings demonstrated that the balance among three biological activities caused variation in replication and pathogenicity of the virus, which was closely related to the origin of the HN protein. Our study highlights the importance of the HN glycoprotein in modulating the virulence of NDV and contributes to a more complete understanding of the virulence of NDV.

The impact of the butterfly effect on human parainfluenza virus haemagglutinin-neuraminidase inhibitor design

Human parainfluenza viruses represent a leading cause of lower respiratory tract disease in children, with currently no available approved drug or vaccine. The viral surface glycoprotein haemagglutinin-neuraminidase (HN) represents an ideal antiviral target. Herein, we describe the first structure-based study on the rearrangement of key active site amino acid residues by an induced opening of the 216-loop, through the accommodation of appropriately functionalised neuraminic acid-based inhibitors. We discovered that the rearrangement is influenced by the degree of loop opening and is controlled by the neuraminic acid’s C-4 substituent’s size (large or small). In this study, we found that these rearrangements induce a butterfly effect of paramount importance in HN inhibitor design and define criteria for the ideal substituent size in two different categories of HN inhibitors and provide novel structural insight into the druggable viral HN protein.

Exploring inhibitor structural features required to engage the 216-loop of human parainfluenza virus type-3 hemagglutinin-neuraminidase.

Human parainfluenza virus type-3 is a leading cause of acute respiratory infection in infants and children. There is currently neither vaccine nor clinically effective treatment for parainfluenza virus infection. Hemagglutinin-neuraminidase glycoprotein is a key protein in viral infection, and its inhibition has been a target for inhibitor development. In this study, we explore the structural features required for Neu2en derivatives to efficiently lock-open the 216-loop of the human parainfluenza virus type-3 hemagglutinin-neuraminidase protein.

Site-specific glycosylation of the Newcastle disease virus haemagglutinin-neuraminidase.

Members of the Avulavirus, Respirovirus and Rubulavirus genera of the Paramyxoviridae family of viruses utilise haemagglutinin-neuraminidase glycoproteins as their attachment proteins. These glycoproteins are oligomeric type II integral membrane proteins, which possess haemagglutination and sialidase activity. Previous studies have shown that the N-linked glycans present on these proteins can modulate the ability of the virus to infect host cells and stimulate the host immune system. However, site-specific heterogeneity of these glycans has not been defined. This study concerns characterisation of the glycan compositions attached to haemagglutinin-neuraminidase of the Avulavirus Newcastle disease virus, which causes Newcastle disease in a range of avian species. Haemagglutinin-neuraminidase was derived from egg propagated virions of V4-VAR, an isolate of the avirulent strain QLD/66. Reverse-phase liquid chromatography tandem mass spectrometry strategies including collision induced dissociation, higher-energy collision dissociation and electron-transfer dissociation were implemented to characterise glycopeptides from the haemagglutinin-neuraminidase protein. Overall 63, 58, and 37 glycan compositions were identified at asparagine residues 341, 433 and 481, respectively. N-linked sites 433 and 481 were observed to contain high mannose glycans with paucimannose glycans also observed at site 481. Asparagine residues 341, 433 and 481 contained complex or hybrid glycans with many of the compositions containing variations of fucose and sulfate or phosphate. Sialyation of complex or hybrid N-linked glycans was additionally observed at sites 341 and 433. In addition, a previously undocumented O-linked glycopeptide was identified from the stalk domain of the haemagglutinin-neuraminidase protein. These finding will form the basis for future quantitative glycomic studies of the distribution of glycan structures across N-linked glycosylation sites of Newcastle disease virus haemagglutinin-neuraminidase and assessment of the functional significance of the O-linked glycan in the stalk domain of this protein.

Recombinant measles virus incorporating heterologous viral membrane proteins for use as vaccines.

Recombinant measles virus (rMV) vectors expressing heterologous viral membrane protein antigens are potentially useful as vaccines. Genes encoding the mumps virus haemagglutinin-neuraminidase (MuV-HN), the influenza virus haemagglutinin (Flu-HA) or the respiratory syncytial virus fusion (RSV-F) proteins were inserted into the genome of a live attenuated vaccine strain of measles virus. Additionally, in this case rMV with the MuV-HN or the influenza HA inserts, chimeric constructs were created that harboured the measles virus native haemagglutinin or fusion protein cytoplasmic domains. In all three cases, sucrose-gradient purified preparations of rMV were found to have incorporated the heterologous viral membrane protein on the viral membrane. The possible utility of rMV expressing RSV-F (rMV.RSV-F) as a vaccine was tested in a cotton rat challenge model. Vaccination with rMV.RSV-F efficiently induced neutralizing antibodies against RSV and protected animals from infection with RSV in the lungs.

Evidence that Receptor Destruction by the Sendai Virus Hemagglutinin-Neuraminidase Protein Is Responsible for Homologous Interference.

Receptor destruction has been considered one of the mechanisms of homologous Sendai virus (SeV) interference. However, direct evidence of receptor destruction upon virus infection and its relevance to interference is missing. To investigate a precise mechanism of homologous interference, we established SeV persistently infected cells. The persistently infected cells inhibited superinfection by homologous SeV but supported replication of human parainfluenza virus 2 (hPIV2) and influenza A virus (IAV). We confirmed that SeV particles could not attach to or penetrate the infected cells and that the hemagglutinin-neuraminidase (HN) protein of SeV was involved in the interference. Lectin blot assays showed that the α2,3-linked sialic acids were specifically reduced in the SeV-infected cells, but the level of α2,6-linked sialic acids had not changed. As infection with IAV removed both α2,3- and α2,6-linked sialic acids, especially α2,3-linked sialic acids, IAV-infected cells inhibited superinfection of SeV. These results provide concrete evidence that destruction of the specific SeV receptor, α2,3-linked sialic acids, is relevant to homologous interference by SeV. Viral interference is a classically observed phenomenon, but the precise mechanism is not clear. Using SeV interference, we provide concrete evidence that reduction of the α2,3-linked sialic acid receptor by the HN of SeV is closely related with viral interference. Since SeV infection resulted in decrease of only α2,3-linked sialic acids, IAV, which also utilized α2,6-linked sialic acids to initiate infection, superinfected the SeV-infected cells. In contrast, SeV could not superinfect the IAV-infected cells because both α2,3- and α2,6-linked sialic acids were removed. These results indicate that receptor destruction critically contributes to viral interference.

Newcastle Disease Virus Hemagglutinin Neuraminidase as a Potential Cancer Targeting Agent

The hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) with its immunotherapeutic activities and sialic acid binding abilities is a promising cancer adjuvant. The HN was surfaced displayed on Lactococcus lactis and its cancer targeting ability was investigated via attachment to the MDA-MB231 breast cancers. To surface display the HN protein on the bacterial cell wall, HN was fused to N-acetylmuraminidase (AcmA) anchoring motif of L. lactis and expressed in Chinese hamster ovary cells. The expressed recombinant fusion proteins were purified and mixed with a culture of L. lactis and Lactobacillus plantarum. Immunofluorescence assay showed the binding of the recombinant HN-AcmA protein on the surface of the bacterial cells. The bacterial cells carrying the HN-AcmA protein interacted with the MDA-MB231 breast cancer cells. Direct and fluorescent microscopy confirmed that L. lactis and Lb. plantarum surface displaying the recombinant HN were attached to the breast cancer MDA-MB231 cells, providing evidence for the potential ability of HN in targeting to cancer cells.

Improved immunogenicity of Newcastle disease virus inactivated vaccine following DNA vaccination using Newcastle disease virus hemagglutinin-neuraminidase and fusion protein genes.

The present study describes the development of DNA vaccines using the hemagglutinin-neuraminidase (HN) and fusion (F) genes from AF2240 Newcastle disease virus strain, namely pIRES/HN, pIRES/F and pIRES-F/HN. Transient expression analysis of the constructs in Vero cells revealed the successful expression of gene inserts in vitro. Moreover, in vivo experiments showed that single vaccination with the constructed plasmid DNA (pDNA) followed by a boost with inactivated vaccine induced a significant difference in enzyme-linked immunosorbent assay antibody levels (p < 0.05) elicited by either pIRES/F, pIRES/F+ pIRES/HN or pIRES-F/HN at one week after the booster in specific pathogen free chickens when compared with the inactivated vaccine alone. Taken together, these results indicated that recombinant pDNA could be used to increase the efficacy of the inactivated vaccine immunization procedure.

Construction and immunological evaluation of recombinant Lactobacillus plantarum expressing HN of Newcastle disease virus and DC- targeting peptide fusion protein

Newcastle disease virus (NDV) has been considered as one of the most severe threats to poultry industry. In this study, we constructed a series of food grade recombinant Lactobacillus plantarum (L. plantarum) strains (RLP) synthesizing either virus hemagglutinin–neuraminidase protein (HN) alone or HN fused with DC cells targeting peptides (DCpep), named RLP(pSIP409-HN) and RLP (pSIP409-HN-DCpep), respectively. The immune responses and protective efficacy were then evaluated in chickens. Results showed that the presence of DCpep in RLP (pSIP409-HN-DCpep) group significantly increased the production of secretory immunoglobulin A (SIgA) in intestines and the percentages of CD3+CD4+ T cells in spleen and peripheral blood leukocytes (P<0.05) compared to chickens immunized with RLP (pSIP409-HN). In addition, the similar enhancement effects were also observed with regard to trachea SIgA, T lymphocytes proliferation and survival rates after NDV challenge, even without significant statics differences. The results demonstrated the possibility to take use of DCpep as an immune adjuvant in the design of NDV vaccine.

The Fusion Protein Specificity of the Parainfluenza Virus Hemagglutinin-Neuraminidase Protein Is Not Solely Defined by the Primary Structure of Its Stalk Domain.

Virus-specific interaction between the attachment protein (HN) and the fusion protein (F) is prerequisite for the induction of membrane fusion by parainfluenza viruses. This HN-F interaction presumably is mediated by particular amino acids in the HN stalk domain and those in the F head domain. We found in the present study, however, that a simian virus 41 (SV41) F-specific chimeric HPIV2 HN protein, SCA, whose cytoplasmic, transmembrane, and stalk domains were derived from the SV41 HN protein, could not induce cell-cell fusion of BHK-21 cells when coexpressed with an SV41 HN-specific chimeric PIV5 F protein, no. 36. Similarly, a headless form of the SV41 HN protein failed to induce fusion with chimera no. 36, whereas it was able to induce fusion with the SV41 F protein. Interestingly, replacement of 13 amino acids of the SCA head domain, which are located at or around the dimer interface of the head domain, with SV41 HN counterparts resulted in a chimeric HN protein, SCA-RII, which induced fusion with chimera no. 36 but not with the SV41 F protein. More interestingly, retroreplacement of 11 out of the 13 amino acids of SCA-RII with the SCA counterparts resulted in another chimeric HN protein, IM18, which induced fusion either with chimera no. 36 or with the SV41 F protein, similar to the SV41 HN protein. Thus, we conclude that the F protein specificity of the HN protein that is observed in the fusion event is not solely defined by the primary structure of the HN stalk domain. It is appreciated that the HN head domain initially conceals the HN stalk domain but exposes it after the head domain has bound to the receptors, which allows particular amino acids in the stalk domain to interact with the F protein and trigger it to induce fusion. However, other regulatory roles of the HN head domain in the fusion event have been ill defined. We have shown in the current study that removal of the head domain or amino acid substitutions in a particular region of the head domain drastically change the F protein specificity of the HN protein, suggesting that the ability of a given HN protein to interact with an F protein is defined not only by the primary structure of the HN stalk domain but also by its conformation. This notion seems to account for the unidirectional substitutability among rubulavirus HN proteins in triggering noncognate F proteins.

The catalytic mechanism of human parainfluenza virus type 3 haemagglutinin-neuraminidase revealed.

Human parainfluenza virus type 3 (hPIV-3) is one of the leading causes for lower respiratory tract disease in children, with neither an approved antiviral drug nor vaccine available to date. Understanding the catalytic mechanism of human parainfluenza virus haemagglutinin-neuraminidase (HN) protein is key to the design of specific inhibitors against this virus. Herein, we used (1) H NMR spectroscopy, X-ray crystallography, and virological assays to study the catalytic mechanism of the HN enzyme activity and have identified the conserved Tyr530 as a key amino acid involved in catalysis. A novel 2,3-difluorosialic acid derivative showed prolonged enzyme inhibition and was found to react and form a covalent bond with Tyr530. Furthermore, the novel derivative exhibited enhanced potency in virus blockade assays relative to its Neu2en analogue. These outcomes open the door for a new generation of potent inhibitors against hPIV-3 HN.

Roles of the highly conserved amino acids in the globular head and stalk region of the Newcastle disease virus HN protein in the membrane fusion process.

Newcastle disease virus (NDV), an avain paramyxovirus, has been assigned to the genus Avulavirus within the family Paramyxoviridae. It causes Newcastle disease (ND) that is a highly contagious and fatal viral disease affecting poultry and most species of birds. The hemagglutinin-neuraminidase (HN) protein of NDV has multiple functions including mediating hemadsorption (HAD), neuraminidase (NA), and fusion promotion activities affecting the process of viral attachment, entry, replication and dissemination. Fusion ability of the NDV was highly correlated to its virulence. Mutations in the HN globular head and headless HN of NDV were constructed to determinate the impact of highly conserved amino acids in the globular head of paramyxovirus HN proteins and the roles of the stalk region of HN in the fusion process. It was found that the interaction between F and HN mutants E401A, G402A, G468A, V469A, Y526A, and T527A was equal to that in F and wt HN. The mutations of G402A, G468A, V469A, and T527A had various effects on cell fusion promotion, receptor binding ability, and NA activity, but the membrane merging rate was comparable to wt HN. The elimination of hemadsorption ability and NA activity of E401A and Y526A resulted in the loss of the fusion promotion function of HN. The conclusion was that receptor binding and NA had a common active site and E401 and Y526 amino acids were essential for virus attachment, entry, and dissemination. In addition, G468A mutation made different contributions to HAD and NA, which indicated that G468 was one of the potential key amino acids in switching the two functions between receptor binding and sialic acid destruction of HN. It was also proven that the headless HN of NDV could promote the fusion event mediated by F. Thus, it revealed a novel mechanism in F activation of NDV.

Molecular diagnosis of Newcastle disease Iraqi Virulent strain virus HN gene by specific primers design

Newcastle disease is considered the most contagious poultry disease and may cause severe economic loss in the poultry industry, The first report in which NDV was consider as zoonotic disease and a human pathogen that was published by Burnet, in 1943. The aims of this study was to detect HN gene of two NDV Iraqi isolates (Najaf APMV1/ Chicken/ Iraq-najaf/ ICCMGR/2012and Baghdad ICCMGR) by designing specific priemrs for the Iraqi isolates. DNV isolates propagated in embryonated chicken eggs and showed to kill embryos in different times intervals ( more than 40 hours and less than 72 hours) with marked sever hemorrhage in infected embryos and it was similar in the two isolates, agglutination activity reflect titer (1024) for Najaf isolate and (512) for Baghdad isolate. The present study successfully detected part of (HN) gene by using specific primers designed for one-step RT-PCR undertaken to use them on molecular diagnosis of the hemagglutinin-neuraminidase (HN) gene in the Iraqi isolates and can be used for NDV virus detection in Iraq.

Structure-guided discovery of potent and dual-acting human parainfluenza virus haemagglutinin-neuraminidase inhibitors.

Human parainfluenza viruses (hPIVs) cause upper and lower respiratory tract disease in children that results in a significant number of hospitalizations and impacts health systems worldwide. To date, neither antiviral drugs nor vaccines are approved for clinical use against parainfluenza virus, which reinforces the urgent need for new therapeutic discovery strategies. Here we use a multidisciplinary approach to develop potent inhibitors that target a structural feature within the hPIV type 3 haemagglutinin-neuraminidase (hPIV-3 HN). These dual-acting designer inhibitors represent the most potent designer compounds and efficiently block both hPIV cell entry and virion progeny release. We also define the binding mode of these inhibitors in the presence of whole-inactivated hPIV and recombinantly expressed hPIV-3 HN by Saturation Transfer Difference NMR spectroscopy. Collectively, our study provides an antiviral preclinical candidate and a new direction towards the discovery of potential anti-parainfluenza drugs.

Analysis of synonymous codon usage in Newcastle disease virus hemagglutinin–neuraminidase (HN) gene and fusion protein (F) gene

Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) is a multifunctional protein, which possesses both the receptor recognition and neuraminidase activities. The fusion (F) protein is a type I membrane glycoprotein that mediates the merger of the viral envelope to the host cell membrane. Although the functions of the HN and F proteins have been well studied, however, the factors shaping synonymous codon usage bias and nucleotide composition in HN and F genes have been few reported. In our study, we analyzed synonymous codon usage using the 69 NDV HN and F genes, respectively. The general correlation between base composition and codon usage bias suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in HN and F genes. In addition, other factors, such as the aromaticity and hydrophobicity, also influence the codon usage variation among HN and F genes. This study represents the most comprehensive analysis to date of NDV HN and F genes codon usage patterns and provides a basic understanding of the mechanisms for codon usage bias.

Recombinant infectious bursal disease virus expressing Newcastle disease virus (NDV) neutralizing epitope confers partial protection against virulent NDV challenge in chickens

In this study, the regions in the infectious bursal disease virus (IBDV) genome that are amenable to the introduction of a sequence encoding a virus-neutralizing epitope of Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) protein were identified. By using the reverse genetics approach, insertions or substitutions of sequences encoding the NDV epitope were engineered in the exposed loops (PBC, PHI and PAA′) of the VP2 capsid protein and the N terminus of the nonstructural VP5 protein as well as the pep7a and pep7b regions of the pVP2 precursor of a commonly used IBDV vaccine strain, Gt. Three recombinant IBDVs expressing the NDV epitopes were successfully rescued in the PBC, pep7b and VP5 regions and the expressed epitope was recognized by anti-HN antibodies. Genetic analysis showed that the IBDV recombinants carrying the NDV epitopes were stable in cell cultures and in chickens. Animal studies demonstrated that the IBDV recombinants were innocuous in chickens. Vaccination with the recombinant viruses generated antibody responses against both IBDV and NDV, and provided 70–80% protection against IBDV and 50–60% protection against NDV. These results indicate that the recombinant IBDV has the potential to serve as a novel vaccine vector for other pathogens. In future studies, it is worth considering research to improve IBDV vector vaccine to get complete protection and safety of animals and humans.

Novel polyvalent live vaccine against varicella–zoster and mumps virus infections

The varicella-zoster virus (VZV) Oka vaccine strain (vOka) is a highly immunogenic and safe live vaccine that has long been used worldwide. Because its genome is large, making it suitable for inserting foreign genes, vOka is considered a candidate vector for novel polyvalent vaccines. Previously, a recombinant vOka, rvOka-HN, that expresses mumps virus (MuV) hemagglutinin-neuraminidase (HN) was generated by the present team. rvOka-HN induces production of neutralizing antibodies against MuV in guinea pigs. MuV also expresses fusion (F) protein, which is important for inducing neutralizing antibodies, in its viral envelope. To induce a more robust immune response against MuV than that obtained with rvOka-HN, here an rvOka expressing both HN and F (rvOka-HN-F) was generated. However, co-expression of HN and F caused the infected cells to form syncytia, which reduced virus titers. To reduce the amount of cell fusion, an rvOka expressing HN and a mutant F, F(S195Y) were generated. Almost no syncytia formed among the rvOka-HN-F(S195Y)-infected cells and the growth of rvOka-HN-F(S195Y) was similar to that of the original vOka clone. Moreover, replacement of serine 195 with tyrosine had no effect on the immunogenicity of F in mice and guinea pigs. Although obvious augmentation of neutralizing antibody production was not observed after adding F protein to vOka-HN, the anti-F antibodies did have neutralizing activity. These data suggest that F protein contributes to induction of immune protection against MuV. Therefore this recombinant virus is a promising candidate vaccine for polyvalent protection against both VZV and MuV.

Quantitative Comparison of Human Parainfluenza Virus Hemagglutinin-Neuraminidase Receptor Binding and Receptor Cleavage

The human parainfluenza virus (hPIV) hemagglutinin-neuraminidase (HN) protein binds (H) oligosaccharide receptors that contain N-acetylneuraminic acid (Neu5Ac) and cleaves (N) Neu5Ac from these oligosaccharides. In order to determine if one of HN's two functions is predominant, we measured the affinity of H for its ligands by a solid-phase binding assay with two glycoprotein substrates and by surface plasmon resonance with three monovalent glycans. We compared the dissociation constant (Kd) values from these experiments with previously determined Michaelis-Menten constants (Kms) for the enzyme activity. We found that glycoprotein substrates and monovalent glycans containing Neu5Acα2-3Galβ1-4GlcNAc bind HN with Kd values in the 10 to 100 μM range. Km values for HN were previously determined to be on the order of 1 mM (M. M. Tappert, D. F. Smith, and G. M. Air, J. Virol. 85:12146-12159, 2011). A Km value greater than the Kd value indicates that cleavage occurs faster than the dissociation of binding and will dominate under N-permissive conditions. We propose, therefore, that HN is a neuraminidase that can hold its substrate long enough to act as a binding protein. The N activity can therefore regulate binding by reducing virus-receptor interactions when the concentration of receptor is high.

A Mutation in the Stalk of the Newcastle Disease Virus Hemagglutinin-Neuraminidase (HN) Protein Prevents Triggering of the F Protein despite Allowing Efficient HN-F Complex Formation

Newcastle disease virus (NDV)-induced membrane fusion requires formation of a complex between the hemagglutinin-neuraminidase (HN) and fusion (F) proteins. Substitutions for NDV HN stalk residues A89, L90, and L94 block fusion by modulating formation of the HN-F complex. Here, we demonstrate that a nearby L97A substitution, though previously shown to block fusion, allows efficient HN-F complex formation and likely acts by preventing changes in the HN stalk required for triggering of the bound F protein.

Antitumor effects of Newcastle disease virus hemagglutinin-neuraminidase used as a molecular adjuvant

Gene therapy is a potentially powerful tool used in cancer therapy. The strength of immune responses induced by some strategies is usually low, therefore, the development of agents capable of enhancing these responses is highlighted. The authors investigated the potential of an approach based on the hemagglutinin-neuraminidase(HN) of Newcastle disease virus(NDV) as a potential immune adjuvant. It was found that recombinant adenovirus(Ad) infected SGC7901 cells expressing HN exhibited both hemagglutinin(HA) and neuraminidase(NA) activities. It was demonstrated that administration of HN induced higher levels of the effector cytokines TNF-α, IFN-α and IFN-γ and increased natural killer(NK) cell activity. Based on the therapeutic tumor model, the results show that the administration of HN with Apoptin led to improved survival and tumor suppression. In conclusion, this study indicates that HN stimulates innate immune responses to make the activity of NK cells increased, which highlights the potential adjuvant activity of HN in cancer gene therapy.