Large-Scale Expression and Purification of Mumps Virus Hemagglutinin-Neuraminidase for Structural Analyses and Glycan-Binding Assays.

Many viruses utilize cell-surface glycans as receptors for host cell entry. Viral surface glycoproteins specifically interact with glycan motifs, which strongly contributes to viral tropism. Recently, the interactions between host cell glycan receptors and the mumps virus (MuV) hemagglutinin-neuraminidase (MuV-HN) protein were characterized by determining the co-crystal structure of MuV-HN in complex with glycan receptors. Here, we describe protocols for large-scale expression, purification and crystallization of MuV-HN proteins for structural analyses and glycan-binding assays with the overarching goal of investigating glycan-protein interactions.

Cloning, expression and characterization of potential immunogenic recombinant hemagglutinin-neuraminidase protein of Porcine rubulavirus.

Blue eye disease caused by Porcine rubulavirus (PorPV) is an endemic viral infection of swine causing neurological and respiratory disease in piglets, and reproductive failure in sows and boars. The hemagglutinin-neuraminidase (HN) glycoprotein of PorPV is the most abundant component in the viral envelope and the main target of the immune response in infected animals. In this study, we expressed the HN-PorPV-recombinant (rHN-PorPV) protein in an Escherichia coli system and analyzed the immune responses in mice. The HN gene was cloned from the reference strain PorPV-La Piedad Michoacan Virus (GenBank accession number BK005918), into the pDual expression vector. The expressed protein was identified at a molecular weight of 61.7 kDa. Three-dimensional modeling showed that the main conformational and functional domains of the rHN-PorPV protein were preserved. The antigenicity of the expressed protein was confirmed by Western blot with a monoclonal antibody recognizing the HN, and by testing against serum samples from pigs experimentally infected with PorPV. The immunogenicity of the rHN-PorPV protein was tested by inoculation of BALB/c mice with AbISCO-100(®) as adjuvant. Analysis of the humoral immune responses in mice showed an increased level of specific antibodies 14 days after the first immunization, compared to the control group (P < 0.0005). The results show the ability of the rHN-PorPV protein to induce an antibody response in mice. Due to its immunogenic potential, the rHN-PorPV protein will be further evaluated in pig trials for its suitability for prevention and control of blue eye disease.

Antigenic validation of recombinant hemagglutinin-neuraminidase protein of Newcastle disease virus expressed in Saccharomyces cerevisiae.

The outer membrane glycoprotein, hemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) is important for virus infection and subsequent immune response by host, and offers target for development of recombinant antigen-based immunoassays and subunit vaccines. In this study, the expression of HN protein of NDV is attempted in yeast expression system. Yeast offers eukaryotic environment for protein processing and posttranslational modifications like glycosylation, in addition to higher growth rate and easy genetic manipulation. Saccharomyces cerevisiae was found to be better expression system for HN protein than Pichia pastoris as determined by codon usage analysis. The complete coding  sequence of HN gene was amplified with the histidine tag, cloned in pESC-URA under GAL10 promotor and transformed in Saccharomyces cerevisiae. The recombinant HN (rHN) protein was characterized by western blot, showing glycosylation heterogeneity as observed with other eukaryotic expression systems. The recombinant protein was purified by affinity column purification. The protein could be further used as subunit vaccine.

Purification of the Porcine rubulavirus attachment protein by liquid isoelectric focusing.

Porcine rubulavirus (PoRV) is an emerging virus responsible for meningoencephalitis, respiratory distress, and reproductive alterations in pigs. The hemagglutinin-neuraminidase (HN) glycoprotein is the most exposed and antigenic of the virus proteins. HN plays central roles in PoRV infection; i.e., it recognizes sialic acid-containing cell receptors that mediate virus attachment and penetration; in addition, its neuraminidase (sialic acid hydrolysis) activity has been proposed to be a virulence factor. So, HN is an ideal target for therapeutic treatment and prevention of this viral infection. This work describes a simple, fast, and sensitive method to purify the active form of HN protein based on its isoelectric point. HN was purified at a pH of 4.4, at which a single protein band of 66 kDa was observed on SDS-PAGE. Pure HN showed a maximal enzymatic activity at pH 3.5 and 37 degrees C using bovine fetuin as substrate. However, it retains circa 80% of its activity at a wide temperature range from 30 to 55 degrees C. We also describe improvements of neuraminidase determination method, which permits analysis in a microplate spectrophotometer, thereby increasing the sensitivity and reducing the costs of valuable reagents and biological samples.

Phylogenetic analysis of the L and HN gene of ophidian paramyxoviruses. Brief report.

Two reptilian paramyxoviruses, isolated from a neotropical rattlesnake (neotropical virus, NTV, ATCC VR-1408) and a bush viper (bush viper virus, BVV, ATCC VR- 1409), respectively, were analysed to determine their taxonomic position among other reptilian paramyxoviruses investigated previously by Ahne et al.. A 679 bp long region of the hemagglutinin-neuraminidase (HN) gene and a 627 bp long region of the large (L) gene were reverse transcribed, amplified by polymerase chain reaction (PCR), and sequenced. The deduced amino acid sequences were compared to mammalian paramyxoviruses belonging to the genera Respirovirus and Rubulavirus. The deduced amino acid sequences revealed 58.9 to 62% homology for the partial L protein and 41% to 47.1% homology for the partial HN protein. For phylogenetic analyses, a 518 bp L gene and a 352 bp HN gene fragment were used, both generating similar trees consisting of two distinct main groups, and some intermediate isolates. BVV clustered within group "b" while NTV clustered together with the intermediate ophidian paramyxovirus isolate Crot2-OH90.

Localization of a new neutralizing epitope on the mumps virus hemagglutinin-neuraminidase protein.

Four protein fragments which span the entire hemagglutinin-neuraminidase protein (HN) of mumps virus were expressed in HeLa cells and cell extracts were tested for their capability to induce neutralizing antibodies in mice. Fragment HN3 (aa 213-372) was able to induce the production of hemagglutination-inhibiting and neutralizing antibodies. When a subfragment of HN3, the synthetic peptide NSTLGVKSAREF (aa 329-340 of HN) was used for immunization, hemagglutination-inhibiting and neutralizing antibodies against mumps wild type virus but not against the Urabe Am9 vaccine virus were raised. The peptide could, therefore, contain a new epitope, which may be critical for protective host humoral immune response.

Crystallization of Newcastle disease virus hemagglutinin-neuraminidase glycoprotein.

The hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus was isolated by cleaving HN (cHN) from reconstituted virosome with chymotrypsin. N-terminal sequence analysis of the purified cHN showed that chymotrypsin cleavage had occurred at amino acid 123, freeing the C-terminal 454 amino acids. The purified cHN retained its neuraminidase and receptor binding activities and reacted with specific monoclonal antibodies, showing that the isolated cHN was biologically and antigenically functional. The crystals of the cHN were obtained in acetate buffer (pH 4.6) containing polyethylene glycol 3350 and ammonium sulfate and belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimension of approximately a = 72 A, b = 78 A, and c = 198 A. Crystals of cHN grown in the presence of sialic acid (Neu5Ac) were grown in HEPES buffer (pH 6.2) containing polyethylene glycol 3350 and belong to the hexagonal space groups P6(1) or P6(5) with unit cell dimensions of a = b = 137.5 A and c = 116.6A. The orthorhombic crystals produced in this study diffract X rays to at least 2.0-A resolution, thereby setting the stage for the solution of the three-dimensional structure of the HN glycoprotein of a paramyxovirus.

Purification and characterization of the hemagglutinin-neuraminidase of Porcine rubulavirus LPMV.

The Hemagglutinin-Neuraminidase (HN) from the LPMV strain of Porcine rubulavirus was purified from virions by ultracentrifugation in a continuous 20-60% sucrose gradient and by ion exchange chromatography. The HN is a glycoprotein of 66 kDa constituted by 50.5, 13.3 and 13.6% of non polar, uncharged polar, and charged polar amino acids, respectively. The HN contains 4% of carbohydrates, its glycannic portion is constituted by Man, Gal, GlcNAc, GalNAc, and Neu5Ac in 3:3:4:1:1 molar ratios. The HN possesses hemagglutinating activity in the presence of erythrocytes from several animal species, including human ABO, and treating the erythrocytes with neuraminidase or pronase abolishes this activity. The binding specificity of the purified HN was determined by hapten inhibition assays, indicating that the hemagglutinating activity of the HN is specific for sialic acid and Neu5Acalpha2,3Gal-containing structures.

Functional interaction of paramyxovirus glycoproteins: identification of a domain in Sendai virus HN which promotes cell fusion.

The cell fusion activity of most paramyxoviruses requires coexpression of a fusion protein (F) and a hemagglutinin-neuraminidase protein (HN) which are derived from the same virus type. To define the domain of the HN protein which interacts with the F protein in a type-specific manner a series of chimeric HN proteins between two different paramyxoviruses, Sendai virus (SN) and human parainfluenza virus type 3 (PI3), was constructed and coexpressed with the SN-F protein by using the vaccinia virus T7 RNA polymerase transient-expression system. Quantitative assays were used to evaluate cell surface expression as well as fusion-promoting activities of the chimeric HN molecules. A chimeric HN protein [SN(140)] containing 140 N-terminal amino acids derived from SN-HN and the remainder (432 amino acids) derived from PI3-HN was found to promote cell fusion with the SN-F protein. In contrast, a second chimeric HN with 137 amino acids from SN-HN at the N terminus could not promote fusion with SN-F, even though the protein was expressed on the cell surface. A construct in which the PI3-HN cytoplasmic tail and transmembrane domain were substituted for those of SN in the SN(140) chimera still maintained the ability to promote cell fusion. These results indicate that a region including only 82 amino acids in the extracellular domain, adjacent to the transmembrane domain of the SN-HN protein, is important for interaction with the SN-F protein and promotion of cell fusion.

Interaction of Sendai viral F, HN, and M proteins with host cytoskeletal and lipid components in Sendai virus-infected BHK cells.

We have studied the interaction of Sendai viral fusion (F), hemagglutinin/neuraminidase (H/N), and matrix (M) proteins with host cytoskeletal and lipid components in Sendai virus-infected BHK cells using two nonionic detergents Triton X-100 (TX-100) and octyl glucoside (OG). Our results show that while M protein acquired resistance to both TX-100 and OG extraction, F and HN exhibited insolubility only to TX-100 but not to OG. Furthermore, in the presence of high salt (1 M NaCl), M, but not F or HN, became TX-100 soluble. Both type I (F) and type II (HN) viral glycoproteins acquired TX-100 insolubility at a late stage during exocytic transport as they acquired endo H resistance. In addition, TX-100 insoluble F and HN exhibited a lighter density compared to TX-100 resistant M by flotation analysis. Using recombinant vaccinia viruses that express Sendai virus HN, F, or M protein individually, we observed that each viral protein (F, HN, or M) was independently capable of acquiring TX-100 insolubility in the absence of other viral components. These results would indicate that while Sendai viral F and HN became bound to TX-100 insoluble lipids, M protein bound ionically to TX-100 insoluble cytoskeletal components and not to TX-100 insoluble lipids.

The human parainfluenza virus type-1 prototypic strain contains a heat-labile hemagglutinin-neuraminidase protein.

The virus yield of human parainfluenza virus type-1 (hPIV-1) in cultured cells at 38 degrees C is reduced more than 100-fold compared to 34 degrees C, while the virus yield of Sendai virus (SV, Enders strain), a murine parainfluenza virus type-1 with high homology to hPIV-1 was almost equal at both temperatures. To understand the basis for the differences in the temperature growth characteristics of the two viruses, we examined the heat-stability of hPIV-1 and SV glycoproteins expressed from cDNAs by pulse-chase experiments. The hemagglutinin-neuraminidase (HN) protein of hPIV-1 was stable after a 6-h chase at 34 degrees C, while at 38 degrees C prominent protein degradation was observed starting at 3 h chase and by 6 h HN was reduced by 65%. In contrast, SV HN protein was stable at both 34 and 38 degrees C. The other hPIV-1 glycoprotein, the fusion (F) protein was stable at both temperatures. To identify the amino acids which are responsible for the heat-lability of hPIV-1 HN, mutant HN proteins were constructed by site-directed mutagenesis. Mutant hPIV-1 HN which had substitutions at positions 461 and 462 became heat-stable at 38 degrees C. These data indicate amino acids around 461 are responsible for the heat-lability of the wild type hPIV-1 HN protein and the reduced yield of the virus at 38 degrees C.

Comparative analysis of the immunoprotective abilities of glycosylated and deglycosylated parainfluenza virus type 3 surface glycoproteins.

The role of carbohydrate moieties on the immunoprotective ability of parainfluenza virus type 3 (PIV-3) haemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins was tested in hamsters. HN and F proteins were purified from detergent-solubilized virus by lentil-lectin affinity chromatography and deglycosylated by treatment with endoglycosidase F (endo F). Immunization of hamsters with either 1 or 5 micrograms of mock-treated (glycosylated) affinity-purified proteins elicited strong haemagglutination inhibition and neutralizing antibody responses 4 weeks after the primary injection. In contrast, titres were significantly lower with endo F-treated (deglycosylated) proteins. However, following the booster doses with at least 5 micrograms of antigen, glycosylated and deglycosylated proteins induced comparable antibody titres. There was no significant difference in the ability of the glycosylated or deglycosylated proteins to protect either the upper or lower respiratory tracts of immunized hamsters against PIV-3 challenge. These results suggest that the carbohydrate moieties of the HN and F proteins are not necessary for eliciting a protective response in hamsters.

Crystals of hemagglutinin-neuraminidase of parainfluenza virus contain triple-stranded helices.

When purified dimers of hemagglutinin-neuraminidase molecules released by protease digestion from three strains of human parainfluenza virus 1 were used in crystallization trials, long thin needle crystals formed. Electron microscopic analysis of these needle crystals revealed that they are composed of stacks of triple-stranded helices with each strand of the helix made up of subunits of hemagglutinin-neuraminidase. To our knowledge, this is the first direct demonstration of the assembly of protein subunits into large triple-stranded helices. An understanding of the organization of these triple helices may shed light on the structural properties of the hemagglutinin-neuraminidase molecules that cause them to form these helices.

Crystallization of biologically active hemagglutinin-neuraminidase glycoprotein dimers proteolytically cleaved from human parainfluenza virus type 1.

We isolated, purified, and characterized the hemagglutinin-neuraminidase (HN) of human parainfluenza virus type 1, with the ultimate goal of producing crystals suitable for three-dimensional X-ray structure analysis. Pronase was used to cleave the globular head of the HN molecule directly from virus particles, forming HN monomers and dimers. The purified dimers retained neuraminidase and hemadsorption activity and were recognized by 14 anti-HN monoclonal antibodies, demonstrating intact HN antigenic structure and function. N-terminal sequence analysis of the dimers showed that cleavage had occurred at amino acid 136 or 137, freeing the C-terminal 438 or 439 amino acids. On electron micrography, the dimer appeared as two box-shaped structures, each approximately 5 by 5 nm. When the purified HN dimers were crystallized in hanging drops by vapor diffusion against 20% polyethylene glycol 3350, they formed both rectangular plates and needlelike crystals. The rectangular crystals diffracted X-rays, indicating an ordered atomic structure. However, the resolution was approximately 10 A (1 nm), insufficient for three-dimensional structural analysis. Experiments to improve the resolution by increasing the size and quality of the crystals are in progress.

Comparative analysis of the immunostimulatory properties of different adjuvants on the immunogenicity of a prototype parainfluenza virus type 3 subunit vaccine.

The immunogenicity of a parainfluenza virus type 3 (PIV-3) subunit vaccine consisting of affinity-purified haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins was tested in guinea-pigs and hamsters. The ability of several different immunopotentiating agents to enhance the antibody response of animals to the PIV-3 surface glycoproteins was evaluated. The immunity induced by HN and F alone was compared with the response elicited by purified proteins combined with Freund's complete adjuvant, aluminium phosphate, Syntex's threonyl-muramyl dipeptide (MDP) SAF-MF formulation, or Ribi's adjuvant formulation containing BCG cell wall skeleton (CWS), trehalose dimycolate (TDM) and monophosphoryl lipid A (MPL) in a 2% squalene-in-water emulsion. Purified proteins were also incorporated into three different liposome formulations prepared by the detergent dialysis procedure. Immunization of guinea-pigs and hamsters with two 15 micrograms doses of the PIV-3 surface glycoproteins administered in the absence of adjuvant elicited high haemagglutination inhibition, neutralization and anti-fusion titres. The liposome preparations failed to enhance the antibody titres. Ribi's adjuvant formulation was effective at inducing a good secondary response to the purified proteins while the immunostimulatory effects of aluminium phosphate, Syntex and Freund's adjuvants were clearly demonstrated in both primary and secondary responses. When administered without adjuvant, a 15 microgram dose of the HN and F mixture was capable of protecting hamsters against live virus challenge. The immunoprotective dose of the purified proteins could be reduced to at least 0.1 microgram by the addition of aluminium phosphate, Syntex or Freund's adjuvants.

Identification of amino acid positions associated with neuraminidase activity of the hemagglutinin-neuraminidase glycoprotein of Sendai virus.

Identification of amino acid positions associated with neuraminidase activity on the hemagglutinin-neuraminidase (HN) glycoprotein of paramyxoviruses has been difficult because neuraminidase-inhibiting antibodies are not neutralizing and thus, escape mutants have not been isolated. Instead, many investigators have correlated an altered neuraminidase (NA) activity of natural virus variants, such as plaque-size variants, with sequence changes in the HN protein. To identify regions on the HN glycoprotein of Sendai virus (SV) that are associated with NA activity, we investigated NA activity of three plaque-size variants which potentially differed from the standard SV (SV/std). NA activity was measured by the ability of virus to elute from chicken erythrocytes as a result of cleaving sialic acid receptors, and by the ability of virus to cleave sialic acid from the small trisaccharide neuraminlactose and the larger substrate fetuin in an in vitro assay. Virions purified from each of the isolated plaques had a HN content and hemagglutinating activity similar to that of SV/std, yet each variant eluted much more rapidly from chicken erythrocytes than SV/std. In vitro NA activity of the plaque-size variants was 1.6 to 3.8 times greater than that of SV/std, providing supporting evidence for the elution data. Although all plaque-size variants showed elevated NA activity, there was no correlation of activity with plaque size. Sequence analysis showed that one of the variants had an amino acid change from glutamic acid to valine at position 165 and from lysine to glutamic acid at position 461, while a second variant had only the change at position 461. A third variant had a nearby change at position 468, from threonine to lysine. Taken together, these data support the conclusion that the amino acid residues at positions 461-468 and 165 are involved in neuraminidase activity of SV.

Defective assembly and intracellular transport of mutant paramyxovirus hemagglutinin-neuraminidase proteins containing altered cytoplasmic domains.

The hemagglutinin-neuraminidase (HN) integral membrane protein of paramyxoviruses is expressed at the cell surface as a tetramer consisting of a pair of disulfide-linked dimers. HN has a large C-terminal ectodomain, a 19-residue uncleaved signal-anchor domain, and a 17-residue N-terminal cytoplasmic tail. Various mutant HN genes were constructed to examine the role of residues flanking the signal-anchor domain, including the cytoplasmic tail, on assembly and intracellular transport of the HN glycoprotein. Expression of the altered genes showed that by 90 min after synthesis the majority of the mutant HN proteins were in a conformationally mature form as assayed by their reactivity with conformation-specific monoclonal antibodies. However, the mutant proteins showed varied endoplasmic reticulum-to-Golgi apparatus transport rates, ranging from that of wild-type HN (t1/2 approximately 90 min) to slowly transported molecules (t1/2 approximately 5 h) and to molecules in which transport was not detected. Pulse-chase experiments indicated that the altered HN molecules had a specific and transient interaction with the resident endoplasmic reticulum protein GRP78-BiP, and thus the altered HN molecules were not retained in the endoplasmic reticulum by a prolonged interaction with GRP78-BiP. Sucrose density gradient sedimentation analysis of the mutant HN molecules indicated that they all had an oligomeric form that differed from that of wild-type HN; most of the molecules were found as disulfide-linked dimers rather than as tetramers. These data suggest that the HN cytoplasmic tail may function in the assembly of the final transport-competent oligomeric form of HN and that mutant HN molecules with seemingly properly folded ectodomains are retained in the endoplasmic reticulum by an as yet unidentified mechanism. The possible role of the HN cytoplasmic tail as a signal for intracellular transport is discussed.

Selective and transient association of Sendai virus HN glycoprotein with BiP.

From 10-min [35S]methionine pulse-labeled Sendai virus-infected BHK cells, an anti-BiP monoclonal antibody precipitated, along with the BiP protein, the hemagglutinin-neuraminidase protein (SV-HN) fivefold better than the fusion protein (SV-Fo). A minimal estimate of 30% of the newly made HN was complexed to BiP. The majority of the HN in the complex was endo-H sensitive and the molar ratio of BiP:HN was estimated to be 1:2. With time, HN dissociated from BiP, and the rate of dissociation was found to be inversely proportional to the rate at which HN acquired its native structure. It is proposed that association with BiP followed by slow release (i) is responsible for the HN slow maturation and (ii) represents a normal step in its maturation pathway.

Proteins of Newcastle disease virus envelope: interaction between the outer hemagglutinin-neuraminidase glycoprotein and the inner non-glycosylated matrix protein.

Using linear sucrose-density ultracentrifugation analysis of Triton-solubilized Newcastle Disease Virus envelopes, we have evidenced, for the first time, the existence of interactions between the outer hemagglutinin-neuraminidase transmembrane glycoprotein and the inner non-glycosylated peripheral matrix protein. Such interactions seem to be electrostatic. These conclusions are based on the behavior of both proteins at different ionic strengths. When in low ionic strength buffer, hemagglutinin-neuraminidase and matrix proteins band together in the sucrose gradient, whereas at high ionic strength both proteins band at different rates in the gradient. The behavior of the inner matrix protein in our conditions was the expected one for a peripheral protein. The results of these 'in vitro' studies are also discussed in terms of the possible 'in vivo' role of such interactions.