Virus Hemagglutinin-neuraminidase Glycoprotein Research Articles

Amino acid change 335 E to K affects the sialic-acid-binding and neuraminidase activities of Urabe AM9 mumps virus hemagglutinin–neuraminidase glycoprotein

A mutation coding for the amino acid change E 335 to K is frequently found in the hemagglutinin-neuraminidase (HN) gene of Urabe AM9 mumps viruses isolated during post-vaccination meningitis cases. To identify if this mutation modifies the biological activities of the HN glycoprotein, two variants of Urabe AM9 vaccine differing at amino acid 335 (HN-E 335 and HN-K 335) were isolated and their receptor-binding specificity was determined by means of competence assays. Pre-incubation of the viruses with sialic acids inhibited both syncytia formation in Vero cells and replication in SH-SY5Y cells. Thus, HN-K 335 showed higher affinity towards sialylα2,6lactose, whereas HN-G 335 preferred sialylα2,3lactose. These results are relevant because a high expression of sialylα2,6lactose in nerve cells was confirmed by means of Sambucus nigra lectin-cytochemistry. In addition, kinetics assays showed that HN-K 335 and HN-E 335 also differ in their hydrolysis rate ( V max values of 37.5 vs. 3.5 nmol min −1 mg −1, respectively). Therefore, HN-K 335 variant presented a neuraminidase activity level 11-fold higher than that of HN-E 335 variant. In conclusion, the mutation affects the receptor-binding and neuraminidase activities of Urabe AM9 mumps virus variants.

Human Parainfluenza Virus 3 Neuraminidase Activity Contributes to Dendritic Cell Maturation

Mechanisms of dendritic cells (DCs) immunomodulation by parainfluenza viruses have not been characterized. We analyzed whether the human parainfluenza 3 (HPF3) virus hemagglutinin-neuraminidase glycoprotein (HN) might influence DC maturation. HN possesses a receptor binding function and a neuraminidase or desialidating activity. To assess whether the neuraminidase activity of HN affects DC maturation, human myeloid DCs were exposed to either live or UV-inactivated HPF3 viruses containing wild type or a mutated form of HN with decreased neuraminidase activity. Exposure of human DCs to either UV-inactivated or live virus induced up-regulation of CD83 and CD86 surface markers, morphological changes, and a cytokine expression pattern consistent with maturation. However, the level of maturation was found to be lower in DCs infected with the neuraminidase deficient variant as compared to the wild type. These results suggest that during the course of viral infection, HN's neuraminidase activity may play an important role contributing to maturation and activation of DCs.

Expression of biologically active and antigenically authentic parainfluenza Type 3 virus hemagglutinin-neuraminidase glycoprotein by a recombinant baculovirus

The hemagglutinin-neuraminidase (HN) gene of human type 3 parainfluenza virus has been inserted into a baculovirus expression vector under the control of the polyhedrin promoter. HN protein produced in insect cells by the recombinant baculovirus appeared to be glycosylated, was transported to the cell surface, and was biologically active. All of the HN epitopes previously mapped functionally to a region(s) involved in neuraminidase and/or hemagglutination activities were conformationally unaltered on the recombinant protein. The HN produced in this system also induced a protective immune response in immunized cotton rats. From these studies we conclude that the HN expressed in insect cells represents a source of authentic HN glycoprotein suitable for structural analysis and immunization.

Membrane vesicles containing the Sendai virus binding glycoprotein, but not the viral fusion protein, fuse with phosphatidylserine liposomes at low pH.

Membrane vesicles containing the Sendai virus hemagglutinin/neuraminidase (HN) glycoprotein were able to induce carboxyfluorescein (CF) release from loaded phosphatidylserine (PS) but not loaded phosphatidylcholine (PC) liposomes. Similarly, fluorescence dequenching was observed only when HN vesicles, bearing self-quenched N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (N-NBD-PE), were incubated with PS but not PC liposomes. Thus, fusion between Sendai virus HN glycoprotein vesicles and the negatively charged PS liposomes is suggested. Induction of CF release and fluorescence dequenching were not observed when Pronase-treated HN vesicles were incubated with the PS liposomes. On the other hand, the fusogenic activity of the HN vesicles was not inhibited by treatment with dithiothreitol (DTT) or phenylmethanesulfonyl fluoride (PMSF), both of which are known to inhibit the Sendai virus fusogenic activity. Fusion was highly dependent on the pH of the medium, being maximal after an incubation of 60-90 s at pH 4.0. Electron microscopy studies showed that incubation at pH 4.0 of the HN vesicles with PS liposomes, both of which are of an average diameter of 150 nm, resulted in the formation of large unilamellar vesicles, the average diameter of which reached 450 nm. The relevance of these observations to the mechanism of liposome-membrane and virus-membrane fusion is discussed.

Intracellular maturation of mumps virus hemagglutinin-neuraminidase glycoprotein: conformational changes detected with monoclonal antibodies

Monoclonal antibodies elicited by immunization with mumps virus glycoproteins were selected with either native or chymotrypsin-treated mumps virus in an enzyme-linked immunosorbent assay. Group I antibodies which preferentially recognized chymotrypsin-treated virus failed to recognize native mumps virus hemagglutinin-neuraminidase (HN). They did react with sodium dodecyl sulfate-denatured HN and the HN chymotryptic fragments HNc2' (molecular weight, 41,000) and HNc1 (molecular weight, 32,000) after transfer to nitrocellulose paper. In contrast, group II antibodies, which preferentially recognized native virus in the enzyme-linked immunosorbent assay, reacted with native HN but failed to bind HN after sodium dodecyl sulfate denaturation. These two groups of monoclonal antibodies were used to define the maturation pathway of the mumps virus HN in infected cells. The HN initially appeared as a 76,000-molecular-weight polypeptide and was recognized only by group I antibodies. A truncated form of HN, HNT (molecular weight, 63,000), was synthesized in the presence of tunicamycin and was also recognized only by group I antibodies. The 76,000-molecular-weight HN was rapidly converted to a 74,000-molecular-weight polypeptide; this form of HN was recognized only by group II antibodies. The oligosaccharide side chains were modified, and intermolecular disulfide bonds were formed as HN was transported to the cell surface. The disulfide-linked oligomers of HN were direct precursors of the HN found in mature virus.

A fusing mumps virus variant selected from a nonfusing parent with the neuraminidase inhibitor 2-Deoxy-2,3-dehydro- N-acetylneuraminic acid

A fusing variant of the nonfusing O'Take strain of mumps virus was obtained by growing virus under the selective pressure of the competitive neuraminidase inhibitor 2-deoxy-2,3-dehydro- N-acetylneuraminic acid (DANA). The variant virus, O'Take(DANA)v1, causes extensive syncytial formation in CV-1 cell cultures in contrast to the relatively noncytopathic infection caused by the O'Take virus parent. Neuraminidase assays indicate that O'take(DANA)v1 has no detectable neuraminidase activity using either fetuin or neuraminlactose as substrate. In addition, the O'Take(DANA)v1 virus can agglutinate red blood cells but cannot elute from these cells once adsorbed. No differences were detected in the biochemical or antigenic structure of the hemagglutinin-neuraminidase (HN) proteins from O'Take virus and O'Take(DANA)v1. These results indicate that the neuraminidase enzyme of the mumps virus HN glycoprotein is involved in modulating the cell fusion cytopathology of mumps virus infections.

Human parainfluenza type 3 virus hemagglutinin-neuraminidase glycoprotein: nucleotide sequence of mRNA and limited amino acid sequence of the purified protein

The nucleotide sequence of mRNA for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 3 virus obtained from the corresponding cDNA clone had a single long open reading frame encoding a putative protein of 64,254 daltons consisting of 572 amino acids. The deduced protein sequence was confirmed by limited N-terminal amino acid microsequencing of CNBr cleavage fragments of native HN that was purified by immunoprecipitation. The HN protein is moderately hydrophobic and has four potential sites (Asn-X-Ser/Thr) of N-glycosylation in the C-terminal half of the molecule. It is devoid of both the N-terminal signal sequence and the C-terminal membrane anchorage domain characteristic of the hemagglutinin of influenza virus and the fusion (F0) protein of the paramyxoviruses. Instead, it has a single prominent hydrophobic region capable of membrane insertion beginning at 32 residues from the N terminus. This N-terminal membrane insertion is similar to that of influenza virus neuraminidase and the recently reported structures of HN proteins of Sendai virus and simian virus 5.

Restriction of cell surface expression of sendai virus hemagglutinin-neuraminidase glycoprotein correlates with its higher instability in persistently and standard plus defective interfering virus infected BHK-21 cells

To gain an understanding of the mechanism(s) by which Sendai virus generates a persistent infection, the expression of the hemagglutinin-neuraminidase (HN) and fusion (Fo) glycoproteins at the surfaces of BHK-21 cells infected with standard virus, a mixture of standard and defective interfering (DI) particles (mixed virus infection), and during persistent infection was investigated. The expression of HN and Fo was measured on the surfaces of infected cells by the binding of anti-HN and anti-Fo monoclonal antibodies. The results show that HN expression was restricted relative to Fo during mixed virus and persistent infections. The decreased levels of HN were investigated further by pulse-chase experiments which revealed that HN has an increased turnover rate in persistently infected cells and, to a lesser extent, in mixed virus infected cells. In analyzing the [35S]methionine-labeled protein composition of virus particles produced during the pulse-chase experiments, the increased turnover of newly synthesized HN was found to correlate with its decreased incorporation into virus particles. Interestingly, the poor HN incorporation also correlates with less efficient incorporation of the matrix M protein into virus particles.

Intracellular processing of the vesicular stomatitis virus glycoprotein and the Newcastle disease virus hemagglutinin-neuraminidase glycoprotein.

The kinetics of intracellular transport of the vesicular stomatitis virus (VSV) glycoprotein (G) and the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) glycoprotein in chicken embryo cells were compared. To assay for the appearance of pulse-labelled glycoprotein at the cell surface, an antibody-binding assay was developed which allowed the precipitation of only those molecules on the outside surfaces of infected cells. Using this assay, it was found that pulse-labelled VSV G protein appeared at the cell surface with a half-time of approximately 27 min, while pulse-labelled NDV HN glycoprotein reached the cell surface with a half-time of approximately 78 min. To determine the transit time of these glycoproteins to trans-Golgi membranes, the kinetics of the acquisition of endoglycosidase H resistance was analyzed. The half-time of the transit of the G protein to the trans-Golgi membranes was found to be approximately 13 min while that of the HN glycoprotein was found to be approximately 60 min. Since the G protein migrates to the trans-Golgi membranes with a half-time of 13 min, and the cell surface with a half-time of 27 min, the half-time for the transit between the trans-Golgi membrane and the plasma membrane must be approximately 14 min. In a similar analysis, the half-time for the transit of the HN glycoprotein from the trans-Golgi membrane to the plasma membrane must be approximately 18 min, a time not significantly different from that of the G protein. Thus the difference in the kinetics of the intracellular transport of these two glycoproteins resides primarily in the transit from the rough endoplasmic reticulum to the trans-Golgi membranes. These results argue against a non-selective mechanism for the transport of plasma membrane glycoproteins to the cell surface.

Component(s) of Sendai virus that can induce interferon in mouse spleen cells.

To identify the active component of Sendai virus that induces interferon in mouse spleen cells, infectious and noninfectious viruses, envelope particles derived from them, and isolated hemagglutinin-neuraminidase (HN) glycoproteins were examined for interferon induction. The interaction between membranous structures containing Sendai virus HN glycoprotein and the receptors on the cell surface was shown to be sufficient for interferon induction in mouse spleen cells, suggesting that the actual inducer of interferon in mouse spleen cells is the HN glycoprotein of Sendai virus. When mouse spleen cells were stimulated in vitro with Sendai virus grown in eggs or LLC-MK2 cells or with membranous structures containing glycoproteins obtained from these viruses, interferon could be detected in the culture fluid. Furthermore, isolated HN glycoprotein per se could induce interferon in the cells. A linear correlation was found between the titer of interferon induced and the hemagglutinating activity of the membranous structure containing the HN glycoprotein. It was concluded from these findings that HN glycoprotein was the active component of Sendai virus responsible for interferon induction in mouse spleen cells and that viral RNA and F glycoprotein were not required. The results also showed that the interaction between HN glycoprotein and receptors on the cell surface triggered production of type I interferon (IFN-alpha and IFN-beta). Although when Sendai virus was incubated at 56 degrees C for 5 min it lost its hemolytic and hemagglutinating activities, it induced a considerable amount of interferon in the culture fluid of mouse spleen cells. The interferon-inducing ability of heat-inactivated virus could be absorbed with mouse spleen cells but not with sheep erythrocytes or mouse erythrocytes, indicating that the inactivated virus retained ability to bind to mouse lymphoid cells.