Biochemical features of mumps virus neuraminidases and their relationship with pathogenicity

The neuraminidases (NANases) of six strains of mumps virus were characterized biochemically. Though the hemagglutinin-neuraminidase (HN) glycoproteins were similar in apparent size and immunological properties, the NANase activities exhibited different behavior. Each NANase had a characteristic pH optimum and thermal stability. Kinetic analyses demonstrated unique K_m catalytic constants, and K_i (Cl^?) values for the different NANases. Three strains, Enders, RW, and O'Take, had active NANases (V′_max = 179–680 nmol/min/mg virus protein) that hydrolyzed 37–50% of the total neuraminic acid (NANA) of fetuin; the other strains, Kilham, MJ, and Jeryl Lynn B, had less active NANases (V′_max = 48–95 nmol/min/mg) capable of hydrolyzing only 10–27% NANA of fetuin. The virus hemagglutination activities (HA) and the elution rates for adsorbed virions were consistent with the NANase properties. Enders, RW, and O'Take strains agglutinated erythrocytes most efficiently at physiological NaCl concentration; HA activities for Kilham, MJ, and Jeryl Lynn B strains were not affected by NaCl concentration. Adsorbed Enders, RW, and O'Take virus eluted more rapidly from erythrocytes than did adsorbed Kilham, MJ, and Jeryl Lynn B strains. These properties, associated with the HN glycoprotein, are correlated with the cytopathogenicity of the strains. The O'Take and RW strains cause no cell fusion and little cytopathology; Kilham, MJ, and Jeryl Lynn B strains produce extensive cell fusion after infection of CV-1 cells. These results suggest that, in concert with the paramyxovirus fusion (F) glycoprotein, the viral NANase contributes to the cytopathology of an infection, an active NANase promoting rapid release of progeny virions, so that no cell fusion occurs, and the action of a less active NANase prolonging the association of progeny virus with the infected cell surface, increasing the likelihood of cell fusion.     

Activation of the alternative complement pathway by mumps infected cells: Relationship to viral neuraminidase activity

Summary An inverse relationship exists between the sialic acid content of a particle and its ability to activate the alternative complement pathway. The present studies were performed to determine if the neuraminidase (NANase) activities of different mumps virus strains could influence the ability of mumps virus infected cells to activate the alternative pathway. CV-1 cells were infected with three different mumps virus strains (RW, O'Take, and Kilham) and after 24 hours, 10 percent guinea pig serum (GPS) treated with EGTA/MgCl₂ or GPS lacking the 4th component of complement (C4DGPS) was added to the cell monolayers. After 30 minutes, the percentage C3 consumed was determined by a functional hemolytic assay. Cells infected with RW (high NANase) consumed significantly more C3 (23.2 per cent) than cells infected with Kilham (5.7 percent, low NANase). Cells infected with O'Take were intermediate in their ability to activate C3. The degree of C3 deposition on the surface of infected cells, detected by fluorescence microscopy, was also greater for cells infected with the RW than the Kilham strain of mumps virus. These studies suggest that the NANase activity of mumps virus can influence the ability of infected cells to activate the alternative pathway and thereby, the ability of complement to participate in host defense against mumps virus infection.With 1 Figure     

Differentiation of mumps virus strains with monoclonal antibody to the HN glycoprotein.

A hybridoma cell line secreting antibody of the immunoglobulin G3 isotype with kappa light chains and with activity against the HN glycoprotein of the Kilham strain of mumps virus was established. The antibody exhibited structural homogeneity in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had a microheterogeneous isoelectric spectrum characteristic of an antibody of monoclonal origin. The specificity of the monoclonal antibody, shown by immunoprecipitation performed with radiolabeled virus and infected cell lysates, was for the larger mumps virus glycoprotein. In functional assays the antibody inhibited the hemagglutinating and neuraminidase activities and neutralized the infectivity of the homologous Kilham strain of virus and clearly differentiated this strain from two heterologous strains, Enders and O'Take. The antibody was markedly less effective with the O'Take strain than with either the Kilham or Enders strain in inhibiting both hemagglutination and neuraminidase activity against the macromolecular substrate fetuin. The inhibition of the neuraminidase activity of the Kilham strain was independent of substrate size, the antibody inhibiting the hydrolysis of both fetuin and the trisaccharide neuraminlactose. By contrast, the antibody did not inhibit the hydrolysis of neuraminlactose by the two heterologous mumps strains. These results provide the first demonstration of antigenic differences between mumps virus strains and highlight the utility of monoclonal antibody in analyzing the structural basis underlying functional activities of the HN glycoproteins.     

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.     

Identification of amino acids involved in the sialidase activity of the mumps virus hemagglutinin-neuraminadase protein

We previously described sialidase-deficient variants of the O'Take strain of mumps virus obtained by growth under the selective pressure of the competitive sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA). In this report, we describe the production of a sialidase-deficient variant of the RW strain of mumps virus using an identical selection protocol. The biologic activities of the RW variant, RW(DANA)v1, were identical to those described for O'Take-(DANA)v1 and included a lack of detectable sialidase activity, unchanged hemagglutination activity, and expression of cell-to-cell fusion in infected cell monolayers. Analysis of the structural proteins of each virus by both two-dimensional tryptic peptide mapping and monoclonal antibody binding assays suggested that limited changes occurred in the hemagglutinin-neuraminidase (HN) proteins and that only the HN proteins were altered. The complete nucleotide sequence of the RW(DANA)v1 HN was determined and compared to the HN sequence of the RW parent. Two nucleotide differences accounting for two nonconservative amino acid differences were noted; an lle to a Thr at amino acid 181 and a Gln to Lys at amino acid 261 from RW to RW(DANA)v1, respectively. By comparing the data presented here with those reported for several other paramyxoviruses, we tentatively identify amino acid 181 as a critical residue in the active site of the mumps virus sialidase enzyme.     

Polypeptide synthesis i mumps virus-infected cells

The synthesis of polypeptides in infected Vero cells has been studied with two tissue culture adapted strains of mumps virus. Minor differences between the strains are found in the mobilities of the virus-specific polypeptides in SDS-PAGE. Eight virus-induced polypeptides have been identified with mol. wt. of 180K, 80K, 71K, 69K, 45 K, 39K, 23K and 17K. These may correspond to the L, HN, N, Fo, P, M, C and S polypeptides identified in other paramyxoviruses. The 69K F0 glycoprotein is probably a precursor for glycopolypeptides F1 and F2 with mol. wt. 61K and 14K, respectively.     

腮腺炎病毒疫苗株与野毒株的部分核苷酸序列分析

目的比较腮腺炎病毒疫苗株与野毒株的核苷酸序列差异。方法腮腺炎病毒疫苗株Ｓ７９株和ＪｅｒｙｌＬｙｎｎ株在鸡胚细胞上从第５代连续传至第２５代,采用逆转录－套式聚合酶链反应（ＲＴ－ｎＰＣＲ）法,从传代前后的腮腺炎病毒疫苗株以及野毒株的基因组中扩增血凝素－神经氨酸酶基因片段（ＨＮ）、融合蛋白基因片段（Ｆ）和小分子疏水蛋白基因（ＳＨ）,并利用双脱氧核苷酸链终止法对ＰＣＲ产物进行测序。结果传代之前第５代的两种疫苗株的目的基因的核苷酸序列完全一致;传至第２５代的Ｓ７９株的目的基因的核苷酸序列比２５代的ＪｅｒｙｌＬｙｎｎ株显示了更高的突变率。比较现行疫苗株与野毒株的核苷酸序列后发现,１９９５年分离的野毒株的核苷酸序列与疫苗株有明显的差异。结论推测Ｓ７９株可能与ＪｅｒｙｌＬｙｎｎ株同源,同一地区可能有两种以上的腮腺炎病毒野毒株导致流行性腮腺炎的发生     

Changes in conformation and charge paralleling proteolytic activation of Newcastle disease virus glycoproteins

The hemagglutinin-neuraminidase and the fusion glycoprotein of strains Italien and Ulster of Newcastle disease virus were isolated by isoelectric focusing after solubilization with n-octylglucoside or other nondenaturing detergents. The isoelectric points of the glycoproteins varied depending on the virus strain; host-specific variations were not observed. When the precursor F₀ of the fusion protein of strain Ulster was converted by proteolytic cleavage into the complex F_{1, 2}, there was a shift in the isoelectric point from pH 6.3 to pH 5.0. When the precursor HN₀ of the same strain was cleaved to HN, its isoelectric point (pH 5.6) did not change. A shift from a more basic to a more acidic pH was also observed, when the isoelectric points of F₀ and F_{1, 2} were analyzed under denaturing conditions. These observations are compatible with the concept that F₁ and F₂ are linked in the precursor by a basic intervening sequence that is eliminated in the cleavage reaction. Circular dichroism spectra of the isolated glycoproteins have been determined. The spectra obtained from glycoprotein F_{1, 2} of strain Italien and of strain Ulster were similar, but there were strain-specific differences with glycoprotein HN. With the hemagglutinin-neuraminidase and the fusion protein of strain Ulster, precursors and cleavage products showed different spectra. This indicates that proteolytic activation of both glycoproteins is paralleled by a conformational change.     

Covalent attachment of psoralen to a single site on vesicular stomatitis virus genome RNA blocks expression of viral genes

Mumps virus was adapted to growth in Vero cells, which yielded virus of high infectivity titers. The structural polypeptides of purified virions grown in Vero cells were similar to those described previously for egg-grown mumps virus: L (200K), HN (79K), NP (72K), F₁ (61K), P (45K), M (40K), and F₂ (16K). We have analyzed the synthesis of viral polypeptides in Vero cells by pulse labeling with radioactive amino acid precursors. The nucleoprotein (NP) was the first to be detected intracellularly above the cellular protein background at 6 h.p.i. By 12 h.p.i., all viral polypeptides were observed except for the glycoproteins F₁ and F₂, which are derived from a precursor designated F₀ (74K). Two low-molecular-weight polypeptides not present in purified virions were also detected in infected cells. They are designated pI (28K) and pII (19K). Peptide mapping revealed that these two polypeptides share regions of their amino acid sequence and that they are also related to the structural protein P. Polypeptides pI and pII were found in several cell types (Vero, CEF, MDBK cells) infected with mumps virus. Infection of Vero cells with other paramyxoviruses (SV5 and Sendai virus) did not induce the synthesis of proteins comparable to pI and pII, whereas in mumps virus-infected cells no counterpart to the nonstructural C protein of Sendai virus was detected. Pulse-chase experiments suggest that pI and pII may not be derived from P by proteolytic cleavage.     

Hemagglutinin-neuraminidase sequence and phylogenetic analyses of mumps virus isolates from a vaccinated population in Singapore

During 1999-2000, a sustained mumps outbreak in the highly vaccinated population in Singapore was attributed to vaccine failure associated with the Rubini vaccine strain. To explain this phenomenon, the complete nucleotide and amino acid sequences of the hemagglutinin-neuraminidase (HN) gene of eight mumps virus isolates from patients with parotitis in Singapore were determined and compared with those of known vaccine strains. Phylogenetic trees constructed on the basis of HN nucleotide and amino acid sequences showed that the Singapore mumps virus isolates were more closely related to the Urabe strain and belonged to a different cluster from the Rubini and Jeryl-Lynn strains. The Rubini vaccine showed only approximately 93% nucleotide and approximately 96% amino acid sequence similarity to Urabe and Singapore isolates. Compared with the vaccine strains, six of the eight isolates lacked the extracellular glycosylation site at residues 400-402. Other significant amino acid disparities (e.g., at residue 354) may also affect the antigenic properties of the HN protein. These findings suggest that the evolution and adaptation of the currently circulating mumps virus strains in the community has led to the emergence of genetically distinct viral strains. The low vaccine efficacy of the Rubini strain represents a major reason for the recent mumps resurgence and failure of mumps immunization in Singapore.     

Activation of precursors to both glycoproteins of Newcastle disease virus by proteolytic cleavage

With strain Ulster of Newcastle disease virus, two precursor glycoproteins, HN₀ and F₀, were identified; these are converted by proteolytic cleavage into glycoproteins HN and F, respectively. Purified virions containing predominantly glycoproteins HN₀ and F₀ together with a small amount of HN are not hemolytic and have reduced levels of hemagglutinating and neuraminidase activity and of infectivity. After in vitro treatment with the appropriate proteolytic enzymes, biological activities are fully expressed in these particles. The precursor glycoprotein HN₀ was isolated and found to be largely devoid of hemagglutinating and neuraminidase activities. High levels of both activities were present, however, when this material was subjected to proteolytic cleavage. These observations demonstrate that cleavage is a precondition for the biological activity not only of glycoprotein F but also of glycoprotein HN. There is a striking difference between glycoproteins HN₀ and F₀ with repsect to their susceptibility to proteolytic enzymes. Cleavage and activation of HN₀ can be accomplished by a variety of proteases, such as chymotrypsin, elastase, thermolysin, and trypsin. In contrast, F₀ shows a specific requirement for trypsin.     

Expression of mumps virus glycoproteins in mammalian cells from cloned cDNAs: both F and HN proteins are required for cell fusion.

Two recombinant plasmids were constructed by inserting the cDNAs of either the fusion (F) or the hemagglutinin-neuraminidase (HN) protein genes of mumps virus into the pcDL-SR alpha expression vector. Both the F and the HN proteins expressed in COS7 cells transfected with their respective recombinant plasmids were indistinguishable in terms of electrophoretic mobility from their counterparts synthesized in mumps virus-infected cells. The F protein was cleaved and expressed on the cell surface, but uncleaved forms were also detected. The expressed HN protein was transported to the cell surface and adsorbed guinea pig erythrocytes. Syncytium formation was induced when COS7 cells were transfected with both recombinant plasmid DNAs together, but not with the recombinant plasmid only carrying the F gene. This observation indicates that cell fusion mediated by mumps virus requires both the F and the HN glycoproteins.     

Characterization of virus obtained from MDBK cells persistently infected with a variant of herpes simplex virus type 1 strain MP [HSV-1(MP)]

Virus clones which express glycoprotein gC (gC+) were obtained from two persistently infected (p.i.) MDBK cell lines which had been independently established by infection with HSV-1(MP)10311, a gC- syncytial (syn) variant of herpes simplex virus type 1 strain MP [HSV-1(MP)]. The gC+ revertants were syn in MDBK, HEp-2, and Vero cell lines and in primary human fibroblasts; this offers further evidence that glycoprotein gC does not inhibit cell fusion. The gC+ revertants represented from 70 to 100 percent of the virions present in the virus populations examined, thus suggesting a possible selective advantage of the gC+ revertants in this system of persistent infection.     

Genomic diversity of mumps virus and global distribution of the 12 genotypes

The WHO recently proposed an updated nomenclature for mumps virus (MuV). WHO currently recognizes 12 genotypes of MuV, assigned letters from A to N (excluding E and M), which are based on the nucleotide sequences of small hydrophobic (SH) and haemagglutinin‐neuraminidase (HN) genes. A total of 66 MuV genomes are available in GenBank, representing eight of the 12 genotypes. To complete this dataset, whole genomes of seven isolates representing six genotypes (D, H, I, J, K and L) and one unclassified strain were sequenced. SH and HN genes of other representative strains were also sequenced. The degree of genetic divergence, predicted amino acid substitutions in the HN and fusion (F) proteins and geographic distributions of MuV strains were analysed based on the updated dataset. Nucleotide heterogeneity between genotypes reached 20% within the SH gene, with a maximum of 9% within the HN gene. The geographic and chronologic distributions of the 12 genotypes were summarised. This review contributes to our understanding of strain diversity for wild type MuV, and the results support the current WHO nomenclature. © 2014 Crown copyright. Reviews in Medical Virology © 2014 John Wiley & Sons, Ltd.     

Comparative clinico-morphological research on measles and mumps infections in an experiment on guinea pigs

The pathological process typical of measles and mumps infections was reproduced clinically and morphologically in 250 experimental guinea pigs infected into the anterior eye chamber with different strains of measles and mumps viruses. The intraocular inoculation of measles virus induced in guinea pigs the signs of iridocyclitis and follicular conjunctivitis, whereas inoculation of mumps virus induced dacryoadenitis as well as iridocyclitis and follicular conjunctivitis. The severity of the reproduced process and its dynamics correlated with the pathogenicity of measles and mumps virus strains used in the experiment. The specificity of pathological changes observed in the eye structures was verified virologically by the isolation of virus from the eye membranes and the presence of virus-neutralizing antibodies in the blood sera of the animals.     

Molecular epidemiology of mumps virus in Japan and proposal of two new genotypes

We isolated 872 strains of mumps virus from naso-pharyngeal secretions in seven different districts of Japan from January 2000 to July 2001. Among them, 57 strains were geno-typed by nucleotide sequencing in part of the hemagglutinin-neuraminidase (HN) and small hydrophobic (SH) protein regions. Four different genotypes (B, G, K, and L) of mumps virus were co-circulating in Japan and the distribution of genotypes varied in geographically different districts. Two new clusters designated as genotypes K and L had more than 7% nucleotide variation in the SH gene. Among the 57 strains, 11 were classified as B, 35 as G, three as K, and eight as L, which was mainly isolated in Tokyo. We also examined 104 stains isolated in a clinic in Mie prefecture from 1993 to 2003. Genotype B was the indigenous strain and genotype K was introduced in 1994. Genotypes B and K co-circulated in the 1990s and were replaced by genotype G in 2000. There was no significant change in neutralizing test antibody titers against genotypes B, G, K, and L using seven post-vaccination sera with Hoshino strain (genotype B) and these four genotypes had a different antigenicity from genotype A. We should continue to watch on mumps virus molecular epidemiology.     

Chemiluminescence: an early event in the interaction of Sendai and influenza viruses with mouse spleen cells. I. The role of the envelope glycoproteins in the stimulation of chemiluminescence

On infection with Sendai virus, non-adherent mouse spleen cells emit a burst of chemiluminescence (CL) starting within a few seconds and peaking at 6–8 min postinfection. The biological reactions leading to CL are not known in mouse spleen cells, but in phagocytic cells are believed to be correlated with the generation of unstable oxygen species by the cells (e.g., H₂0₂, 0₂^?, OH·, singlet oxygen). In this paper, we have investigated the mechanism of CL induction by the virus. Envelope particles, possessing the hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins stimulated CL, suggesting that the biochemical reactions leading to light emission are triggered by the interaction of the envelope “spike” glycoproteins with the cell surface. The individual contributions of HN and F to CL stimulation were investigated by removing F from egg-grown virus and by using Sendai virus (grown in MDBK cells) which possesses F₀, the biologically inactive precursor of F. Both viral preparations still induced CL. However, CL was reduced and the peak of light emission shifted from 6–8 to 2.5 min postinfection. In MDBK cell-grown Sendai virus, cleavage of F₀ into F resulted in the increase of CL and shift back of the peak CL to 6–8 min postinfection. These results suggest that the bulk of light emission by the spleen cells is correlated with the action of the F protein. In addition, HN correlates with CL in the initial period following the addition of the virus to the spleen cell suspension, while F is important for the subsequent further increase in light emission. The mechanism of CL induction by the F glycoprotein was investigated using egg-grown nonhemolytic “early harvest” and hemolytic “late harvest” Sendai virus, respectively. “Early harvest” virus, known to possess F and to have fusion activity, was less active in CL induction than “late harvest” Sendai virus which expresses both the fusion and hemolytic activities. This suggests that F stimulates CL by a mechanism related to hemolysis, rather than by envelope-cell membrane fusion. In addition, we show that influenza virus (strain A/RI/5-) also induces CL. The kinetics and extent of CL induction by this virus were similar to those induced by Sendai virus lacking the fusion protein and by nonhemolytic Sendai virus possessing F. As with Sendai virus, Pronase treatment resulted in the loss of CL stimulation while uv-inactivation of the virus did not affect its CL-inducing activity, suggesting that influenza virus also triggers CL by a mechanism involving the envelope glycoproteins.     

Characterization of haemagglutinin-neuraminidase glycoprotein of Newcastle disease virus expressed by a recombinant baculovirus

A recombinant baculovirus containing a cDNA which encodes haemagglutinin-neuraminidase (HN) of Newcastle disease virus (NDV) was constructed. Spodoptera frugiperda cells infected with this recombinant virus produced a large amount of HN glycoprotein similar to the authentic HN in size. The recombinant HN glycoprotein was localized on the surface of the infected cells and conserved its haemadsorption and neuraminidase activities. The antigenic properties of the recombinant HN glycoprotein seemed to be slightly different from the authentic one, as judging by the reactivity with a panel of monoclonal antibodies specific to the antigenic sites responsible for neutralization of viral infectivity. Chickens inoculated with the cells infected with the recombinant virus developed haemagglutination-inhibition and virus neutralization antibodies, and were completely protected from the NDV challenge.     

Fusion protein of the paramyxovirus SV5: destabilizing and stabilizing mutants of fusion activation

The fusion (F) protein of the paramyxovirus SV5 strain W3A causes syncytium formation without coexpression of the SV5 hemagglutinin-neuraminidase (HN) glycoprotein, whereas the F protein of the SV5 strain WR requires coexpression of HN for fusion activity. SV5 strains W3A and WR differ by three amino acid residues at positions 22, 443, and 516. The W3A F protein residues P22, S443, and V516 were changed to amino acids found in the WR F protein (L22, P443, and A516, respectively). Three single-mutants, three double-mutants, and the triple-mutant were constructed, expressed, and assayed for fusion using three different assays. Mutant P22L did not cause fusion under physiological conditions, but fusion was activated at elevated temperatures. Compared with the W3A F protein, mutant S443P enhanced the fusion kinetics with a faster rate and greater extent, and had a lower activation temperature. Mutant V516A had little effect on F protein-mediated fusion. The double-mutant P22L,S443P was capable of causing fusion, suggesting that the two mutations have opposing effects on fusion activation. The WR F protein requires coexpression of HN to cause fusion at 37 degrees C, and does not cause fusion at 37 degrees C when coexpressed with influenza virus hemagglutinin (HA); however, at elevated temperatures coexpression of WR F protein with HA resulted in fusion activation. In the crystal structure of the core trimer of the SV5 F protein (Baker, K. A., Dutch, R. E., Lamb, R.A., and Jardetzky, T. S. (1999). Mol. Cell 3, 309-319), S443 is the last residue (with interpretable electron density) in an extended chain region and the temperature factor for S443 is high, suggesting conformational flexibility at this point. Thus, the presence of prolines at residues 22 and 443 may destabilize the F protein and thereby decrease the energy required to trigger the presumptive conformational change to the fusion-active state.