A Conserved Basic Patch and Central Kink in the Nipah Virus Phosphoprotein Multimerization Domain Are Essential for Polymerase Function.

Nipah virus is a highly lethal zoonotic pathogen found in Southeast Asia that has caused human encephalitis outbreaks with 40%-70% mortality. NiV encodes its own RNA-dependent RNA polymerase within the large protein, L. Efficient polymerase activity requires the phosphoprotein, P, which tethers L to its template, the viral nucleocapsid. P is a multifunctional protein with modular domains. The central P multimerization domain is composed of a long, tetrameric coiled coil. We investigated the importance of structural features found in this domain for polymerase function using a newly constructed NiV bicistronic minigenome assay. We identified a conserved basic patch and central kink in the coiled coil that are important for polymerase function, with R555 being absolutely essential. This basic patch and central kink are conserved in the related human pathogens measles and mumps viruses, suggesting that this mechanism may be conserved.

Functional properties and genetic relatedness of the fusion and hemagglutinin-neuraminidase proteins of a mumps virus-like bat virus.

UNLABELLED: A bat virus with high phylogenetic relatedness to human mumps virus (MuV) was identified recently at the nucleic acid level. We analyzed the functional activities of the hemagglutinin-neuraminidase (HN) and the fusion (F) proteins of the bat virus (batMuV) and compared them to the respective proteins of a human isolate. Transfected cells expressing the F and HN proteins of batMuV were recognized by antibodies directed against these proteins of human MuV, indicating that both viruses are serologically related. Fusion, hemadsorption, and neuraminidase activities were demonstrated for batMuV, and either bat-derived protein could substitute for its human MuV counterpart in inducing syncytium formation when coexpressed in different mammalian cell lines, including chiropteran cells. Cells expressing batMuV glycoproteins were shown to have lower neuraminidase activity. The syncytia were smaller, and they were present in lower numbers than those observed after coexpression of the corresponding glycoproteins of a clinical isolate of MuV (hMuV). The phenotypic differences in the neuraminidase and fusion activity between the glycoproteins of batMuV and hMuV are explained by differences in the expression level of the HN and F proteins of the two viruses. In the case of the F protein, analysis of chimeric proteins revealed that the signal peptide of the bat MuV fusion protein is responsible for the lower surface expression. These results indicate that the surface glycoproteins of batMuV are serologically and functionally related to those of hMuV, raising the possibility of bats as a reservoir for interspecies transmission. IMPORTANCE: The recently described MuV-like bat virus is unique among other recently identified human-like bat-associated viruses because of its high sequence homology (approximately 90% in most genes) to its human counterpart. Although it is not known if humans can be infected by batMuV, the antigenic relatedness between the bat and human forms of the virus suggests that humans carrying neutralizing antibodies against MuV are protected from infection by batMuV. The close functional relationship between MuV and batMuV is demonstrated by cooperation of the respective HN and F proteins to induce syncytium formation in heterologous expression studies. An interesting feature of the glycoproteins of batMuV is the downregulation of the fusion activity by the signal peptide of F, which has not been reported for other paramyxoviruses. These results are important contributions for risk assessment and for a better understanding of the replication strategy of batMuV.

Protein domain definition should allow for conditional disorder.

Proteins are often classified in a binary fashion as either structured or disordered. However this approach has several deficits. Firstly, protein folding is always conditional on the physiochemical environment. A protein which is structured in some circumstances will be disordered in others. Secondly, it hides a fundamental asymmetry in behavior. While all structured proteins can be unfolded through a change in environment, not all disordered proteins have the capacity for folding. Failure to accommodate these complexities confuses the definition of both protein structural domains and intrinsically disordered regions. We illustrate these points with an experimental study of a family of small binding domains, drawn from the RNA polymerase of mumps virus and its closest relatives. Assessed at face value the domains fall on a structural continuum, with folded, partially folded, and near unstructured members. Yet the disorder present in the family is conditional, and these closely related polypeptides can access the same folded state under appropriate conditions. Any heuristic definition of the protein domain emphasizing conformational stability divides this domain family in two, in a way that makes no biological sense. Structural domains would be better defined by their ability to adopt a specific tertiary structure: a structure that may or may not be realized, dependent on the circumstances. This explicitly allows for the conditional nature of protein folding, and more clearly demarcates structural domains from intrinsically disordered regions that may function without folding.

Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae.

BACKGROUND: The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN) and measles hemagglutinin (MeH) in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. RESULTS: Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A) and is closely associated with small heat shock proteins (sHsps) that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto) in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. CONCLUSIONS: Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of these recombinant proteins induces the UPR's cytosolic counterpart, the UPR-Cyto, which represent a subset of proteins involved in the heat-shock response. The involvement of eEF1A may explain the mechanism by which only large chaperones, but not small Hsps are upregulated during this stress response. Our study highlights important differences between viral surface protein expression in yeast and mammalian cells at the first stage of secretory pathway.

Structure of the nucleocapsid-binding domain from the mumps virus polymerase; an example of protein folding induced by crystallization.

The human pathogen mumps virus, like all paramyxoviruses, encodes a polymerase responsible for virally directed RNA synthesis. The template for the polymerase is the nucleocapsid, a filamentous protein-RNA complex harboring the viral genome. Interaction of the polymerase and the nucleocapsid is mediated by a small domain tethered to the end of the phosphoprotein (P), one of the polymerase subunits. We report the X-ray crystal structure of this region of mumps virus P (the nucleocapsid-binding domain, or NBD, amino acids 343-391). The mumps P NBD forms a compact bundle of three alpha-helices within the crystal, a fold apparently conserved across the Paramyxovirinae. In solution, however, the domain exists in the molten globule state. This is demonstrated through application of differential scanning calorimetry, circular dichroism spectroscopy, NMR spectroscopy, and dynamic light scattering. While the mumps P NBD is compact and has persistent secondary structure, it lacks a well-defined tertiary structure under normal solution conditions. It can, however, be induced to fold by addition of a stabilizing methylamine cosolute. The domain provides a rare example of a molten globule that can be crystallized. The structure that is stabilized in the crystal represents the fully folded state of the domain, which must be transiently realized during binding to the viral nucleocapsid. While the intermolecular forces that govern the polymerase-nucleocapsid interaction appear to be different in measles, mumps, and Sendai viruses, for each of these viruses, polymerase translocation involves the coupled binding and folding of protein domains. In all cases, we suggest that this will result in a weak-affinity protein complex with a short lifetime, which allows the polymerase to take rapid steps forward.

Functional consequences of attenuating mutations in the haemagglutinin neuraminidase, fusion and polymerase proteins of a wild-type mumps virus strain.

Wild-type mumps viruses (MuVs) are highly neurotropic and, prior to widespread vaccination programmes, were a major cause of viral meningitis and encephalitis in most developed countries. At present, there are no markers for virus attenuation, apart from the failure of a passaged isolate to produce clinical symptoms in vaccinees. Indeed, some MuV vaccines have retained residual neurovirulence properties and have caused aseptic meningitis in vaccinees. Three amino acid changes associated with the neuroattenuation of a wild-type MuV strain were identified previously. This study evaluated the impact of these changes on the function of the respective proteins. The data demonstrated that the Ser-->Asp amino acid substitution at position 466 in the haemagglutinin-neuraminidase protein resulted in decreased receptor binding and neuraminidase activity, the Ala/Thr-->Thr selection in the fusion protein resulted in decreased fusion activity, and the Ile-->Val substitution in the polymerase resulted in increased replicative/transcriptional activity. These data suggest a polygenic component (i.e. specific and inter-related roles of these amino acid changes) to MuV neuroattenuation.

An attempt to analyse the functional difference between various mumps virus strains.

Enzymatic activity of N-acetylneuraminidase of ten various strains of mumps virus was compared. From the viewpoint of their biological properties, these strains could be classified as laboratory, neurovirulent, and vaccinal (attenuated) ones. The enzymatic activity was evaluated by Scatchard's plot which enables to interpret it according to polarity of the cooperativity of enzymes' binding sites. Laboratory strains of the mumps virus demonstrated an independent type of cooperativity, while vaccinal (attenuated) ones showed a positive type of cooperation.

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.

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.

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

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.

Substrate specificity of the neuraminidase of different mumps virus strains.

The hydrolysis of neuraminlactose, fetuin, ovomucoid, kappa-caseinglycopeptide and mucine from the bovine submaxillary gland with the neuraminidase (NA) of mumps virus strains Jeryl Lynn, Enders and Berlin 9/76 was investigated to determine the pH-dependence of the reaction with the different substrates. The corresponding curves showed splitting into several peaks with reaction maxima ranging from pH 4.7 to pH 6.7. This occurred probably due to the heterogeneity of the virus samples. The NA of each virus strain had the best reaction affinity to neuraminlactose followed by decreasing affinities to ovomucoid, fetuin and kappa-caseinglycopeptide. The mucine from bovine submaxillary gland was not digested at all. The highest hydrolysis of each substrate was found with the Jeryl Lynn strain, which also possessed the lowest substrate specificity. It was followed by that of strain Enders and finally by isolate Berlin 9/76 which, in turn, had the highest substrate specificity. The lower substrate specificity of mumps virus NA seemed to correlate with a lower degree of its cytopathogenicity for hamster ependyma cells.

Conversion of nonfusing mumps virus infections to fusing infections by selective proteolysis of the HN glycoprotein.

Mumps virus strains differ in their ability to induce cell fusion following an infection: strains with active neuraminidase (NANase) fail to cause cell fusion, while strains with less active NANase cause cell fusion. When chymotrypsin is added to infected cells, cell fusion is amplified in a concentration-dependent manner for all mumps virus strains. Virions produced in such infections do not express HN glycoprotein-associated activities. Chymotrypsin treatment of purified mumps virus in vitro results in sequential cleavage of the HN glycoprotein without affecting F glycoprotein structure. Initially, HN is cleaved into two glycopolypeptides, HNc1 (32K) and HNc2' (41K), with concomitant loss of hemagglutinating and NANase activities, and infectivity. Further incubation with chymotrypsin causes complete degradation of HNc1 and digestion of HNc2' to HNc2 (13K-19K). Both HNc2' and HNc2 contain the [3H]palmitic acid label found in the HN polypeptide, which suggests that these fragments are associated with the viral membrane. Analyses of infected cells and released virions indicate that chymotrypsin acts similarly on HN exposed at the cell surface. Exogenous NANase does not abolish the protease-augmented cell fusion, though it does reduce cell fusion of untreated fusing strain infections. These results confirm that mumps virus HN glycoprotein is critically linked to cell fusion cytopathology and show that cryptic cell fusion activity in nonfusing strain infections can be unmasked by the proteolytic removal of the HN glycoprotein.

Simple detection of mumps virus neuraminidase and anti-neuraminidase with the lectin neuraminidase test.

The lectin neuraminidase (LN) test is based on specific interaction between Arachis lectin and the glycoprotein receptors treated with the neuraminidase (NA) of myxoviruses. The test is suitable for detection of mumps virus NA and of corresponding antibodies and is more sensitive than the conventional neuraminidase inhibition test.

Neuraminidase activity and antigenicity of the egg adapted strains of mumps virus.

Two freshly isolated mumps virus strains were adapted to embryonated eggs. The activity of their neuraminidase (NA) and their antigenicity were examined in the 5th, 10th and 15th passages. The NA was characterized by estimation of the Michaelis constant, while the antigenicity of virus strains was assessed by induction of antibodies as detected in complement fixation (CF) tests. The KM value for strain Berlin 9/76 NA in its 15th passage was 5.65 mmol/l and exceeded 4.2 times that of strain Berlin 1/76 (KM = 1.31 mmol/l). The KM values of both strains increased in the course of passaging by 55 or 260%, respectively. The antigenicity of both egg passaged strains remained unchanged. Under the same conditions, the Jeryl Lynn vaccine strain revealed a seroconversion rate of 44%; the KM value of the NA from this strain was closely similar to that of Berlin 1/76 virus in the 15th passage (= 1.34 mmol/l).

Some properties of mumps virus neuraminidase.

Mumps virus strains were characterized by the neuraminidase activity of infected allantoic and amniotic fluids of chick embryos. Ovomucoid was used as substrate for determination of neuraminidase activity. Storage of virus samples at 4 degrees C led to great losses of enzyme activity. Alkali chlorides, divalent cations, EDTA and N-ethylmaleimide exerted a low to medium inhibitory activity. Surfactants, depending on their type, caused inhibition, activation or no effect on the enzyme--substrate reactions. The pH optimum was 5.6 and 5.3 with mumps virus strains Enders and Jeryl Lynn, respectively. The Michaelis constants of 9.43 X 10(-3) and 7.57 X 10(-3) mol/1 allowed a differentiation of the two mumps virus strains based on their neuraminidase.

Simple test for detection of virus neuraminidase and antineuraminidase using lectins (lectin-neuraminidase test system).

The fast and sensitive detection of virus neuraminidase effectiveness (influenza virus A and B or mumps virus) and of antibodies against virus neuraminidase in human serum is described. Lectin mainly obtained from Arachis hypogaea and Helix pomatia is used in the lectin-neuraminidase test (LN-test). The lectins are capable of agglutinating erythrocytes after virus incubation because virus neuraminidase reveals the T-antigen to be situated on erythrocytes. The presence of anti-neuraminidase in human sera can be detected with the aid of lectins after incubation of the virus suspension with human serum as a result of the agglutination having failed to take place. Neuraminidase activities of the influenza virus A and B are detectable up to a virus dilution of 1: 40,000 and anti-neuraminidase could be determined in human serum samples with titres of up to 1: 10,000. All materials required for the LN-test can be arranged in a set.

RNA polymerase in mumps virion.

Mumps virions of the Enders' strain were examined for polymerase activity in vitro. An RNA-dependent RNA polymerase was found to be associated with the virion. The general properties of the reaction appear to be similar to those described for other paramyxoviruses.

Leukotactic factors elaborated by virus-infected tissues.

Infection of chick embryos wih either Newcastle disease virus or mumps virus and infection of BGM cell cultures with mumps virus result in the elaboration of chemotactic activity for neutrophils and macrophages. These factors cannot be found in lysates of uninfected cells. They do not appear to be associated with the viral particles per se, but rather are present in virus-free supernates from infected fluids. Ultracentrifugal studies of the neutrophil chemotactic activity in allantoic fluid of embryos infected with the two different viruses indicate a similar biphasic distribution of activity, while fluid from the mammalian cell cultures shows a single zone of leukotactic activity, further suggesting that the infected cell, rather than the virus, is responsible for the leukotactic activity. Virus-infected cells also release a substance(s) which is itself not leukotactic but which can interact with human C3 or C5 to generate such activity. This leukotactic factor-generating substance is similar to that reported in another virus-infected cell system. It is postulated that the leukotactic factors elaborated as a result of virus infection of cells may play a protective role in vivo.