Short communicationIdentification of amino acid positions associated with neuraminidase activity of the hemagglutinin-neuraminidase glycoprotein of sendai virus
Abstract
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.
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Cited by (10)
The role of the cytoplasmic domain (cytd) of the Sendai virus HN and F glycoproteins in the process of virus assembly and budding are evaluated. Recombinant Sendai virus (rSeV) mutants are generated carrying modifications in the cytd of each of the glycoprotein separately. The modifications include increasing truncations and/or amino acid sequence substitutions. Following steady-state 35[S]methionine/cysteine labeling of the infected cells, the virus particle production is estimated. The radioactive virions in the cell supernatants are measured relative to the extent of the infection, assessed by the intracellular N protein signal. For both the F and HN cytd truncation mutants, the largest cytd deletions lead to a 20- to 50-fold reduction in virion production. This reduction cannot be explained by a reduction of the cell surface expression of the glycoproteins. For the F protein mutants, the virions produced in reduced amount always exhibit a normal F protein composition. It is then concluded that a threshold level of F is required for SeV assembly and budding. The rate or the efficiency with which this threshold is reached up appears to depend on the nature of the F cytd. A minimal cytd length is required as well as a specific sequence. The analysis of HN protein mutants brings to light an apparent paradox. The larger cytd truncations result in significant reduction of virion production. On the other hand, a normal virion production can take place with an underrepresentation of or, even, an undetectable HN in the particles. The HN uptake in virion is confirmed to depend on the previously proposed cytd SYWST signal (T. Takimoto, T. Bousse, E. C. Coronel, R. A Scroggs, and A. Portner. 1998. J. Virol. 72, 9747–9754.).
The use of a quantitative fusion assay to evaluate HN-receptor interaction for human parainfluenza virus type 3
1999, VirologySialic acid is the receptor determinant for the human parainfluenza virus type 3 (HPF3) hemagglutinin–neuraminidase (HN) glycoprotein, the molecule responsible for binding of the virus to cell surfaces. In order for the fusion protein (F) of HPF3 to promote membrane fusion, HN must interact with its receptor. In addition to its role in receptor binding and fusion promotion, the HPF3 HN molecule contains receptor-destroying (sialidase) activity. The putative active sites are in the extracellular domain of this type II integral membrane protein. However, HN is not available in crystalline form; the exact locations of these sites, and the structural requirements for binding to the cellular receptor, which has not yet been isolated, are unknown. Nor have small molecular synthetic inhibitors of attachment or fusion that would provide insight into these processes been identified. The strategy in the present study was to develop an assay system that would provide a measure of a specific step in the viral cycle—functional interaction between viral glycoproteins and the cell during attachment and fusion—and serve to screen a variety of substances for inhibitory potential. The assay is based on our previous finding that CV-1 cells persistently infected (p.i.) with HPF3 do not fuse with one another but that the addition of uninfected CV-1 cells, supplying the critical sialic acid containing receptor molecules that bind HN, results in rapid fusion. In the present assay two HeLa cell types were used: we persistently infected HeLa-LTR-βgal cells, assessed their fusion with uninfected HeLa-tat cells, and then quantitated the β-galactosidase (βgal) produced as a result of this fusion. The analog α-2-S-methyl-5-N-thioacetylneuraminic acid (α-Neu5thioAc2SMe) interfered with fusion, decreasing βgal production by 84% at 50 mM and by 24% at 25 mM. In beginning to extend our studies to different types of molecules, we tested an unsaturated derivative of sialic acid, 2,3-dehydro-2-deoxy-n-acetyl neuraminic acid (DANA), which is known to inhibit influenza neuraminidase by virtue of being a transition-state analog. We found that 10 mM DANA inhibited neuraminidase activity in HPF3 viral preparations. More significantly, this compound was active in our assay of HN–receptor interaction; 10 mM DANA completely blocked fusion and βgal production, and hemadsorption inhibition by DANA suggested that DANA blocks attachment. In plaque reduction assays performed with the compounds, the active analog α-Neu5thioAc2SMe reduced plaque formation by 50% at a 50 mM concentration; DANA caused a 90% inhibition in the plaque reduction assay at a concentration of 25 mM. Our results indicate that specific sialic acid analogs that mimic the cellular receptor determinant of HPF3 can block virus cell interaction and that an unsaturated n-acetyl-neuraminic acid derivative with affinity to the HN site responsible for neuraminidase activity also interferes with HN–receptor binding. Strategies suggested by these findings are now being pursued to obtain information regarding the relative locations of the active sites of HN and to further elucidate the relationship between the receptor-binding and receptor-destroying activities of HN during the viral life cycle. The quantitative assay that we describe is of immediate applicability to large-scale screening for potential inhibitors of HPF3 infection in vivo.
Hemagglutinin-Neuraminidase of Human Parainfluenza 3: Role of the Neuraminidase in the Viral Life Cycle
1995, VirologyThe function of neuraminidase in the life cycle and pathogenesis of human parainfluenza virus type 3 (HPF3) was studied by analyzing a variant of HPF3 that has decreased neuraminidase enzymatic activity. The variant virus is more fusogenic than the wild-type virus during an acute infection. Cloning and sequencing of the fusion (F) and hemagglutinin–neuraminidase (HN) genes from this variant revealed a single amino acid change in the HN protein and no alterations in the F protein sequence. Analysis of the growth properties of this variant revealed a delay in release of virus particles into the supernatant. Addition of exogenous neuraminidase to the culture resulted in increased release of infectious viral particles, suggesting that the viral neuraminidase is important for release of HPF3 from the infected cell surface. In addition, the behavior of the variant virus during high-multiplicity infection and in the presence of exogenous neuraminidase provided evidence that the neuraminidase of HPF3 determines the outcome of viral infection (cytopathic versus persistent) in cell culture.
The human parainfluenza virus type-1 prototypic strain contains a heat-labile hemagglutinin-neuraminidase protein
1994, Virus ResearchThe virus yield of human parainfluenza virus type-1 (hPIV-1) in cultured cells at 38°C is reduced more than 100-fold compared to 34°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°C, while at 38°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°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°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°C.
A study was made to elucidate the effect of host cells on the HANA protein of Sendai virus. Two strains of Sendai virus were isolated from an epidemic in an animal laboratory by inoculating the lung homogenate of a moribund mouse either into LLC-MK2 cells (Oh-L) or into the allantoic cavity of embryonated eggs (Oh-E). Oh-E agglutinated chicken red blood cells at 37° (HA37+), while Oh-L did not (HA37-). When Oh-L was passaged in eggs, conversion of the HA37- virus to the HA37+ virus readily occurred. A single point mutation was recognized on the HANA protein of the HA37+ virus either at position 525 (Gin → Arg) or at position 198 (Leu → Phe). HI test with monoclonal antibody revealed conformational changes around the receptor binding site. Neuraminidase activity was also affected by these mutations. The changes in these biological activities of the HANA protein seemed to allow the HA37+ virus to replicate in eggs. On the contrary, the HA37+ virus replicates as efficiently as the HA37- virus in LLC-MK2 cells and no reversion to the HA37- virus was observed. The overall results indicate that the passage of Sendai virus in eggs resulted in selection of viruses possessing a specific mutation on the HANA protein. The pneumopathogenicity in mice was not significantly different between the HA37- virus and the HA37+ virus, suggesting the existence of genes other than the HANA gene that determine mouse pathogenicity.
Cloned cDNA encoding the Sendai virus (SV) hemagglutinin-neuraminidase (HN) envelope glycoprotein was expressed in cultured cells in two ways: (I) infection with HN-expressing recombinant vaccinia virus, or (II) transfection with a plasmid with T7 promoter and termination sequences flanking the HN gene, with intracellular T7 RNA polymerase supplied by coinfection with recombinant vaccinia virus that expresses the enzyme. The HN expressed was indistinguishable from the authentic SV protein in antigenicity, cell surface location, and formation of oligomeric structures. In addition, HN expressed from cDNA functioned normally in both hemadsorption and neuraminidase activities. The usefulness of cDNA expression for analyzing HN structure and function was evaluated by mutating the HN cDNA and observing the consequences for HN protein activity. Since previous work indicated that the lysine residue at position 461 is important for the neuraminidase activity of HN, we used site-directed mutation to produce HN protein with this lysine residue changed to glutamic acid. The mutated HN had neuraminidase activity with significantly increased thermal stability, indicating that residue 461 may be essential to the protein's conformation.
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Present address: Veterans Administration Medical Center, 1030 Jefferson Ave, Memphis, TN 38104.