Targeting Human Parainfluenza Virus Type-1 Haemagglutinin-Neuraminidase with Mechanism-Based Inhibitors.

Human parainfluenza virus (hPIV) infections are a major cause of respiratory tract illnesses in children, with currently no available vaccine or drug treatment. The surface glycoprotein haemagglutinin-neuraminidase (HN) of hPIV has a central role in the viral life cycle, including neuraminic acid-recognising receptor binding activity (early stage) and receptor-destroying activity (late stage), which makes it an ideal target for antiviral drug disovery. In this study, we showed that targeting the catalytic mechanism of hPIV-1 HN by a 2α,3ß-difluoro derivative of the known hPIV-1 inhibitor, BCX 2798, produced more potent inhibition of the neuraminidase function which is reflected by a stronger inhibition of viral replication. The difluorosialic acid-based inhibitor efficiently blocked the neuraminidase activity of HN for a prolonged period of time relative to its unsaturated neuraminic acid (Neu2en) analogue, BCX 2798 and produced a more efficient inhibition of the HN neuraminidase activity as well as in vitro viral replication. This prolonged inhibition of the hPIV-1 HN protein suggests covalent binding of the inhibitor to a key catalytic amino acid, making this compound a new lead for a novel class of more potent hPIV-1 mechanism-based inhibitors.

Rapid Fluorescent Detection Assay for Human Parainfluenza Viruses.

Human parainfluenza virus type 1 (hPIV1) does not form clear plaque by the conventional plaque formation assay because of slightly a cytopathic effects in many cell lines infected with hPIV1, thus making in virus titration, isolation and inhibitor evaluation difficult. We have succeeded in fluorescent histochemical visualization of sialidase activities of influenza A and B viruses, Newcastle disease virus and Sendai virus by using a novel fluorescent sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac). In this study, we applied the BTP3-Neu5Ac assay for rapid detection of hPIV1 and hPIV type 3. The BTP3-Neu5Ac assay could histochemically visualize dot-blotted hPIVs on a membrane and hPIV-infected cells as local fluorescence under UV irradiation. We succeeded in distinct fluorescent visualization of hPIV1-infected cells in only 3 d using the BTP3-Neu5Ac assay. Due to there being no fixation, hPIV1 was isolated directly from fluorescent stained focus cells by the BTP3-Neu5Ac assay. Establishment of a sensitive, easy, and rapid fluorescent focus detection assay for hPIV, hPIV1 in particular will contribute greatly to progress in hPIV studies.

Quantitative comparison of human parainfluenza virus hemagglutinin-neuraminidase receptor binding and receptor cleavage.

The human parainfluenza virus (hPIV) hemagglutinin-neuraminidase (HN) protein binds (H) oligosaccharide receptors that contain N-acetylneuraminic acid (Neu5Ac) and cleaves (N) Neu5Ac from these oligosaccharides. In order to determine if one of HN's two functions is predominant, we measured the affinity of H for its ligands by a solid-phase binding assay with two glycoprotein substrates and by surface plasmon resonance with three monovalent glycans. We compared the dissociation constant (Kd) values from these experiments with previously determined Michaelis-Menten constants (Kms) for the enzyme activity. We found that glycoprotein substrates and monovalent glycans containing Neu5Acα2-3Galß1-4GlcNAc bind HN with Kd values in the 10 to 100 µM range. Km values for HN were previously determined to be on the order of 1 mM (M. M. Tappert, D. F. Smith, and G. M. Air, J. Virol. 85:12146-12159, 2011). A Km value greater than the Kd value indicates that cleavage occurs faster than the dissociation of binding and will dominate under N-permissive conditions. We propose, therefore, that HN is a neuraminidase that can hold its substrate long enough to act as a binding protein. The N activity can therefore regulate binding by reducing virus-receptor interactions when the concentration of receptor is high.

Syntheses of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid analogues modified by α-acylaminoamido groups at the C-4 position using isocyanide-based four-component coupling and biological evaluation as inhibitors of human parainfluenza virus type 1.

Novel sialidase inhibitors 11 having an α-acylaminoamido group at the C-4 position of Neu5Ac2en 1 against human parainfluenza virus type 1 (hPIV-1) were synthesized using one-pot isocyanide-based four-component condensation, and their inhibitory activities against hPIV-1 sialidase were studied. Compound 11b showed inhibitory activity (IC(50)=5.1 mM) against hPIV-1 sialidase. The degree of inhibition of 11b was much weaker than that of 1 (IC(50)=0.3 mM).

Syntheses of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid analogues modified by N-sulfonylamidino groups at the C-4 position and biological evaluation as inhibitors of human parainfluenza virus type 1.

Eleven novel sialidase inhibitors 9 and 10 with an N-sulfonylamidino group at the C-4 position of Neu5Ac2en 1 against human parainfluenza virus type 1 (hPIV-1) were synthesized using copper-catalyzed three-component coupling reactions, and their inhibitory activities against hPIV-1 sialidase were studied.

2-Deoxy-2,3-didehydro-N-acetylneuraminic acid analogues structurally modified at the C-4 position: synthesis and biological evaluation as inhibitors of human parainfluenza virus type 1.

To explore the influence of binding to human parainfluenza virus type 1 (hPIV-1), a series of 4-O-substituted Neu5Ac2en derivatives 6a-e was synthesized and tested for their ability to inhibit hPIV-1 sialidase. Among compounds 6a-e, the 4-O-ethyl-Neu5Ac2en derivative 6b showed the most potent inhibitory activity (IC50 6.3 microM) against hPIV-1 sialidase.

2beta,3beta-Difluorosialic acid derivatives structurally modified at the C-4 position: synthesis and biological evaluation as inhibitors of human parainfluenza virus type 1.

A series of 4-O-substituted 2beta,3beta-difluorosialic acid derivatives (3a-d) has been synthesized. A key intermediate was synthesized efficiently by the electrophilic syn-addition of fluorine to the double bond of a glycal precursor using molecular fluorine or xenon difluoride in the presence of BF(3).OEt(2). Among compounds 3a-d, the 4-O-thiocarbamoylmethyl derivative 3c showed the most potent inhibitory activity against sialidase of human parainfluenza virus type 1. [structure: see text].

Codon substitution mutations at two positions in the L polymerase protein of human parainfluenza virus type 1 yield viruses with a spectrum of attenuation in vivo and increased phenotypic stability in vitro.

The Y942H and L992F temperature-sensitive (ts) and attenuating amino acid substitution mutations, previously identified in the L polymerase of the HPIV3cp45 vaccine candidate, were introduced into homologous positions of the L polymerase of recombinant human parainfluenza virus type 1 (rHPIV1). In rHPIV1, the Y942H mutation specified the ts phenotype in vitro and the attenuation (att) phenotype in hamsters, whereas the L992F mutation specified neither phenotype. Each of these codon mutations was generated by a single nucleotide substitution and therefore had the potential to readily revert to a codon specifying the wild-type amino acid residue. We introduced alternative amino acid assignments at codon 942 or 992 as a strategy to increase genetic stability and to generate mutants that exhibit a range of attenuation. Twenty-three recombinants with codon substitutions at position 942 or 992 of the L protein were viable. One highly ts and att mutant, the Y942A virus, which had a difference of three nucleotides from the codon encoding a wild-type tyrosine, also possessed a high level of genetic and phenotypic stability upon serial passage in vitro at restrictive temperatures compared to that of the parent Y942H virus, which possessed a single nucleotide substitution. We obtained mutants with substitutions at position 992 that, in contrast to the L992F virus, possessed the ts and att phenotypes. These findings identify the use of alternative codon substitution mutations as a method that can be used to generate candidate vaccine viruses with increased genetic stability and/or a modified level of attenuation.

Inhibition of human parainfluenza virus type 1 sialidase by analogs of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid.

Eleven novel analogs of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en) modified at the C-4 and C-9 positions were designed and tested for their ability to inhibit sialidase of human parainfluenza virus type 1 (hPIV-1). The analogs modified by the cyanomethyl, amidinomethyl, and thiocarbamoylmethyl groups at the C-4 position exhibited potent inhibition against hPIV-1 sialidase compared with Neu5Ac2en. The most effective compound was thiocarbamoylmethyl analog (4-O-thiocarbamoylmethyl-Neu5Ac2en). The activity of 4-O-thiocarbamoylmethyl-Neu5Ac2en causing 50% enzyme inhibition at a concentration of approximately 1.0x10(-5) M was 30-fold larger than Neu5Ac2en. While, the analogs of Neu5Ac2en modified by the azido and N-acetyl groups at the C-9 showed a decrease in inhibition of sialidase compared with the 9-hydroxy analogs. In addition, 4-O-thiocarbamoylmethyl-Neu5Ac2en strongly inhibited hPIV-1 infections of Lewis lung carcinoma-monkey kidney cells in comparison with Neu5Ac2en. The present findings would provide useful information for the development of anti-human parainfluenza virus compounds.

Virus-receptor interactions of human parainfluenza viruses types 1, 2 and 3.

Human parainfluenza viruses types 1, 2 and 3 (HPF 1, 2 and 3) are important pathogens in children. While these viruses share common structures and replication strategies, they target different parts of the respiratory tract; the most common outcomes of infection with HPF3 are bronchiolitis and pneumonia, while HPF 1 and 2 are associated with croup. While the HPF3 fusion protein (F) is critical for membrane fusion, our previous work revealed that the receptor binding hemagglutinin-neuraminidase (HN) is also essential to the fusion process; interaction between HN and its sialic acid-containing receptor on cell surfaces is required for HPF3 mediated cell fusion. Using our understanding of HPF3 HN's functions in the cell-binding and viral entry process, we are investigating the ways in which these processes differ in HPF 1 and 2, in part by manipulating receptor availability. Three experimental treatments were used to compare the HN-receptor interaction of HPF 1, 2 and 3: infection at high multiplicity of infection (m.o.i.); bacterial neuraminidase treatment of cells infected at low m.o.i.; and viral neuraminidase treatment of cells infected at low m.o.i. (using Newcastle disease virus [NDV] neuraminidase or UV irradiated HPF3 as sources of neuraminidase). In cells infected with HPF3, we have shown that infection with high m.o.i. blocks fusion, by removing sialic acid receptors for the viral HN. However, in cells infected with HPF 1 and 2, infection with high m.o.i. did not block fusion; the fusion increases with increasing m.o.i. In cells infected with HPF 1 and 2, neither bacterial nor NDV neuraminidase blocked cell fusion, using amounts of neuraminidase that completely block fusion of HPF3 infected cells. However, when inactivated HPF3 was used as a source of viral neuraminidase, the treatment inhibited fusion of cells infected with HPF 1 and 2 as well as 3. The differences found between these viruses in terms of their interaction with the cell, ability to modulate cell-cell fusion and response to exogenous neuraminidases of various specificities, may reflect salient differences in biological properties of the three viruses.

Cooperative neuraminidase activity in a paramyxovirus.

Paramyxoviruses possess neuraminidase (NA) activity, i.e., the ability to cleave sialic acid from membrane-bound and soluble glycoconjugates. The activity is associated with a homotetrameric, surface glycoprotein spike, called the hemagglutinin-neuraminidase. This structure also mediates viral attachment to sialic acid-containing receptors and constitutes the major neutralizing antigen for the virus. Cooperativity has been demonstrated for the NA activity of an isolate of one of the paramyxoviruses, Newcastle disease virus. Although all known viral NA proteins are homooligomeric, this is the first demonstration of cooperativity in this family of proteins. A variant virus, selected by escape from neutralization by a monoclonal antibody thought to bind close to the NA active site, has lost cooperativity. Conversion to the noncooperative state correlates with increases in both avidity for cellular receptors and fusogenic activity.

Inhibition of Sendai virus hemagglutinin neuraminidase by the fusion protein.

The Sendai virus envelope contains two glycoproteins: the fusion (F) protein and the hemagglutinin-neuraminidase (HN). Inactivation of F causes the loss of fusogenic activity and an increase of the neuraminidase activity of HN. After inactivation of F, HN can be inhibited by fetuin or asialofetuin, as already observed on the water-soluble, C-terminal fragment of HN (Dallocchio, F., Bellini, T., Martuscelli, G., Baiocchi, M., & Tomasi, M. (1991) Biochem. Int. 25, 663-668). Disruption of viral envelopes by detergents does not affect the neuraminidase activity of virions containing inactive F, while it causes an increase of the neuraminidase activity in native virions. Reconstitution of HN into liposomes is accompanied by a decrease of enzymatic activity, due to the random inside-outside distribution of the protein. However, the decrease of the neuraminidase activity is higher in liposomes containing both HN and F. These data suggest that F inhibits the neuraminidase activity of HN.

Simple purification and secondary structure evaluation of Sendai virus neuraminidase water soluble fragment.

We have developed a quick and simple method to purify the water soluble fragment of Sendai virus neuraminidase (cHN). We used the trifluoperazine (TFP) as detergent because of its ability to selectively solubilize the HN (Baiocchi et al., (1988) FEBS Lett. 238, 171-174). Here we describe conditions by which trypsin produces the cHN from the TFP treated virions. The cHN is further purified by size exclusion chromatography and it fully retains the neuraminidase activity and the allosteric inhibitory site as described in (Dallocchio et al., (1991) Biochem. Int. 25, 663-668). Both circular dichroism (CD) spectra, analyzed by deconvolution methods and secondary structure prediction were used to assess the secondary structure of cHN.

Targeted delivery of hygromycin B using reconstituted Sendai viral envelopes lacking hemagglutinin-neuraminidase.

Hygromycin B was encapsulated in reconstituted Sendai viral envelopes containing only the fusion (F) protein (F-virosomes). Incubation of loaded F-virosomes with cultured HepG2 cells resulted in fusion mediated delivery of hygromycin B to the cell cytoplasm, as was inferred from inhibition of DNA synthesis. Binding of the F-virosomes to HepG2 cells was mediated by the interaction of terminal beta-galactose residues of fusion protein with asialoglycoprotein receptor on HepG2 cells, subsequently leading to fusion between the two membranes. The cytotoxic effect of hygromycin B enclosed in F-virosomes was comparable with that of F,HN-virosomes containing both hemagglutinin-neuraminidase (HN) and F protein and F,HNred-virosomes containing HN whose disulfide bonds were irreversibly reduced (HNred). Hygromycin B loaded fusogenic liposomes were prepared by coreconstituting the viral envelope containing only fusion protein with exogenous lipids. These fusogenic liposomes were found to be more active than F-virosomes at the same fusion protein concentrations.

Antibody against the carboxyl terminus of the F2 subunit of Sendai virus fusion glycoprotein inhibits proteolytic activation.

Rabbit antibody, F2C12, was prepared against a synthetic peptide of 13 amino acids corresponding to the C-terminus of the F2 subunit of Sendai virus F protein. F2C12 was shown to bind specifically to the F2 subunit, irrespective of proteolytic cleavage of the F protein. F2C12 did not affect the infectivity, hemolytic, and cell fusion activities of activated virus, but F2C12 did inhibit proteolytic cleavage of the precursor F0 to subunits, F1 and F2, by trypsin, factor Xa from chick embryos and Tryptase Clara from rat lungs. When F2C12 was added to the chorioallantoic cavity of chick embryos, cleavage activation of Sendai virus was inhibited. Intranasal administration of F2C12 to Sendai virus-infected rats suppressed activation and multiplication of progeny virus in the lungs. These results indicate that proteolytic cleavage of the F protein, with a single arginine residue at the cleavage site, occurs extracellularly in the allantoic cavity of chick embryos and in the bronchial lumen of rats.

Selective modification of Sendai virus hemagglutinin neuraminidase by pyridoxal 5'-phosphate: evidence for an allosteric modulation of neuraminidase activity.

Incubation of Sendai virus with pyridoxal 5'-phosphate (PLP) causes inhibition of hemolytic activity, a slight reduction of hemagglutinating activity, and an increase in neuraminidase activity. The effects on hemagglutination and neuraminidase are prevented by the presence in the incubation mixture of sialyl lactose, a substrate of hemagglutinin-neuraminidase. Incubation with PLP of the water-soluble enzymatic domain of the neuraminidase has no effect on enzymatic activity, while the allosteric inhibition (Dallocchio et al. (1991) Biochem. Int. 25, 663-668) disappears. Both virus-bound and solubilized neuraminidase are selectively modified by PLP at the lysine-553. Our data suggest that PLP inactivates a previously undetected inhibitory site on the viral neuraminidase, and that a physiological effector is present on the viral envelope.

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.

Genetic variation and evolution of human parainfluenza virus type 1 hemagglutinin neuraminidase: analysis of 12 clinical isolates.

The extent of genetic variation and evolution in a population of human parainfluenza virus type 1 was investigated. The hemagglutinin neuraminidase genes of 13 isolates collected over a 26-year period were sequenced and compared. All isolates except the 1957 type strain were from a single geographic location and demonstrated significant consistent genetic change from the type strain (47/7 [nucleotide/amino acid] substitutions). Antigenic subgroup A isolates demonstrated minor intragroup differences (9/1 substitutions). However, 18/7 unique substitutions separated subgroup A from B regardless of geographic location or year of isolation. Multiple strains of both subgroups appeared and reappeared over decades with only minor variation. There may be significant genetic differences between clinical isolates based on geographic location, and progressive mutational change may occur. Previously defined antigenic and now genetic subgroups were stable and at least regional in distribution over the period studied. The biologic implications and extent of this variation need further evaluation.

Allosteric inhibition of the water-soluble C-terminal fragment of Sendai virus neuraminidase.

Trypsin solubilized hemagglutinin-neuraminidase of Sendai virus (cHN) displays michaelian kinetics, with native fetuin as substrate, at 37 degrees C. Vmax and Km values are only marginally altered, as compared to intact viral neuraminidase. At lower temperatures, cHN follows non-michaelian kinetics, with marked substrate inhibition at 4 degrees C. With denaturated fetuin, michaelian kinetics are observed in all conditions, while asialo fetuin was an uncompetitive inhibitor of cHN, with native fetuin or sialyl lactose as substrates. These results can be explained assuming that the protein moiety of fetuin acts as an allosteric inhibitor of cHN.

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.