Receptor-binding specificity of the human parainfluenza virus type 1 hemagglutinin-neuraminidase glycoprotein

The hemagglutinin-neuraminidase (HN) glycoprotein is utilized by human parainfluenza viruses for binding to the host cell. By the use of glycan array assays, we demonstrate that, in addition to the first catalytic-binding site, the HN of human parainfluenza virus type 1 has a second site for binding covered by N-linked glycan. Our data suggest that attachment of the first site to sialic acid (SA)-linked receptors triggers exposure of the second site. We found that both sites bind to α2-3-linked SAs with a preference for a sialyl-Lewis(x) motif. Binding to α2-3-linked SAs with a sulfated sialyl-Lewis motif as well as to α2-8-linked SAs was unique for the second binding site. Neither site recognizes α2-6-linked oligosaccharides.     

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.     

Relative affinity of the human parainfluenza virus type 3 hemagglutinin-neuraminidase for sialic acid correlates with virus-induced fusion activity.

The ability of enveloped viruses to cause disease depends on their ability to enter the host cell via membrane fusion events. An understanding of these early events in infection, crucial for the design of methods of blocking infection, is needed for viruses that mediate membrane fusion at neutral pH, such as paramyxoviruses and human immunodeficiency virus. Sialic acid is the receptor 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, the HN must interact with its receptor. In the present report, two variants of HPF3 with increased fusion-promoting phenotypes were selected and used to study the function of the HN glycoprotein in membrane fusion. Increased fusogenicity correlated with single amino acid changes in the HN protein that resulted in increased binding of the variant viruses to the sialic acid receptor. These results suggest that the avidity of binding of the HN protein to its receptor regulates the level of F protein-mediated fusion and begin to define one role of the receptor-binding protein of a paramyxovirus in the membrane fusion process.     

mRNA sequence and deduced amino acid sequence of the mumps virus small hydrophobic protein gene.

The mRNA of a putative small hydrophobic protein (SH) of mumps virus was identified in mumps virus-infected Vero cells, and its complete nucleotide sequence was determined by sequencing the genomic RNA and cDNA clones and partial sequencing of mRNA. The SH mRNA is 310 nucleotides long excluding the poly(A) and contains a single open reading frame encoding a protein of 57 amino acids with a calculated molecular weight of 6,719. The predicted protein is highly hydrophobic and contains a stretch of 25 hydrophobic amino acids near the amino terminus which could act as a membrane anchor region. There is no homology between the putative SH protein of mumps virus and the SH protein of simian virus 5, even though the SH genes are located in the same locus in the corresponding genome. One interesting observation is that the hydrophobic domain of simian virus 5 SH protein is at the carboxyl terminus, whereas that of mumps virus putative SH protein is near the amino terminus.     

Conserved epitopes on the hemagglutinin-neuraminidase proteins of human and bovine parainfluenza type 3 viruses: nucleotide sequence analysis of variants selected with monoclonal antibodies.

We have previously identified 11 epitopes located in two topologically nonoverlapping antigenic sites (A and B) and a third bridging site (C) on the human type 3 parainfluenza virus (PIV3) hemagglutinin-neuraminidase (HN) glycoprotein by using monoclonal antibodies (MAbs) which inhibit hemagglutination and virus infectivity (K. L. Coelingh, C. C. Winter, and B. R. Murphy, Virology 143:569-582, 1985). We have identified three additional antigenic sites (D, E, and F) on the HN molecule by competitive-binding assays of anti-HN MAbs which have no known biological activity. Epitopes in sites A, D, and F are conserved on the bovine PIV3 HN glycoprotein and also among a wide range of human isolates. The dideoxy method was used to identify nucleotide substitutions in the HN genes of antigenic variants selected with neutralizing MAbs representing epitopes in site A which are shared by human and bovine PIV3. The deduced amino acid substitutions in the variants were located in separate hydrophilic stretches of HN residues which are conserved in the primary structures of the HN proteins of both human and bovine PIV3 strains.     

Homooligomerization of the hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3 occurs before the acquisition of correct intramolecular disulfide bonds and mature immunoreactivity.

The posttranslational maturation of the hemagglutinin-neuraminidase (HN) glycoprotein of human parainfluenza type 3 virus (PIV3) was investigated in pulse-chase experiments in which folding was monitored by immunoprecipitation with conformation-dependent antibodies and gel electrophoresis under nonreducing conditions and oligomerization was monitored by chemical cross-linking and sedimentation in sucrose gradients. The acquisition of mature immunoreactivity and the formation of correct intramolecular disulfide bonds were concurrent events, with half-times of approximately 10 to 15 min. The finding that newly synthesized HN had little reactivity with postinfection cotton rat serum or with most of the members of a panel of HN-specific monoclonal antibodies indicated that the major epitopes of the PIV3 HN protein are highly conformational in nature. Chemical cross-linking studies indicated that the mature HN protein is present in homoligomers, which are probably tetramers. These findings are consistent with recent observations for the HN protein of Sendai virus (S.D. Thompson, W.G. Laver, K.G. Murti, and A. Portner, J. Virol. 62:4653--4660, 1988; S. Vidal, G. Mottet, D. Kolakofsky, and L. Roux, J. Virol. 63:892--900, 1989). Surprisingly, analysis of pulse-labeled HN protein by sedimentation on sucrose gradients after labeling periods of as little as 2 min indicated that it was present intracellularly only in oligomeric form. The same results were obtained when the labeling period was preceded by a 1.5-h cycloheximide treatment to clear the endoplasmic reticulum of presynthesized HN protein, which indicated that the oligomerization did not involve the incorporation of newly synthesized monomers into partially assembled oligomers. Subsequent chase incubations did not significantly alter the sedimentation profile or stability of the oligomeric forms, suggesting that oligomers detected after short labeling periods were tetramers. Association with cellular proteins did not appear to be responsible for the sedimentation of newly synthesized HN protein as an oligomer. The absence of a detectable monomeric form of intracellular HN protein raised the possibility that oligomerization is cotranslational, and it is possible that the type II membrane orientation of the HN protein might be an important factor in its mode of oligomerization.     

Respiratory syncytial virus fusion glycoprotein: nucleotide sequence of mRNA, identification of cleavage activation site and amino acid sequence of N-terminus of F1 subunit.

The amino acid sequence of respiratory syncytial virus fusion protein (Fo) was deduced from the sequence of a partial cDNA clone of mRNA and from the 5' mRNA sequence obtained by primer extension and dideoxysequencing. The encoded protein of 574 amino acids is extremely hydrophobic and has a molecular weight of 63371 daltons. The site of proteolytic cleavage within this protein was accurately mapped by determining a partial amino acid sequence of the N-terminus of the larger subunit (F1) purified by radioimmunoprecipitation using monoclonal antibodies. Alignment of the N-terminus of the F1 subunit within the deduced amino acid sequence of Fo permitted us to identify a sequence of lys-lys-arg-lys-arg-arg at the C-terminus of the smaller N-terminal F2 subunit that appears to represent the cleavage/activation domain. Five potential sites of glycosylation, four within the F2 subunit, were also identified. Three extremely hydrophobic domains are present in the protein; a) the N-terminal signal sequence, b) the N-terminus of the F1 subunit that is analogous to the N-terminus of the paramyxovirus F1 subunit and the HA2 subunit of influenza virus hemagglutinin, and c) the putative membrane anchorage domain near the C-terminus of F1.     

Antigenic and functional organization of human parainfluenza virus type 3 fusion glycoprotein.

Twenty-six monoclonal antibodies (MAbs) (14 neutralizing and 12 nonneutralizing) were used to examine the antigenic structure, biological properties, and natural variation of the fusion (F) glycoprotein of human type 3 parainfluenza virus (PIV3). Analysis of laboratory-selected antigenic variants and of PIV3 clinical isolates indicated that the panel of MAbs recognizes at least 20 epitopes, 14 of which participate in neutralization. Competitive binding assays indicated that the 14 neutralization epitopes are organized into three nonoverlapping antigenic sites (A, B, and C) and one bridge site (AB) and that the 6 nonneutralization epitopes form four sites (D, E, F, and G). Most of the neutralizing MAbs were involved in nonreciprocal competitive binding reactions, suggesting that they induce conformational changes in other neutralization epitopes. Fusion-inhibition and complemented-enhanced neutralization assays indicated that antigenic sites AB, B, and C may correspond to functional domains of the F molecule. Our results indicated that antibody binding alone is not sufficient for virus neutralization and that many anti-F MAbs neutralize by mechanisms not involving fusion-inhibition. The degree of antigenic variation in the F epitopes of clinical strains was examined by binding and neutralization tests. It appears that PIV3 frequently develops mutations that produce F epitopes which efficiently bind antibodies, but are completely resistant to neutralization by these antibodies.     

Antigenic and structural properties of the hemagglutinin-neuraminidase glycoprotein of human parainfluenza virus type 3: sequence analysis of variants selected with monoclonal antibodies which inhibit infectivity, hemagglutination, and neuraminidase activities.

The hemagglutinin-neuraminidase (HN) gene sequence was determined for 16 antigenic variants of human parainfluenza virus type 3 (PIV3). The variants were selected by using monoclonal antibodies (MAbs) to the HN protein which inhibit neuraminidase, hemagglutination, or both activities. Each variant had a single-point mutation in the HN gene, coding for a single amino acid substitution in the HN protein. Operational and topographic maps of the HN protein correlated well with the relative positions of the substitutions. There was little correlation between the cross-reactivity of a MAb with the bovine PIV3 HN and the amount of amino acid homology between the human and bovine PIV3 HN proteins in the regions of the epitopes, suggesting that many of the epitopes are conformational in nature. Computer-assisted analysis of the HN protein predicted a secondary structure composed primarily of hydrophobic beta sheets interconnected by random hydrophilic coil structures. The HN epitopes were located in predicted coil regions. Epitopes recognized by MAbs which inhibit neuraminidase activity of the virus were located in a region which appears to be structurally conserved among several paramyxovirus HN proteins and which may represent the sialic cid-binding site of the HN molecule.     

Nucleotide and predicted amino acid sequence analysis of the fusion protein and hemagglutinin-neuraminidase protein genes among Newcastle disease virus isolates. Phylogenetic relationships among the Paramyxovirinae based on attachment glycoprotein sequences

Highly virulent Newcastle disease virus (NDV) isolates are List A pathogens for commercial poultry, and reports of their isolation among member nations must be made to the Office of International Epizootes (OIE). The virus is classified as a member of the order Mononegavirales in the family Paramyxoviridae of the subfamily Paramyxovirinae. Two interactive surface glycoproteins, the fusion (F) and hemagglutinin-neuraminidase (HN) proteins, play essential roles in NDV attachment and fusion of cells during infection. Antibodies to the F or HN proteins are capable of virus neutralization; however, no full-length sequences are available for these genes from recently obtained virulent isolates. Therefore, nucleotide and predicted amino acid sequences of the F and HN protein genes from 16 NDV isolates representing highly virulent viruses from worldwide sources were obtained for comparison to older virulent isolates and vaccine strains. The F protein amino acid sequence was relatively conserved among isolates maintaining potential glycosylation sites and C residues for disulfide bonds. A dibasic amino acid motif was present at the cleavage site among more virulent isolates, while the low virulence viruses did not have this sequence. However, a Eurasian collared dove virus had a K114Q substitution at the F cleavage site unique among NDV isolates. The HN protein among NDV isolates maintained predicted catalytic and active site residues necessary for neuraminidase activity and hemagglutination. Length of the HN for the Eurasian collared dove isolate and a previously reported heat resistant virulent isolate were longer relative to other more recent virulent isolates. Phylogenetically NDV isolates separated into four groups with more recent virulent isolates forming a diverse branch, while all the avian paramyxoviruses formed their own clade distinct from other members of the Paramyxoviridae.     

Effects of hemagglutinin-neuraminidase protein mutations on cell-cell fusion mediated by human parainfluenza type 2 virus

The monoclonal antibody M1-1A, specific for the hemagglutinin-neuraminidase (HN) protein of human parainfluenza type 2 virus (HPIV2), blocks virus-induced cell-cell fusion without affecting the hemagglutinating and neuraminidase activities. F13 is a neutralization escape variant selected with M1-1A and contains amino acid mutations N83Y and M186I in the HN protein, with no mutation in the fusion protein. Intriguingly, F13 exhibits reduced ability to induce cell-cell fusion despite its multistep replication. To investigate the potential role of HPIV2 HN protein in the regulation of cell-cell fusion, we introduced these mutations individually or in combination to the HN protein in the context of recombinant HPIV2. Following infection at a low multiplicity, Vero cells infected with the mutant virus H-83/186, which carried both the N83Y and M186I mutations, remained as nonfused single cells at least for 24 h, whereas most of the cells infected with wild-type virus mediated prominent cell-cell fusion within 24 h. On the other hand, the cells infected with the mutant virus, carrying either the H-83 or H-186 mutation, mediated cell-cell fusion but less efficiently than those infected with wild-type virus. Irrespective of the ability to cause cell-cell fusion, however, every virus could infect all the cells in the culture within 48 h after the initial infection. These results indicated that both the N83Y and M186I mutations in the HN protein are involved in the regulation of cell-cell fusion. Notably, the limited cell-cell fusion by H-83/186 virus was greatly promoted by lysophosphatidic acid, a stimulator of the Ras and Rho family GTPases.     

Human C4-binding protein: N-terminal amino acid sequence analysis and limited proteolysis by trypsin

Fres isolated blood cells recombined with normal heparinized plasma and then incubated with endotoxin, induced a 100-fold increase in monocyte tissue thromboplastin synthesis. In contrast, recombination of these cells with heat inactivated plasma, cobra venom factor-treated plasma, Ca²⁺-free plasma, or BioRex 70-treated plasma (plasma free of Clq and D) before incubation with endotoxin, failed to induce monocyte synthesis of tissue thromboplastin. These results strongly support the hypothesis that complement is required for endotoxin stimulation of blood monocyte synthesis of tissue thromboplastin.     

Prostatic spermine-binding protein. Cloning and nucleotide sequence of cDNA, amino acid sequence, and androgenic control of mRNA level

cDNA for mRNA of an androgen-dependent spermine-binding protein (SBP) of rat ventral prostate was cloned by inserting cDNA into a dG-tailed expression vector, pUC8, and screening the expression library with anti-SBP antibodies. Hybrid-selected translation using plasmid DNA from positive clones yielded a 34-kDa protein which was immunoprecipitated by affinity-purified anti-SBP antibodies. SBP mRNA is about 1260 bases long as measured by Northern blot hybridization. An amino acid sequence deduced from the nucleotide sequence of the cDNA was identical to an amino acid sequence found in SBP. SBP is extremely rich in acidic residues. Aspartic and glutamic acids, which make up about 33% of the total sequence, comprise 89 of a stretch of 126 amino acids at the carboxyl-terminal end. By dot hybridization analysis, SBP mRNA was not detected in rat liver, kidney, brain, submaxillary gland, or uterus. The prostate levels of SBP mRNA were measured by mRNA translation and dot hybridization. SBP mRNA level decreased to less than 20% of normal 2 days after castration of rats, and this decrease was reversed by 5 alpha-dihydrotestosterone injection into castrated rats.     

Naturally occurring human parainfluenza type 3 viruses exhibit divergence in amino acid sequence of their fusion protein neutralization epitopes and cleavage sites.

Many human parainfluenza type 3 virus (PIV3) strains isolated from children with respiratory illness are resistant to neutralization by monoclonal antibodies (MAbs) which recognize epitopes in antigenic site A or B of the fusion (F) protein of the prototype 1957 PIV3 strain. The F protein genes of seven PIV3 clinical isolates were sequenced to determine whether their neutralization-resistant phenotypes were associated with specific differences in amino acids which are recognized by neutralizing MAbs. Several clinical strains which were resistant to neutralization by site A or B MAbs had amino acid differences at residues 398 or 73, respectively. These specific changes undoubtedly account for the neutralization-resistant phenotype of these isolates, since identical substitutions at residues 398 or 73 in MAb-selected escape mutants confer resistance to neutralization by site A or B MAbs. The existence of identical changes in naturally occurring and MAb-selected neutralization-resistant PIV3 strains raises the possibility that antigenically different strains may arise by immune selection during replication in partially immune children. Three of the seven clinical strains examined had differences in their F protein cleavage site sequence. Whereas the prototype PIV3 strain has the cleavage site sequence Arg-Thr-Lys-Arg, one clinical isolate had the sequence Arg-Thr-Arg-Arg and two isolates had the sequence Arg-Thr-Glu-Arg. The different cleavage site sequences of these viruses did not affect their level of replication in either continuous simian or bovine kidney cell monolayers (in the presence or absence of exogenous trypsin or plasmin) or in the upper or lower respiratory tract of rhesus monkeys. We conclude that two nonconsecutive basic residues within the F protein cleavage site are sufficient for efficient replication of human PIV3 in primates.