Masato Tsurudome

Extensive antigenic diversity among human parainfluenza type 2 virus isolates and immunological relationships among paramyxoviruses revealed by monoclonal antibodies

A panel of 128 monoclonal antibodies (MAbs) directed against hemagglutinin-neuraminidase (HN), fusion (F), matrix (M), and polymerase (P) proteins, and nucleoprotein (NP) of the Toshiba strain of human parainfluenza type 2 virus (PIV2) was prepared to examine the antigenic relationships among clinical isolates of PIV2 and among paramyxoviruses by indirect enzyme-linked immunosorbent assays. The HN proteins of 18 clinical isolates of PIV2 showed extensive antigenic diversity: 23 of 33 anti-HN MAbs showed no or limited reactivity to many isolates, while other structural proteins were antigenically well conserved. Some anti-HN MAbs recognizing conserved epitopes of the isolates exhibited two types of neutralizing activity, that is, these antibodies inhibited viral infectivity through attachment inhibition or fusion inhibition. This result also showed the presence of a potential third function of the HN protein which might affect the fusing activity of the F protein besides the hemagglutinating and neuraminidase activities. Many of the anti-NP and anti-P MAbs reacted with simian virus 41 (SV41) and simian virus 5 (SV5), whereas a few reacted with mumps virus or PIV4. Two of 6 anti-F MAbs reacted with SV41. None of the 128 MAbs showed reactivity with PIV1, PIV3, Newcastle disease virus (NDV), and measles virus. This result confirmed antigenic proximity of SV5 and SV41 to PIV2 and revealed comparatively restricted immunological relatedness among PIV2, PIV4, and mumps virus.

Molecular and biological characterization of fusion regulatory proteins (FRPs): anti-FRP mAbs induced HIV-mediated cell fusion via an integrin system.

Anti‐FRP mAbs induced polykaryocyte formation of U2ME‐7 cells (CD4+U937 cells transfected with the HIV gp160 gene). Anti‐FRP‐1 mAb immunoprecipitated gp80‐85, gp120 and homodimers of these peptides, and anti‐FRP‐2 mAb reacted with gp135 identically to the alpha 3 subunit of integrin. Both anti‐FRP‐1 and anti‐FRP‐2 mAb‐induced cell fusion was blocked by anti‐beta 1 integrin antibody, fibronectin or inhibiting anti‐FRP‐1 antibody. Therefore, anti‐FRP mAbs were thought to induce the fusion via an integrin system(s). FRP‐mediated fusion was temperature, cytoskeleton, energy and Ca2+ dependent. These experiments showed a possible regulatory function of cell fusion by an integrin system(s).

Sequence analysis of P gene of human parainfluenza type 2 virus: P and cysteine-rich proteins are translated by two mRNAs that differ by two nontemplated G residues

We cloned and sequenced the cDNAs against genomic RNA and mRNA for phosphoprotein (P) of human parainfluenza type 2 virus (PIV-2). cDNA clone from genomic RNA was 1439 nucleotides in length excluding poly(A) and was found to have two small open reading frames encoding proteins of 233 and 249 amino acids. Two different mRNA cDNA clones were obtained; that is, one mRNA contained a smaller reading frame coding 225 amino acids, V protein, and the other mRNA contained a larger reading frame coding 395 amino acids, P protein. Both mRNAs had G cluster in coding frame. The former mRNA contained seven G residues, and two extra G residues were inserted in the latter mRNA. Ten cDNA clones from the genomic RNA were identical and were composed of seven G residues, indicating that genomes analyzed here were a homogeneous population. Therefore, V protein is encoded by faithfully copied mRNA and P protein is translated from mRNA in which two additional G residues are nontemplately inserted immediately after seven genomically encoded G residues. The V and P proteins are amino coterminal proteins and have different C termini. The C terminus of V protein is cysteine-rich and bears some resemblance to metal-binding protein of the zinc finger-type motif. P protein sequence of PIV-2 showed high homologies with SV 5 (40.4%) and mumps virus (35.5%), and a moderate homology with Newcastle disease virus (20.6%). On the other hand, very little homology was found between PIV-2 and other paramyxoviruses including Sendai virus, PIV-3, and measles virus. The cysteine-rich region in V protein was found to be highly conserved in PIV-2, SV 5, and measles virus, suggesting that V protein of paramyxoviruses plays important roles in transcription and/or replication. The predicted cysteine-rich V protein was detected in virus-infected cells using antiserum directed against an oligopeptide specific for the predicted V polypeptide.

Sequence determination of the hemagglutinin-neuraminidase (HN) gene of human parainfluenza type 2 virus and the construction of a phylogenetic tree for HN proteins of all the paramyxoviruses that are infectious to humans

The nucleotide sequence of the hemagglutinin-neuraminidase (HN) gene of human parainfluenza type 2 virus (PIV-2) was determined. The PIV-2 HN gene was 2112 nucleotides excluding poly(A) tail. There was a single large open reading frame in the mRNA which encoded a protein of 571 amino acids with a calculated molecular weight of 63,262. Analysis of the deduced amino acid sequence revealed that there were fourteen potential glycosylation sites and a major hydrophobic region near the N-terminus, which would anchor the protein in the viral membrane. Comparisons of the HN protein sequences of PIV-2 with those of Simian virus 5 (SV5), Sendai virus (SV, parainfluenza virus type 1), human parainfluenza virus type 3 (PIV-3), type 4 (PIV-4), bovine parainfluenza virus type 3 (BPIV-3), mumps virus (MuV), and Newcastle disease virus (NDV) showed definite amino acid sequence relatedness, indicating a common ancestor for these viruses. Furthermore, statistical analysis of the protein sequences suggested a possible evolutionary relatedness among the paramyxoviruses. This is the first time that a phylogenetic tree has been constructed for all the parainfluenza viruses and mumps virus which are infectious to humans. In addition, amino acid sequences involved in hemagglutinating and neuraminidase activities of paramyxovirus were discussed.

Immunological Interrelationships Among Human and Non-human Paramyxoviruses Revealed by Immunoprecipitation

Antigenic interrelationships among paramyxoviruses were examined by immunoprecipitation of isotope-labelled virus-infected cell lysates with specific antisera against virions or virus components. Sendai virus, human parainfluenza virus type 1 (Pa-1) and human parainfluenza virus type 3 (Pa-3) belonged to one antigenic group, and human parainfluenza virus type 2 (Pa-2), human parainfluenza virus type 4 (Pa-4), mumps virus (MuV) and simian virus 5 to a second group. Furthermore, the human paramyxoviruses Pa-1, Pa-2, Pa-3, Pa-4 and MuV formed a single antigenic group which overlapped the above groups. Although Newcastle disease virus (NDV) belonged to a separate group it showed some cross-reaction with the human paramyxoviruses. In particular, certain batches of anti-NDV antisera reacted with the fusion (F) polypeptide of Pa-2 and the reciprocal reaction of anti-Pa-2 antiserum with NDV F was also found. The nucleoprotein showed the broadest cross-reactions among these paramyxoviruses, whereas the matrix polypeptide exhibited antigenic individuality. The nucleoprotein of MuV was most cross-reactive. Pa-2 haemagglutinin-neuraminidase (HN) and anti-MuV HN serum showed cross-reactivity with various antisera and antigens.

Sequence analysis of the phosphoprotein (P) genes of human parainfluenza type 4A and 4B viruses and RNA editing at transcript of the P genes: The number of G residues added is imprecise

We cloned and sequenced the cDNAs against genomic RNAs and mRNAs for phosphoproteins (Ps) of human parainfluenza virus types 4A (PIV-4A) and 4B (PIV-4B). The PIV-4A and -4B P genes were 1535 nucleotides including poly(A) tract and were found to have two small open reading frames, neither of which was apparently large enough to encode the P protein. A cluster of G residues was found in genomic RNA and the number of G residues was 6 in both PIV-4A and -4B. However, the number of G residues at the corresponding site in the mRNAs to the genomic RNA was not constant. Three different mRNA cDNA clones were obtained; the first type of mRNA encodes a larger (P) protein of 399 amino acids, the second type encodes V protein of 229 or 230 amino acids, and the third type encodes the smallest protein (156 amino acids). Comparisons on the nucleotide and the amino acid sequences P and V proteins between these two subtypes revealed extensive homologies. However, these homology degrees are lower than that of NP protein. The C-terminal regions of the P and V proteins of PIV-4s could be aligned with all other Paramyxoviruses, PIV-2, mumps virus (MuV), simian virus 5 (SV 5), Newcastle disease virus (NDV), measles virus (MV), canine distemper virus (CDV), Sendai virus (SV), and PIV-3. On the other hand, the P-V common (N-terminal) regions showed no homology with MV, CDV, SV, and PIV-3. Seven phylogenetic trees of Paramyxoviruses were constructed from the entire and partial regions of P and V proteins.

Monoclonal antibodies against the glycoproteins of mumps virus: fusion inhibition by anti-HN monoclonal antibody

Six monoclonal antibodies (MAbs) against the haemagglutinin-neuraminidase (HN) protein and three against the fusion (F) protein of mumps virus were obtained. Anti-HN MAbs were classified into three antigen-specific groups in competitive binding assays. All of the anti-HN MAbs showed virus-neutralizing and haemolysis-inhibiting activities and inhibited fusion from without (FFWO). Three of them inhibited fusion from within (FFWI) and the cell-to-cell spread of infection. The most effective MAb in inhibiting FFWI possessed neuraminidase-inhibiting activity and little haemagglutination-inhibiting activity. All anti-F MAbs inhibited virus-induced haemolysis but none of them inhibited FFWO, FFWI or the cell-to-cell spread of infection.

Isolation and characterization of monoclonal antibodies to human parainfluenza virus type 4 and their use in revealing antigenic relation between subtypes 4A and 413

Eighty monoclonal antibodies (MAbs) against parainfluenza virus type 4(PIV-4) were isolated and characterized. Of 50 MAbs against PIV-4A, 14 reacted with the nucleocapsid (NP) protein, 11 with the hemagglutinin-neuraminidase (HN) glycoprotein, 6 with the fusion (F) glycoprotein, and 19 with the matrix (M) protein. With the aid of the PIV-4A and PIV-2 specific MAbs showing cross-reactivity with PIV-4B, the structural proteins of PIV-4B were identified. gp72, p65, gp65, gp55, p53, and p40 of PIV-4B were assigned to HN, NP, Fo, F1, P, and M proteins, respectively. Based on the results, specificities of the MAbs against PIV-4B were determined. Of 30 hybridoma clones against PIV-4B, 13 clones were found to produce antibodies against the NP protein, 7 against the HN protein, and 10 against the F protein. Epitope mapping of these MAbs was performed with competitive binding assays in ELISA. According to their biological activities, the MAbs against the HN protein of either PIV-4A or 4B could be divided into three groups. The first group showed high hemagglutination inhibition (HI), hemolysis inhibition (HLI), and neutralizing (NT) activities. The second group showed high NT activity, but could not block hemagglutination. The final group showed a lower level of all activities. The MAbs against the F protein of PIV-4A and against PIV-4B were divided into two groups. Some MAbs against the F protein had high titer of NT, suggesting that the F protein had neutralizing-related epitopes. Antigenicity of the NP protein was highly conserved among subtypes of PIV-4. On the other hand, the MAbs against the HN and the F proteins showed high reactivity with the homologous subtype viruses, but low reactivity with the heterologous subtype viruses, indicating that the external glycoproteins exhibited antigenic variations between two subtypes of PIV-4. When the immunological interrelationship among various paramyxoviruses was analyzed. PIV-4 was found to be antigenically related to PIV-2, SV 5, and mumps virus.

Complete nucleotide sequence of the matrix gene of human parainfluenza type 2 virus and expression of the M protein in bacteria

The sequence of the M gene of human parainfluenza virus type 2 (PIV-2) has been determined. The sequence contained a large open reading frame with 1131 nucleotides encoding a protein with a calculated molecular weight of 42,312. Comparison of M protein sequence indicated that PIV-2 was more closely related to mumps virus and Newcastle disease virus than to other parainfluenza viruses, Sendai virus (SV), and parainfluenza virus type 3 (PIV-3), indicating a possible subdividing of the Paramyxovirus into two groups. This grouping is consistent with that obtained from analysis of the HN gene. Measles virus and canine distemper virus definitely belong to the subgroup composed of SV and PIV-3. No homology region was found in all the paramyxoviruses compared. However, a tertiary structure may be conserved in each subgroup of paramyxovirus. The M protein of PIV-2 was expressed in bacteria, and the product was recognized by a monoclonal antibody specific for the PIV-2 M protein. The bacterial-expressed protein, however, was heterogeneous and smaller in size.

Nucleotide sequence of the leader and nucleocapsid protein gene of mumps virus and epitope mapping with the in vitro expressed nucleocapsid protein

The nucleotide sequence of the leader and the gene encoding the nucleocapsid protein (NP) of mumps virus Miyahara strain have been determined. The leader sequence is 55 nucleotides in length and the NP gene is 1845 nucleotides in length, exclusive of poly(A). The NP gene codes for a protein of 549 amino acids, with a calculated molecular weight of 61,365. For epitope mapping, a series of NPs from which C-termini were serially deleted were expressed in vitro from five mRNA constructs and were examined by radioimmunoprecipitation assay (RIPA) with eight nonoverlapping monoclonal antibodies (MoAbs) against the mumps virus NP. It was found that seven out of eight MoAbs reacted with the NP synthesized in vitro. Five recognized the epitopes located within the C-terminal 74 amino acids region and one within the adjacent 64 amino acids upstream. The epitope of the remaining one was in the N-terminal half of the NP.