Shigeru Kusagawa

Gene expression during osteoclast-like cell formation induced by antifusion regulatory protein-1/CD98/4F2 monoclonal antibodies (MAbs): c-src is selectively induced by anti-FRP-1 MAb

Human blood monocytes can differentiate into osteoclast-like cells when they are cultured in the presence of anti-FRP-1. Messenger (mRNA) expression of markers related to osteoclasts was analyzed during differentiation of osteoclasts from monocytes. As markers related to osteoclasts, we selected cathepsin-K, carbonic anhydrase (CA) II, vacuolar H(+)-ATPase (v-ATPase), vitronectin receptor (VNR), tartrate-resistant acid phosphatase (TRAP), osteopontin (OPN), galectin-3, c-src, c-fos, and c-fms. The mRNAs other than c-src mRNA were expressed in freshly isolated monocytes or monocytes incubated with control antibody or anti-FRP-1 monoclonal antibody (MAb) for 14 days. Of these mRNAs, cathepsin-K, CA II, v-ATPase, VNR, TRAP, OPN, and c-fms mRNAs were expressed at higher levels in the osteoclast-like cells than those in monocytes cultured with control antibody. On the other hand, galectin-3 mRNA was expressed at lower levels in the osteoclast-like cells, and there was no significant difference in c-fos mRNA expression between the monocytes cultured with control antibody and anti-FRP-1 MAb. c-src mRNA could not be detected in monocytes freshly isolated or incubated with control antibody. Surprisingly, expression of c-src mRNA was induced in monocytes by anti-FRP-1 MAb and was detectable as early as 3 h after anti-FRP-1 MAb treatment, indicating that c-src is selectively induced by anti-FRP-1 MAb treatment. Furthermore, the osteoclast-like cells expressed calcitonin receptor. Receptor activator of NF-kappaB (RANK) mRNA was detectable in freshly isolated monocytes or monocytes cultured with control antibody or anti-FRP-1 MAbs. Maximal expression of RANK was observed in osteoclast-like cells. On the other hand, no receptor activator of NF-KB ligand (RANKL) mRNA was detectable in any of the samples, suggesting that anti-FRP-1 mAb can induce osteoclast-like cells from blood monocytes without RANKL.

Identification of regions on the fusion protein of human parainfluenza virus type 2 which are required for haemagglutinin-neuraminidase proteins to promote cell fusion

Using a plasmid expression system in HeLa cells, we have previously shown that the fusion (F) protein of simian virus 41 (SV-41) induces cell fusion when coexpressed with the haemagglutinin-neuraminidase (HN) protein of human parainfluenza virus type 2 (PIV-2), while the PIV-2 F protein does not induce cell fusion with the SV-41 HN protein. In the present study, we found that the PIV-2 F protein induced extensive cell fusion with the HN protein of mumps virus (MuV), whereas the SV-41 F protein did not. Chimaeric analyses of the F proteins of PIV-2 and SV-41 identified two regions (designated M1 and M2) on the PIV-2 F protein, either of which was necessary for chimaeric F proteins to show fusogenic activity with the MuV HN protein. Subsequently, two additional regions (P1 and P2) were identified on the PIV-2 F protein, both of which were necessary for chimaeric F proteins to prevent induction of cell fusion with the SV-41 HN protein. Consequently, it was proved that a given chimaeric F protein, harbouring regions P1 and P2 together with either of region M1 or M2, induced cell fusion specifically with HN proteins of PIV-2 and MuV, the same as the PIV-2 F protein. Region M2 was located at the membrane proximal end of the PIV-2 F1 ectodomain, while regions P1, M1 and P2 clustered together in the middle of the ectodomain. These regions on the PIV-2 F protein may be involved in a putative functional interaction with HN proteins, which is considered to be a prerequisite for cell fusion.

Mapping of domains on the human parainfluenza virus type 2 nucleocapsid protein (NP) required for NP-phosphoprotein or NP-NP interaction

The epitopes recognized by 41 monoclonal antibodies directed against the nucleocapsid protein (NP) of human parainfluenza virus type 2 (hPIV-2) were mapped on the primary structure of the hPIV-2 NP protein by testing their reactivities with deletion mutants. By Western immunoblotting using these monoclonal antibodies, the analysis of deletion mutants of the hPIV-2 NP protein was performed to identify the region essential for NP-NP interaction and phosphoprotein (P)-binding sites on the NP protein. The results indicate that the N-terminal 294 aa of the NP protein are all required for NP-NP self-assembly, and that two C-terminal parts of the NP protein are essential for NP-P binding: one region, aa 295-402, is required for binding to the C-terminal part of the P protein and another region, aa 403-494, to the N-terminal part of the P protein.

Identification of the sequences responsible for nuclear targeting of the V protein of human parainfluenza virus type 2.

In human parainfluenza virus type 2 (hPIV-2)-infected cells, anti-phosphoprotein (P)-specific monoclonal antibody (MAb) densely stained the perinuclear regions of infected cells throughout infection, indicating that the P protein was localized exclusively in the cell cytoplasm. By contrast, antigens recognized by MAbs directed against the P-V-common domain of hPIV-2 were located predominantly in the cytoplasm, but in some hPIV-2-infected cells they were also found in the nuclei, suggesting that a fraction of hPIV-2 V protein is localized there. hPIV-2 V protein expressed from a cDNA clone was localized in the nuclei of transfected cells. By using indirect immunofluorescence analyses, we examined the intracellular localization of various sequentially deleted V proteins, to determine the nuclear localization signals (NLS) of the V protein. Two noncontiguous regions in the V protein were required for nuclear localization and retention, since deletion of these regions [region I (aa 1-46) and region II (aa 175-196)] resulted in cytoplasmic localization. Both regions resulted in nuclear localization independently. A nucleoplasmin-like NLS was identified in region II but no consensus targeting sequence could be found in region I. When NP protein was co-expressed with V protein or the N-terminal fragment (aa 1-46) of V protein, a fraction of the NP protein was translocated into cell nuclei.

Antigenic diversity of human parainfluenza virus type 1 isolates and their immunological relationship with Sendai virus revealed by using monoclonal antibodies

Fifty-six monoclonal antibodies (MAbs) directed against human parainfluenza virus type 1 (hPIV-1) were prepared in order to identify the structural proteins of hPIV-1, to examine the immunological relationship between hPIV-1 and Sendai virus (SV), and to determine the antigenic diversity of clinical isolates of hPIV-1. In addition, 41 MAbs characterized previously and directed against SV were used for immunological comparison of SV and hPIV-1 isolates. Of the MAbs against hPIV-1, two reacted with phospho (P) protein, 11 with nucleocapsid protein (NP), 24 with haemagglutinin-neuraminidase (HN) protein and 19 with fusion (F) protein. With the aid of MAbs against hPIV-1 and those against SV showing cross-reactivity with hPIV-1, the structural proteins of hPIV-1 were identified; p83, p56, p34, gp74 and gp60 of hPIV-1 were identified as the P, NP, M, HN and F proteins, respectively. The MAbs against the P protein and NP of hPIV-1 showed limited cross-reactivity with SV, whereas they had high reactivity with clinical isolates of hPIV-1. Interestingly, one MAb against the NP of hPIV-1 lacked reactivity with clinical isolates which were isolated in the 1970s and 1980s. The MAbs against the HN of hPIV-1 also exhibited quite limited reactivity with SV and the clinical isolates; two groups of HN-specific MAbs showed almost no reactivity with the clinical isolates from the 1970s and 1980s, similarly to the NP-specific MAb. However, anti-HN MAbs belonging to the two groups showing specific activities (neuraminidase inhibition and haemolysis inhibition) reacted with almost all clinical isolates. On the other hand, although anti-F protein MAbs had limited reactivity with SV, they showed reactivity with almost all hPIV-1 isolates. The MAbs against the P, NP, M, HN and F proteins of SV also showed limited cross-reactivity with the clinical hPIV-1 isolates, and this reactivity was independent of the time and place of isolation, except for that of the F protein. These results confirm that although hPIV-1 is related to SV, it is antigenically distinct from it.

Molecular evolution of human paramyxoviruses. Nucleotide sequence analyses of the human parainfluenza type 1 virus NP and M protein genes and construction of phylogenetic trees for all the human paramyxoviruses.

SummaryThe nucleotide sequences of the NP and M genes of human parainfluenza type 1 virus (HPIV-1) were determined. The NP gene was 1677 nucleotides long excluding polyadenylic acid. The NP gene contained a single large open reading frame (ORF), which encoded a polypeptide of 524 amino acids with a calculated molecular weight of 57,736. The M gene 1173 nucleotides long excluding the poly(A) tract and the sequence also contained a single large ORF which encoded a polypeptide of 348 amino acid with a molecular weight of 38,445, which was inconsistent with 28 kDa previously determined by SDS-PAGE. We aligned the deduced HPIV-1 NP and M protein sequences with 12 and 13 other paramyxoviruses, respectively, suggesting that a common tertiary structure was found in the NPs or Ms of HPIV-1, Sendai virus (SV), HPIV-3 and BPIV-3 and that other common structure was also maintained in these proteins of HPIV-2, SV 41 and 5, MuV, HPIV-4. Phylogenetic trees were constructed for the NP and M proteins of all the paramyxoviruses of which nucleotide sequences had been previously reported. Paramyxoviruses could be subdivided into two groups, i.e., PIV-1 group and PIV-2 group; the former group is composed of HPIV-1, SV, HPIV-3 and BPIV-3, and the latter group consists of HPIV-2, SV 41, SV 5, MuV, HPIV-4 A and HPIV-4 B.

Isolation of monoclonal antibodies directed against the V protein of human parainfluenza virus type 2 and localization of the V protein in virus-infected cells

Abstract Two monoclonal antibodies (mAbs) specific for the human parainfluenza virus type 2 (hPIV-2) V protein were obtained by immunizing mice with the V protein recombinantly expressed in Escherichia coli. Both mAbs were found to react with the V protein in ELISA and in Western blot analysis. Using these mAbs and previously obtained mAbs specific for hPIV-2 nucleoprotein (NP) or hPIV-2 phospho-(P) protein, we examined the intracellular distributions of the V, P and NP proteins in hPIV-2-infected cells by indirect immunofluorescence analyses. The P and NP proteins were organized in numerous granules in the cytoplasm of hPIV-2 infected cells. In contrast, the V protein showed diffuse nuclear and cytoplasm distributions.

HN proteins of human parainfluenza type 4A virus expressed in cell lines transfected with a cloned cDNA have an ability to induce interferon in mouse spleen cells

Primary monkey kidney cells infected with human parainfluenza type 4A virus (HPIV-4A) were treated with various concentrations of formaldehyde. Formaldehyde (0.275%) treatment completely blocked virus production. However, when mouse spleen cells were cocultured with the fixed virus-infected cells, interferon was produced in the culture fluid. On the other hand, when mouse spleen cells were incubated with the fixed virus-infected cells in the presence of anti-HPIV-4A antiserum or a mixture of anti-HN protein monoclonal antibodies, interferon activity could scarcely be detected in the culture fluid. These findings indicated that the fixed virus-infected cells had an ability to induce interferon in mouse spleen cells and that the HN protein was related to interferon induction. Subsequently, a recombinant plasmid was constructed by inserting the cDNA of the HN gene of HPIV-4A into a pcDL-SR alpha expression vector. Mouse spleen cells produced interferon when cocultured with COS7 cells transfected with the recombinant plasmid, but did not when cocultured with COS7 cells transfected with the vector alone. Furthermore, we established HeLa cells constitutively expressing HPIV-4A HN (HeLa-4aHN cells) or F protein (HeLa-4aF cells). Type I (alpha/beta) interferon was detected in culture fluids of mouse spleen cells with HeLa-4aHN cells, but was not detected in those with HeLa-4aF cells. Therefore, it was concluded that the HN glycoproteins on the cell surface were sufficient for interferon induction to occur.

Sequence determination of the P gene of simian virus 41: presence of irregular deletions near the RNA-editing sites of paramyxoviruses

The complete nucleotide sequence of the P gene of simian virus 41 (SV41) was determined. The gene was found to be 1406 nucleotides long and to contain a relatively small open reading frame encoding a cysteine-rich V protein with a calculated M(r) of 24076. We have demonstrated that RNA-editing events occur in SV41 P gene transcripts and that the ratio of edited mRNAs to faithfully copied mRNA (P-mRNA:V-mRNA) is about 1:5 at either 24 or 40 h post-infection. The mRNA with two G insertions was capable of encoding a P protein of 395 amino acids with a predicted M(r) of 41,992. A kinetic study of P and V proteins by Western blot analysis showed that in virus-infected cells the amounts of both proteins were almost equal although the V-mRNA was considerably more abundant than the P-mRNA. Alignment of the SV41 P and V proteins with those of nine other paramyxoviruses demonstrated that irregular gaps were present around the RNA-editing sites.

Isolation and characterization of monoclonal antibodies directed against murine FRP-1/CD98/4F2 heavy chain: Murine FRP-1 is an alloantigen and amino acid change at 129 (P←→R) is related to the alloantigenicity

Nineteen mAb directed against murine fusion regulatory protein‐1 (mFRP‐1)/4F2/CD98 were isolated and their biological properties were analysed. Intriguingly, mFRP‐1 was found to be an alloantigen, namely, FRP‐1.1 (DBA/2 and CBA mice type) and FRP‐1.2 (BALB/c, C57BL/6 and C3H/He mice type). The nucleotide sequences of FRP‐1.1 and FRP‐1.2 were determined, demonstrating that amino acid change at 129 (P←→R) is related to the alloantigenicity. mFRP‐1 is expressed on thymocytes, on spleen cells, on peripheral lymphocytes and on blood monocytes, suggesting that the physiological role in vivo of murine FRP‐1 is different from that of human FRP‐1. The biological activities of antimFRP‐1 mAbs showed by the present study are: (i) enhancement of Newcastle disease virus‐induced cell fusion; (ii) suppression of HIVgp160‐mediated cell fusion; and (iii) induction of aggregation and multinucleated giant cells of monocytes/macrophages.