Haruo Matsumura

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).

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.

Transcripts of simian virus 41 (SV41) matrix gene are exclusively dicistronic with the fusion gene which is also transcribed as a monocistron.

The complete nucleotide sequences of the matrix (M) and fusion (F) genes of simian virus 41 (SV41) were determined. Deduced amino acid sequences confirmed the close relationship of SV41 with human parainfluenza type 2 virus (PIV2). Analyses of noncoding regions between the F and the hemagglutinin-neuraminidase (HN) genes suggested the absence of the small hydrophobic gene, which is present between the F and the HN genes of simian virus 5 and mumps virus. It was striking that there was no apparent consensus gene end sequence between the M and the F genes and that the M gene was transcribed exclusively as a dicistron with the F gene. The number of monocistronic transcripts of the F gene was approximately half that of the dicistronic transcripts. However, the F protein of SV41 seemed to be efficiently translated, since viral multiplication and fusion from within were as efficient as in PIV2. These results suggest that the lack of a consensus gene end sequence resulted in the readthrough of viral RNA polymerases between the M and the F genes and that the initiation of F gene transcription could occur by newly entered polymerases independently of the polymerases that started the upstream M gene transcription.

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.

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.

Fusion properties of cells constitutively expressing human parainfluenza virus type 4A haemagglutinin-neuraminidase and fusion glycoproteins.

We established HeLa cell lines that constitutively expressed the fusion (F) and/or haemagglutinin-neuraminidase (HN) glycoproteins of human parainfluenza virus type 4A (PIV-4A) and used them to analyse the roles of these glycoproteins in virus-induced cell fusion. No syncytium formation occurred, even in HeLa cells expressing both the F and HN proteins (HeLa-4aF+HN cells). Also no syncytium was found in a mixed culture of cells expressing the F protein (HeLa-4aF) and the HN protein (HeLa-4aHN). Syncytia were observed in HeLa-4aF cells transfected with the HN gene, but no syncytium formation was found in HeLa-4aHN cells transfected with the F gene. Co-cultivation of HeLa-4aF+HN cells with HeLa-4aF cells generated large polykaryocytes, whereas co-cultivation with HeLa-4aHN cells induced no cell fusion. Infection of HeLa-4aF cells with PIV-4A generated large syncytia and degenerated nuclei, whereas little or no polykaryocytes were found in HeLa-4aHN cells infected with PIV-4A. From the above findings, the following conclusions were drawn: (i) the expression of both the F and HN proteins in the same cell is necessary for cell fusion; (ii) the expression of the F protein alone enhances susceptibility to cell fusion; (iii) the constitutive expression of the HN protein promotes resistance to paramyxovirus-induced cell fusion.

Sequence analysis of the large (L) protein of simian virus 5

The complete nucleotide sequence of the large (L) protein gene of simian virus 5 (SV5) was determined from cDNA of the genomic RNA and mRNA, and found to be 6804 bases in length, exclusive of a poly(A) tract. The sequence contained an open reading frame of 6765 nucleotides encoding 2255 amino acids. Results of dot matrix comparisons of the L protein of SV5 with those of human parainfluenza type 3 virus and Sendai virus indicated that there are five conserved domains, and that each domain contains characteristic sequence(s). The L protein of SV5 was detected in purified virions using antiserum directed against an oligopeptide corresponding to the N-terminal region.

RNA EDITING-LIKE PHENOMENON IN PARAMYXOVIRUS V GENE MRNA OBSERVED IN INSECT CELLS INFECTED WITH A RECOMBINANT BACULOVIRUS

The V gene of the paramyxovirus human parainfluenza virus type 2 (hPIV2) is transcribed into both V and P mRNA. The V mRNA is a faithful transcript of the V gene; however, the P mRNA is transcribed by an RNA-editing mechanism in hPIV2-infected mammalian cells. Recombinant baculoviruses (rBV) were constructed containing the wild-type V gene, which has seven G residues at its editing site, and a manipulated V gene with ten G residues at its editing site. A small amount of the P protein was synthesized, in addition to the V protein, when the wild-type V gene was expressed in rBV-infected insect cells. Furthermore, synthesis of the P protein increased when rBV containing the manipulated V gene was used to infect insect cells. Both the P and V proteins were detected after in vitro translation of mRNA from rBV-infected cells. Moreover, G-residue insertions and a deletion were detected in mRNA. Since the P protein was not detected after in vitro translation of V RNA that had been transcribed in vitro by T7 RNA polymerase, these results suggest that the non-encoded G residues were inserted and deleted during transcription in insect cells. This RNA editing-like phenomenon and the implications of the length of the G cluster are discussed.