The Fusion Protein Specificity of the Parainfluenza Virus Hemagglutinin-Neuraminidase Protein Is Not Solely Defined by the Primary Structure of Its Stalk Domain.

UNLABELLED: Virus-specific interaction between the attachment protein (HN) and the fusion protein (F) is prerequisite for the induction of membrane fusion by parainfluenza viruses. This HN-F interaction presumably is mediated by particular amino acids in the HN stalk domain and those in the F head domain. We found in the present study, however, that a simian virus 41 (SV41) F-specific chimeric HPIV2 HN protein, SCA, whose cytoplasmic, transmembrane, and stalk domains were derived from the SV41 HN protein, could not induce cell-cell fusion of BHK-21 cells when coexpressed with an SV41 HN-specific chimeric PIV5 F protein, no. 36. Similarly, a headless form of the SV41 HN protein failed to induce fusion with chimera no. 36, whereas it was able to induce fusion with the SV41 F protein. Interestingly, replacement of 13 amino acids of the SCA head domain, which are located at or around the dimer interface of the head domain, with SV41 HN counterparts resulted in a chimeric HN protein, SCA-RII, which induced fusion with chimera no. 36 but not with the SV41 F protein. More interestingly, retroreplacement of 11 out of the 13 amino acids of SCA-RII with the SCA counterparts resulted in another chimeric HN protein, IM18, which induced fusion either with chimera no. 36 or with the SV41 F protein, similar to the SV41 HN protein. Thus, we conclude that the F protein specificity of the HN protein that is observed in the fusion event is not solely defined by the primary structure of the HN stalk domain. IMPORTANCE: It is appreciated that the HN head domain initially conceals the HN stalk domain but exposes it after the head domain has bound to the receptors, which allows particular amino acids in the stalk domain to interact with the F protein and trigger it to induce fusion. However, other regulatory roles of the HN head domain in the fusion event have been ill defined. We have shown in the current study that removal of the head domain or amino acid substitutions in a particular region of the head domain drastically change the F protein specificity of the HN protein, suggesting that the ability of a given HN protein to interact with an F protein is defined not only by the primary structure of the HN stalk domain but also by its conformation. This notion seems to account for the unidirectional substitutability among rubulavirus HN proteins in triggering noncognate F proteins.

Full conversion of the hemagglutinin-neuraminidase specificity of the parainfluenza virus 5 fusion protein by replacement of 21 amino acids in its head region with those of the simian virus 41 fusion protein.

For most parainfluenza viruses, a virus type-specific interaction between the hemagglutinin-neuraminidase (HN) and fusion (F) proteins is a prerequisite for mediating virus-cell fusion and cell-cell fusion. The molecular basis of this functional interaction is still obscure partly because it is unknown which region of the F protein is responsible for the physical interaction with the HN protein. Our previous cell-cell fusion assay using the chimeric F proteins of parainfluenza virus 5 (PIV5) and simian virus 41 (SV41) indicated that replacement of two domains in the head region of the PIV5 F protein with the SV41 F counterparts bestowed on the PIV5 F protein the ability to induce cell-cell fusion on coexpression with the SV41 HN protein while retaining its ability to induce fusion with the PIV5 HN protein. In the study presented here, we furthered the chimeric analysis of the F proteins of PIV5 and SV41, finding that the PIV5 F protein could be converted to an SV41 HN-specific chimeric F protein by replacing five domains in the head region with the SV41 F counterparts. The five SV41 F-protein-derived domains of this chimera were then divided into 16 segments; 9 out of 16 proved to be not involved in determining its specificity for the SV41 HN protein. Finally, mutational analyses of a chimeric F protein, which harbored seven SV41 F-protein-derived segments, revealed that replacement of at most 21 amino acids of the PIV5 F protein with the SV41 F-protein counterparts was enough to convert its HN protein specificity.

Ribavirin inhibits human parainfluenza virus type 2 replication in vitro.

The antiviral activities of eight nucleoside analog antiviral drugs (ribavirin, acyclovir, lamivudine, 3'-azido-3'-deoxythymidine, emtricitabine, tenofovir, penciclovir and ganciclovir) against human parainfluenza virus type 2 (hPIV-2) were investigated. Only ribavirin (RBV) inhibited both cell fusion and hemadsorption induced by hPIV-2. RBV considerably reduced the number of viruses released from the cells. Virus genome synthesis was inhibited by RBV, as determined by real time PCR. An indirect immunofluorescence study showed that RBV largely inhibited viral protein synthesis. mRNAs of the proteins were not detected, indicating that inhibition of protein synthesis was caused by transcription inhibition by RBV. Using a recombinant green fluorescence protein-expressing hPIV-2 without matrix protein, it was found that RBV did not completely inhibit virus entry into the cells; however, it almost completely blocked multinucleated giant cell formation. RBV did not disrupt actin microfilaments and microtubules. These results indicate that the inhibitory effect of RBV is caused by inhibition of both virus genome and mRNA synthesis, resulting in inhibition of virus protein synthesis, viral replication and multinucleated giant cell formation (extensive cell-to-cell spreading of the virus).

Inhibitory effects of kaempferol, quercetin and luteolin on the replication of human parainfluenza virus type 2 in vitro.

The eight flavonoids, apigenin, chrysin, hesperidin, kaempferol, myricetin, quercetin, rutin and luteolin were tested for the inhibition of human parainfluenza virus type 2 (hPIV-2) replication. Three flavonoids out of the eight, kaempferol, quercetin and luteolin inhibited hPIV-2 replication. Kaempferol reduced the virus release (below 1/10,000), partly inhibited genome and mRNA syntheses, but protein synthesis was observed. It partly inhibited virus entry into the cells and virus spreading, and also partly disrupted microtubules and actin microfilaments, indicating that the virus release inhibition was partly caused by the disruption of cytoskeleton. Quercetine reduced the virus release (below 1/10,000), partly inhibited genome, mRNA and protein syntheses. It partly inhibited virus entry and spreading, and also partly destroyed microtubules and microfilaments. Luteolin reduced the virus release (below 1/100,000), largely inhibited genome, mRNA and protein syntheses. It inhibited virus entry and spreading. It disrupted microtubules and microfilaments. These results indicated that luteolin has the most inhibitory effect on hPIV-2 relication. In conclusion, the three flavonoids inhibited virus replication by the inhibition of genome, mRNA and protein syntheses, and in addition to those, by the disruption of cytoskeleton in vitro.

Toxicologic pathological mechanism of acute lung injury induced by oral administration of benzalkonium chloride in mice.

Benzalkonium chloride (BAC) intoxication causes fatal lung injuries, such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). However, the pathogenesis of ALI/ARDS induced by BAC ingestion is poorly understood. This study aimed to clarify the mechanism of lung toxicity after BAC ingestion in a mouse model. BAC was orally administered to C57BL/6 mice at doses of 100, 250, and 1250 mg/kg. After administration, BAC concentrations in the blood and lungs were evaluated via liquid chromatography with tandem mass spectrometry. Lung tissue injury was evaluated via histological and protein analyses. Blood and lung BAC concentration levels after oral administration increased in a dose-dependent manner, with the concentrations directly proportional to the dose administered. The severity of lung injury worsened over time after the oral administration of 1250 mg/kg BAC. An increase in the terminal transferase dUTP nick end labeling-positive cells and cleaved caspase-3 levels was observed in the lungs after 1250 mg/kg BAC administration. In addition, increased cleaved caspase-9 levels and mitochondrial cytochrome c release into the cytosol were observed. These results suggest that lung tissue injury with excessive apoptosis contributes to BAC-induced ALI development and exacerbation. Our findings provide useful information for developing an effective treatment for ALI/ARDS induced by BAC ingestion.

Identification of domains on the fusion (F) protein trimer that influence the hemagglutinin-neuraminidase specificity of the f protein in mediating cell-cell fusion.

For most paramyxoviruses, virus type-specific interaction between fusion (F) protein and attachment protein (hemagglutinin-neuraminidase [HN], hemagglutinin [H], or glycoprotein [G]) is a prerequisite for mediating virus-cell fusion and cell-cell fusion. Our previous cell-cell fusion assay using the chimeric F proteins of human parainfluenza virus 2 (HPIV2) and simian virus 41 (SV41) suggested that the middle region of the HPIV2 F protein contains the site(s) that determines its specificity for the HPIV2 HN protein. In the present study, we further investigated the sites of the F protein that could be critical for determining the HN protein specificity. By analyzing the reported structure of the F protein of parainfluenza virus 5 (PIV5), we found that four major domains (M1, M2, M3, and M4) and five minor domains (A to E) in the middle region of the PIV5 F protein were exposed on the trimer surface. We then replaced these domains with the SV41 F counterparts individually or in combination and examined whether the resulting chimeras could mediate cell-cell fusion when coexpressed with the SV41 HN protein. The results showed that a chimera designated M(1+2), which harbored SV41 F-derived domains M1 and M2, mediated cell-cell fusion with the coexpressed SV41 HN protein, suggesting that these domains are involved in determining the HN protein specificity. Intriguingly, another chimera which harbored the SV41 F-derived domain B in addition to domains M1 and M2 showed increased specificity for the SV41 HN protein compared to that of M(1+2), although it was capable of mediating cell-cell fusion by itself.

Human parainfluenza virus type 2 L protein regions required for interaction with other viral proteins and mRNA capping.

The large RNA polymerase (L) protein of human parainfluenza virus type 2 (hPIV2) binds the nucleocapsid, phosphoprotein, and V protein, as well as itself, and these interactions are essential for transcription and replication of the viral RNA genome. Although all of these interactions were found to be mediated through the domains within the N terminus of L, the C terminus of the L protein was also required for minigenome reporter gene expression. We have identified a highly conserved rubulavirus domain near the C terminus of the L protein that is required for mRNA synthesis but not for genome replication. Remarkably, this region of L shares homology with a conserved region of cellular capping enzymes that binds GTP and forms a lysyl-GMP enzyme intermediate, the first step in the cellular capping reaction. We propose that this conserved region of L also binds GTP (or GDP) to carry out the second step of the unconventional nonsegmented negative-strand virus capping reaction.

The role of CD98hc in mouse macrophage functions.

CD98hc is a type II transmembrane protein that covalently links to one of several L-type amino acid transporters. CD98hc was first identified as a lymphocyte activation marker. In this study, we examined the role that CD98hc plays in the functions of macrophages using tissue specific knock-out miceCD98hc (CD98hc(flox/-)LysM-cre mice). When isolated peritoneal macrophages were incubated for 48 h, the macrophages obtained from the knock-out mice showed round-shaped morphologies, while almost all of the cells obtained from the control mice were spindle-shaped. The macrophage functions such as the antigen-presenting, phagocytic, and fusion activities, have been reported to decrease in CD98hc-deficient peritoneal macrophages. In addition, when the CD98hc deficient macrophages were stimulated with either IFN-γ/LPS or IL-4, the production of NO(2) or arginase-I decreased in comparison to that observed in the control macrophages. These findings show that the CD98hc molecules play an important role in the activation and functions of macrophages.

Completion of the full-length genome sequence of human parainfluenza virus types 4A and 4B: sequence analysis of the large protein genes and gene start, intergenic and end sequences.

We have already reported the nucleotide sequences of the NP, P/V, M, F and HN genes of human parainfluenza virus type 4A (hPIV-4A) and type 4B (hPIV-4B). Here, we have determined the sequences of the L protein genes as well as the gene start, intergenic and end sequences, thereby completing the full-length genome sequence of hPIV-4A and 4B. hPIV-4A and 4B have 17,052 and 17,304 nucleotides, respectively. The end sequence of hPIV-4, especially 4B, was extraordinarily long. In a comparison with members of the genus Rubulavirus, the hPIV-4 L proteins were closely related to those of mumps virus (MUV) and hPIV-2, less closely related to those of Menangle virus and Tioman virus, and more distantly related to those of Mapuera virus and porcine rubulavirus.

Inhibitory effect of traditional herbal (kampo) medicines on the replication of human parainfluenza virus type 2 in vitro.

Thirteen herbal medicines, Kakkonto (TJ-001), Kakkontokasenkyushin'i (TJ-002), Hangekobokuto (TJ-016), Shoseiryuto (TJ-019), Maoto (TJ-027), Bakumondoto (TJ-029), Hochuekkito (TJ-041), Goshakusan (TJ-063), Kososan (TJ-070), Chikujountanto (TJ-091), Gokoto (TJ-095), Saibokuto (TJ-096), and Ryokankyomishingeninto (TJ-119) were tested for human parainfluenza virus type 2 (hPIV-2) replication. Eight (TJ-001, TJ-002, TJ-019, TJ-029, TJ-041, TJ-063, TJ-095 and TJ-119) out of the thirteen medicines had virus growth inhibitory activity. TJ-001 and TJ-002 inhibited virus release, and largely inhibited genome, mRNA and protein syntheses. TJ-019 slightly inhibited virus release, inhibited gene and mRNA syntheses, and largely inhibited protein synthesis. TJ-029 slightly inhibited virus release, largely inhibited protein synthesis, but gene and mRNA syntheses were unaffected. TJ-041 only slightly inhibited virus release, the gene and mRNA syntheses, but largely inhibited protein synthesis. TJ-091 largely inhibited gene, mRNA and protein syntheses. TJ-095 largely inhibited gene synthesis, but NP and HN mRNAs were slightly detected, and protein syntheses were observed. TJ-119 inhibited gene, mRNA and protein syntheses. TJ-001, TJ-002, TJ-091, TJ-095 and TJ-119 inhibited multinucleated giant cell formation derived from cell-to-cell spreading of virus. However, in TJ-019, TJ-029 and TJ-041 treated infected cells, only small sized fused cells with some nuclei were found. TJ-019 and TJ-041 slightly disrupted actin microfilaments, and TJ-001 and TJ-002 destroyed them. TJ-041 slightly disrupted microtubules, and TJ-001 and TJ-002 disrupted them. In general, the medicines effective on common cold and bronchitis inhibited hPIV-2 replication.

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.

Investigation of the Effective Concentration of Ozonated Water for Disinfection in the Presence of Protein Contaminants.

Ozonated water (OW) is presently used as a chemical disinfectant in many fields, due to its versatile antimicrobial properties. As ozone rapidly decomposes to oxygen, especially in the presence of organic matter, it is important to estimate the authentic antimicrobial activity of OW in the presence of contaminants. However, the effect of contaminants on the antimicrobial activity of OW has not been fully investigated. To address this, we evaluated the effect of protein contaminants on the antimicrobial activity of OW. The survival rate of each tested microorganism excluding Bacillus subtilis spores was reduced to less than 0.1%, when the microorganism suspension was exposed to 9.1 ppm of OW for 15 s in the presence of 0.0045% protein. Our study therefore suggests that approximately 10 ppm of OW can reduce the survival rates of almost all microorganisms in the presence of proteins.

Inhibitions of human parainfluenza virus type 2 replication by ribavirin and mycophenolate mofetil are restored by guanosine and S-(4-nitrobenzyl)-6-thioinosine.

The antiviral activities of a nucleoside analog antiviral drug (ribavirin) and a non-nucleoside drug (mycophenolate mofetil) against human parainfluenza virus type 2 (hPIV-2) were investigated, and the restoration of the inhibition by guanosine and S-(4-nitrobenzyl)-6-thioinosine (NBTI: equilibrative nucleoside transporter 1 inhibitor) were also investigated. Ribavirin (RBV) and mycophenolate mofetil (MMF) inhibited cell fusion induced by hPIV-2. Both RBV and MMF considerably reduced the number of viruses released from the cells. Virus genome synthesis was inhibited by RBV and MMF as determined by polymerase chain reaction (PCR) and real time PCR. mRNA syntheses were also reduced. An indirect immunofluorescence study showed that RBV and MMF largely inhibited viral protein syntheses. Using a recombinant green fluorescence protein (GFP)-expressing hPIV-2 without matrix protein (rhPIV-2ΔMGFP), it was found that virus entry into the cells and multinucleated giant cell formation were almost completely blocked by RBV and MMF. RBV and MMF did not disrupt actin microfilaments or microtubules. Both guanosine and NBTI completely or partially reversed the inhibition by RBV and MMF in the viral replication, syntheses of genome RNA, mRNA and protein, and multinucleated giant cell formation. NBTI caused a little damage in actin microfilaments, but had no effect on microtubules. Both RBV and MMF inhibited the replication of hPIV-2, mainly by inhibiting viral genome RNA, mRNA and protein syntheses. The inhibition was almost completely recovered by guanosine. These results indicate that the major mechanism of the inhibition is the depletion of intracellular GTP pools.

Glycyrrhizin inhibits human parainfluenza virus type 2 replication by the inhibition of genome RNA, mRNA and protein syntheses.

The effect of glycyrrhizin on the replication of human parainfluenza virus type 2 (hPIV-2) was examined. Cell fusion induced by hPIV-2 was inhibited by glycyrrhizin, and glycyrrhizin reduced the number of viruses released from the cells. Glycyrrhizin did not change cell morphology at 1 day of culture, but caused some damage at 4 days, as determined by the effect on actin microfilaments. However, it affected the cell viability at 1 day: about 20% of the cells were not alive by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 1 day of culture. Real-time polymerase chain reaction (PCR) and PCR showed that virus genome synthesis was largely inhibited. mRNA synthesis was also inhibited by glycyrrhizin. Viral protein synthesis was largely inhibited as observed by an indirect immunofluorescence study. Multinucleated giant cell formation was studied using a recombinant green fluorescence protein (GFP)-expressing hPIV-2 without matrix protein (rhPIV-2ΔMGFP). A few single cells with fluorescence were observed, but the formation of giant cells was completely blocked. Taken together, it was shown that viral genome, mRNA and protein syntheses, including F and HN proteins, were inhibited by glycyrrhizin, and consequently multinucleated giant cell formation was not observed and the infectious virus was not detected in the culture medium.

Possible activation of murine T lymphocyte through CD98 is independent of interleukin 2/interleukin 2 receptor system.

CD98 is a widely expressed cell surface heterodimeric protein of 125 kDa. Its expression is upregulated during lymphocyte activation induced by mitogen, superantigen, conventional antigen, and a combination of phorbol myristate acetate (PMA) and ionomycin. However, the role of CD98 in the immune system is not so well understood. The role of CD98 in murine T lymphocyte proliferation was investigated, especially in correlation with the interleukin 2 (IL-2)/interleukin 2 receptor (IL-2R) system. Monoclonal antibody (mAb) directed against murine CD98 heavy chain (mCD98HC) suppressed the proliferation of lymphocytes stimulated with concanavalin A (Con A). Anti-mCD98HC mAb did not suppress the expression of IL-2Ralpha. Anti-IL-2Ralpha mAb, which suppressed DNA synthesis, did not inhibit the expression of CD98HC. Murine IL-2 (recombinant), which induced considerable DNA synthesis by lymphocytes stimulated with a sub-optimal dose of Con A, did not induce CD98HC expression in lymphocytes. In addition, anti-mCD98HC mAb did not inhibit the production of IL-2 by lymphocyte stimulated with Con A. Taken together with these findings, it was speculated that the CD98 system is independent of the IL-2/IL-2R system in murine T lymphocyte activation.

Cytological properties of stromal cells derived from giant cell tumor of bone (GCTSC) which can induce osteoclast formation of human blood monocytes without cell to cell contact.

When human blood monocytes were cocultured with stromal cells derived from human giant cell tumor of bone (GCTSC) and a Millipore filter (0.4 microm) was interposed between monocytes and GCTSC, multinucleated giant cell formation of monocytes was induced. The multinucleated giant cells have characters as osteoclast-like cells, indicating that a soluble osteoclast-inducing factor(s) is secreted from GCTSC expressing RANK, RANKL/ODF/OPGL and TACE mRNA. Furthermore, OCIF/OPG inhibited GCTSC-induced osteoclastogenesis, showing that the RANK-RANKL system is involved in GCTSC-induced osteoclastogenesis and that soluble form of ODF/RANKL induces osteoclasts from monocytes. GCTSC expressed the cytokine mRNAs such as M-CSF, GM-CSF, IL-3, IL-4, IL-6, and IFN-gamma mRNAs. None of IL-1ralpha, IL-1alpha, IL-1beta, IL-2, IL-4, IL-10, IL-18, TNF-alpha, G-CSF and IFN-gamma could be detected in all culture media. A significant amount of IL-6 could be detected in the culture media of all GCTSC. IL-8 was found in the culture media of two GCTSC and two osteosarcoma-derived cells. M-CSF was detected in all culture media. GCTSC express CaSR, and stimulation of GCTSC with either extracellular Ca(2+) or neomycin, agonist of CaSR, augmented the expression of RANKL. Some lines of GCTSC expressed alkaline phosphatase, osteocalcin and Cbfa1, suggesting that GCTSC are intimately related to osteoblastic lineage.

Up-regulated expression of a novel gene in activated human peripheral blood mononuclear cells that is a truncated paralog of the human system L-amino acid transporter 1.

INTRODUCTION AND AIMS: The human system L-amino acid transporter1 (hLAT1) is one of the CD98 light chains and its gene has been mapped to chromosome 16q24. Our preliminary findings have indicated that in HeLa S3 cells there are transcripts whose nucleotide sequences are very similar but not identical to that of the amino acid transporter. This study intends to examine whether these novel transcripts have biological significance through elucidating their genetic aspects and expression profiles in human cells. METHODS: The expression levels of the transcripts were quantified by real-time PCR analysis. Chromosomal mapping of the gene was performed by fluorescence in situ hybridization (FISH). RESULTS: Three types of transcripts were identified and their nucleotide sequences were aligned with the chromosome 16p12 clone with high identity. They encoded 180- or 190-amino acid proteins, showing 92-94% of amino acid identity to the amino-terminal region of the hLAT1 (507 amino acids). However, their 3' non-coding sequences did not show homology to the nucleotide sequence of the amino acid transporter. Their genes were mapped to chromosome 16p11.2-p13.1 as low-copy repeats (LCRs). The transcription of one of these genes in peripheral blood mononuclear cells was significantly up-regulated when the cells were stimulated with concanavalin A. CONCLUSION: We have characterized the three truncated paralogs of the hLAT1 gene. It is suggested that the expression of one of these paralogs may play an important role in the activation of peripheral blood mononuclear cells.

Intranasally administered antigen 85B gene vaccine in non-replicating human Parainfluenza type 2 virus vector ameliorates mouse atopic dermatitis.

Atopic dermatitis (AD) is a refractory and recurrent inflammatory skin disease. Various factors including heredity, environmental agent, innate and acquired immunity, and skin barrier function participate in the pathogenesis of AD. T -helper (Th) 2-dominant immunological milieu has been suggested in the acute phase of AD. Antigen 85B (Ag85B) is a 30-kDa secretory protein well conserved in Mycobacterium species. Ag85B has strong Th1-type cytokine inducing activity, and is expected to ameliorate Th2 condition in allergic disease. To perform Ag85B function in vivo, effective and less invasive vaccination method is required. Recently, we have established a novel functional virus vector; recombinant human parainfluenza type 2 virus vector (rhPIV2): highly expressive, replication-deficient, and very low-pathogenic vector. In this study, we investigated the efficacy of rhPIV2 engineered to express Ag85B (rhPIV2/Ag85B) in a mouse AD model induced by repeated oxazolone (OX) challenge. Ear swelling, dermal cell infiltrations and serum IgE level were significantly suppressed in the rhPIV2/Ag85B treated mouse group accompanied with elevated IFN-γ and IL-10 mRNA expressions, and suppressed IL-4, TNF-α and MIP-2 mRNA expressions. The treated mice showed no clinical symptom of croup or systemic adverse reactions. The respiratory tract epithelium captured rhPIV2 effectively without remarkable cytotoxic effects. These results suggested that rhPIV2/Ag85B might be a potent therapeutic tool to control allergic disorders.

Legume lectins inhibit human parainfluenza virus type 2 infection by interfering with the entry.

Three lectins with different sugar binding specificities were investigated for anti-viral activity against human parainfluenza virus type 2 (hPIV-2). The lectins, concanavalin A (Con A), lens culinaris agglutinin (LCA) and peanut agglutinin (PNA), inhibited cell fusion and hemadsorption induced by hPIV-2. Virus nucleoprotein (NP) gene synthesis was largely inhibited, but fusion (F) and hemagglutinin-neuraminidase (HN) gene syntheses were not. An indirect immunofluorescence study showed that Con A inhibited virus NP, F and HN protein syntheses, but LCA did not completely inhibit them, and that PNA inhibited only NP protein synthesis. Using a recombinant green fluorescence protein-expressing hPIV-2, without matrix protein (rghPIV-2ΔM), it was found that virus entry into the cells was not completely prevented. The lectins considerably reduced the number of viruses released compared with that of virus infected cells. The lectins bound to cell surface within 10 min, and many aggregates were observed at 30 min. Con A and LCA slightly disrupted actin microfilaments and microtubules, but PNA had almost no effect on them. These results indicated that the inhibitory effects of the lectins were caused mainly by the considerable prevention of virus adsorption to the cells by the lectin binding to their receptors.

Effects of multiple amino acids of the parainfluenza virus 5 fusion protein on its haemagglutinin-neuraminidase-independent fusion activity.

The fusion (F) protein of parainfluenza virus 5 (PIV-5) strain W3A is able to induce cell fusion when it is expressed alone in baby hamster kidney cells, whilst the F protein of PIV-5 strain WR induces cell fusion only when co-expressed with the haemagglutinin-neuraminidase (HN) protein. It has been shown previously that when Leu-22 of the WR F protein is replaced with the W3A F counterpart (Pro-22), the resulting mutant L22P exhibits HN-independent fusion activity. Furthermore, previous chimeric analysis between L22P and the F protein of PIV-5 strain T1 has suggested that Glu-132 also contributes to the HN-independent fusion activity of L22P. It was shown here that substitution of Glu-132 of L22P with various amino acids including the T1 F protein counterpart (Lys-132) resulted in a reduction in fusion activity, whereas substitution with Asp was the exception in being tolerated. Interestingly, reduced fusion activity of an L22P mutant that harboured the E132K substitution could be restored by an additional D416K substitution but not by a D416E mutation, suggesting that the presence of the same charge at positions 132 and 416 is important for the HN-independent fusion activity. In contrast, substitution of Leu-22 of the WR F protein with various amino acids except those with aliphatic side chains resulted in acquisition of fusion activity, suggesting that the HN dependence of the WR F protein in the induction of cell fusion is attributable to the hydrophobicity of Leu-22. These results indicate that at least three amino acids are involved in the HN-independent fusion activity of the PIV-5 F protein.