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.

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.

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.

Immunological association of inducible bronchus-associated lymphoid tissue organogenesis in Ag85B-rHPIV2 vaccine-induced anti-tuberculosis mucosal immune responses in mice.

We previously reported that Ag85B-expressing human parainfluenza type 2 virus (Ag85B-rHPIV2) was effective as a nasal vaccine against tuberculosis in mice; however, the mechanism by which it induces an immune response remains to be investigated. In the present study, we found that organogenesis of inducible bronchus-associated lymphoid tissue (iBALT) played a role in the induction of antigen-specific T cells and IgA antibody responses in the lung of mice intra-nasally administered Ag85B-rHPIV2. We found that expression of Ag85B was dispensable for the development of iBALT, suggesting that HPIV2 acted as an iBALT-inducing vector. When iBALT organogenesis was disrupted in Ag85B-rHPIV2-immunized mice, either by neutralization of the lymphotoxin pathway or depletion of CD11b+ cells, Ag85B-specific immune responses (i.e. IFN γ-producing T cells and IgA antibody) were diminished in the lung. Furthermore, we found that immunization with Ag85B-rHPIV2 induced neutrophil and eosinophil infiltration temporally after the immunization in the lung. Thus, our results show that iBALT organogenesis contributes to the induction of antigen-specific immune responses by Ag85B-rHPIV2 and that Ag85B-rHPIV2 provokes its immune responses without inducing long-lasting inflammation.

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.

Improvement in early diagnosis of Japanese spotted fever by using a novel Rick PCR system.

Rickettsia diseases, including Japanese spotted fever (JSF), are serious infections. Delayed diagnosis occasionally results in life-threatening liver disorders and disseminated intravascular coagulation (DIC). Because of the shortness of the latent period, serological diagnosis is not preferable for early diagnosis of JSF. Until now, a polymerase chain reaction (PCR)-based diagnosis method has been used for early diagnosis, and the sensitivity reaches as high as 90% using skin biopsy samples as we previously reported. On the other hand, the sensitivity of the same PCR method using blood samples is limited at less than 50%. In the present study, using peripheral blood samples, we developed a novel diagnostic method for JSF using a Rick PCR system with original PCR primers, showing improved sensitivity compared with the conventional nested PCR. It may constitute a preferable diagnostic tool for early and sensitive diagnosis of Rickettsia infection.

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).

Recombinant Ag85B vaccine by taking advantage of characteristics of human parainfluenza type 2 virus vector showed Mycobacteria-specific immune responses by intranasal immunization.

Viral vectors are promising vaccine candidates for eliciting suitable Ag-specific immune response. Since Mycobacterium tuberculosis (Mtb) normally enters hosts via the mucosal surface of the lung, the best defense against Mtb is mucosal vaccines that are capable of inducing both systemic and mucosal immunity. Although Mycobacterium bovis bacille Calmette-Guérin is the only licensed tuberculosis (TB) vaccine, its efficacy against adult pulmonary forms of TB is variable. In this study, we assessed the effectiveness of a novel mucosal TB vaccine using recombinant human parainfluenza type 2 virus (rhPIV2) as a vaccine vector in BALB/c mice. Replication-incompetent rhPIV2 (M gene-eliminated) expressing Ag85B (rhPIV2-Ag85B) was constructed by reverse genetics technology. Intranasal administration of rhPIV2-Ag85B induced Mtb-specific immune responses, and the vaccinated mice showed a substantial reduction in the number of CFU of Mtb in lungs and spleens. Unlike other viral vaccine vectors, the immune responses against Ag85B induced by rhPIV2-Ag85B immunization had an advantage over that against the viral vector. In addition, it was revealed that rhPIV2-Ag85B in itself has an adjuvant activity through the retinoic acid-inducible gene I receptor. These findings provide further evidence for the possibility of rhPIV2-Ag85B as a novel TB vaccine.

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.

Human parainfluenza virus type 2 vector induces dendritic cell maturation without viral RNA replication/transcription.

The dendritic cell (DC), a most potent antigen-presenting cell, plays a key role in vaccine therapy against infectious diseases and malignant tumors. Although advantages of viral vectors for vaccine therapy have been reported, potential risks for adverse effects prevent them from being licensed for clinical use. Human parainfluenza virus type 2 (hPIV2), one of the members of the Paramyxoviridae family, is a nonsegmented and negative-stranded RNA virus. We have developed a reverse genetics system for the production of infectious hPIV2 lacking the F gene (hPIV2ΔF), wherein various advantages for vaccine therapy exist, such as cytoplasmic replication/transcription, nontransmissible infectivity, and extremely high transduction efficacy in various types of target cells. Here we demonstrate that hPIV2ΔF shows high transduction efficiency in human DCs, while not so high in mouse DCs. In addition, hPIV2ΔF sufficiently induces maturation of both human and murine DCs, and the maturation state of both human and murine DCs is almost equivalent to that induced by lipopolysaccharide. Moreover, alkylating agent ß-propiolactone-inactivated hPIV2ΔF (BPL-hPIV2ΔF) elicits DC maturation without viral replication/transcription. These results suggest that hPIV2ΔF may be a useful tool for vaccine therapy as a novel type of paramyxoviral vector, which is single-round infectious vector and has potential adjuvant activity.

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.

Single systemic administration of Ag85B of mycobacteria DNA inhibits allergic airway inflammation in a mouse model of asthma.

The immune responses of T-helper (Th) and T-regulatory cells are thought to play a crucial role in the pathogenesis of allergic airway inflammation observed in asthma. The correction of immune response by these cells should be considered in the prevention and treatment of asthma. Native antigen 85B (Ag85B) of mycobacteria, which cross-reacts among mycobacteria species, may play an important biological role in host-pathogen interaction since it elicits various immune responses by activation of Th cells. The current study investigated the antiallergic inflammatory effects of DNA administration of Ag85B from Mycobacterium kansasii in a mouse model of asthma. Immunization of BALB/c mice with alum-adsorbed ovalbumin followed by aspiration with aerosolized ovalbumin resulted in the development of allergic airway inflammation. Administration of Ag85B DNA before the aerosolized ovalbumin challenge protected the mice from subsequent induction of allergic airway inflammation. Serum and bronchoalveolar lavage immunoglobulin E levels, extent of eosinophil infiltration, and levels of Th2-type cytokines in Ag85B DNA-administered mice were significantly lower than those in control plasmid-immunized mice, and levels of Th1-and T-regulatory-type cytokines were enhanced by Ag85B administration. The results of this study provide evidence for the potential utility of Ag85B DNA inoculation as a novel approach for the treatment of asthma.

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.

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.

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.

A quantitative method for analyzing establishing-efficiency of persistent viral infection.

A quantitative method for analyzing establishing-efficiency of persistent infection was devised. The efficiency of hPIV2 CA and SV5 T1 strains was found to be high, that is, 0.1 approximately 0.3 (an efficiency of 1.0 indicates that 100% of the virus-infected cells became persistently infected). The efficiency of the SV5 WR strain was also high, approximately 0.1, though the virus had no ability to immediately establish a steady state of persistent infection in whole cell-culture systems. At about 0.0007, the efficiency of SV41 was almost the same as that of the hPIV2 Toshiba strain. The establishing efficiencies of various rSeV were further analyzed in detail. The efficiencies of the rSeV(PA), rSeV(Ppi) and rSeV(HNpi) were below the limit of detection, while that of rSeV(Lpi) was nearly 1. Although the efficiency was around 0.001, the rSeV(Mpi) and the rSeV(Fpi) were unexpectedly found to be capable of forming persistently-infected cells, indicating that both the Fpi and Mpi proteins contribute to the establishing efficiency of persistent infection of SeVpi.

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.

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.