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.

University students' living conditions during the COVID-19 pandemic and predictors of their subjective health views: A cross-sectional survey.

This study aimed to explore the factors influencing subjective health views based on the living conditions and concerns of university students during the coronavirus infection 2019 (COVID-19) pandemic. From March to April 2021, a questionnaire survey was administered to 8,547 Japanese university students, and logistic regression analysis was used to explore factors related to subjective health views. The results showed that satisfaction with quality of sleep (OR = 2.651, 95% Cl 2.370-2.966,p < 0.001), satisfaction with university life (OR = 2.486, 95%Cl 2.215-2.789, p < 0.001), satisfaction with diet (OR = 1.849, 95% CI: 1.496-2.285, p < 0.001), regular exercise (OR = 1.759, 95% CI: 1.594-1.941, p < 0.001), consciousness of nutritional balance (OR = 1.276, 95% CI: 1.147-1.420,p < 0.001), eating breakfast every day (OR = 1.247, 95% CI: 1.121-1.387, p < 0.001), and consuming soft drinks at least once a week (OR = 0.865, 95% CI: 0.755-0.966, p = 0.010) were positive factors for subjective views of health. On the other hand, anxiety about whether the necessary credits can be obtained (OR = 0.885, 95% CI: 0.799-0.980, p = 0.019), infection from minimal outings (OR = 0.881, 95% CI: 0.794-0.976, p = 0.016) building and maintaining friendships on campus (OR = 0.867, 95% CI: 0.767-0.980, p = 0.023), and being able to continue working (OR = 0.713, 95% CI: 0.640-0.795, p < 0.001) were identified as negative factors. To ensure a healthy university life during the COVID-19 pandemic or future pandemic, supports tailored to students' living conditions and measures to address their anxieties are required.

Nicotine Dependence among College Students Uninterested in Smoking Cessation during the COVID-19 Pandemic: A Cross-Sectional Survey.

This study investigated nicotine dependence among Japanese university students who had reached the smoking age (20 years or older) by the time of the coronavirus disease 2019 (COVID-19) pandemic and examined factors that encourage early smoking cessation. Social dependence on nicotine was evaluated using the Kano Total Social Nicotine Dependence Level (KTSND), and physiological dependence was evaluated using the Fagerström Nicotine Dependence Index (FTND). Of the 356 college students who smoked (4.4% of the total), 182 (51.1%) stated that they were not interested in quitting. Furthermore, 124 (68.1%) of those with no interest in quitting smoking were aware that smoking is a high-risk factor for COVID-19, and 58 (31.9%) were unaware. The group not aware of this risk had significantly higher KTSND scores than the group aware of it. The examination of cigarette type that indicated the users of non-conventional cigarette products and dual-user groups scored significantly higher than the cigarette group on FTND items. Overall, the smokers scored above the normal range for social nicotine dependence, suggesting the need to reduce nicotine dependence to encourage college students who continue to smoke to quit smoking.

Factors affecting health-related quality of life among firefighters during the COVID-19 pandemic: A single-center study.

During the coronavirus disease 2019 (COVID-19) outbreak, firefighters have been working in an environment that is both physically and mentally taxing. This study aimed to investigate factors affecting health-related quality of life (HRQOL) among firefighters in Japan during the COVID-19 pandemic. A total of 227 firefighters from a single firefighting organization were surveyed in June 2021, during the fourth infection spread period of COVID-19 in Japan. Regression analysis was performed to examine factors affecting HRQOL of firefighters measured with the SF-8. In the present study, factors affecting HRQOL among firefighters during the COVID-19 pandemic were lack of sleep, physical abnormalities due to infection control measures, exercise habits, living with family members, and history of suspected COVID-19 infection. The present findings may help develop support services for first responders, including firefighters during the COVID-19 pandemic.

Willingness and Predictors of Bystander CPR Intervention in the COVID-19 Pandemic: A Survey of Freshmen Enrolled in a Japanese University.

The coronavirus disease 2019 (COVID-19) pandemic has decreased bystander cardiopulmonary resuscitation (BCPR) intervention rates. The purpose of this study was to elucidate the willingness of university freshmen to provide BCPR during the COVID-19 pandemic and the predictors thereof. A cross-sectional survey of 2789 newly enrolled university students was conducted after the end of the sixth wave of the COVID-19 epidemic in Japan; predictors of willingness to provide BCPR were assessed by regression analysis. Of the 2534 participants 1525 (60.2%) were willing to intervene and provide BCPR during the COVID-19 pandemic. Hesitancy due to the anxiety that CPR intervention might result in poor prognosis was a negative predictor of willingness. In contrast, anxiety about the possibility of infection during CPR intervention did not show a negative impact. On the other hand, interest in CPR and willingness to participate in a course, confidence in CPR skills, awareness of automated external defibrillation, and knowledge of CPR during the COVID-19 pandemic, were also positive predictors. This study suggests that the barrier to willingness to intervene with BCPR during a COVID-19 pandemic is not fear of infection, but rather hesitation due to the possibility of poor prognosis from the intervention. The significance of conducting this study during the COVID-19 epidemic is great, and there is an urgent need for measures to overcome hesitation regarding BCPR.

Cross-infection risks of SARS-CoV-2 while playing catch using a baseball: Creating a safe sporting environment during the COVID-19 pandemic.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease (COVID-19), is found primarily in the respiratory tract secretions of infected individuals with one of the main routes of transmission being direct or indirect contact. In this study, using fluorescent paint, we evaluated the spread of contaminants while playing catch with a baseball. Fluorescent paint was applied to the right hand of a right-handed baseball player who then engaged in playing catch with 10 other right-handed players (partners) for 5 min each. The fluorescent paint was detected on the right hands (inside) and gloves (inside) of all the 10 partners as well as on the ball; in some partners it was also detected on the back of the right hands or the back of the gloves. However, except for their right hands, fluorescent paint was not detected on the surface of the bodies of the partners. These observations indicated that the fluorescent paint (mimicking virus-containing contaminants) on the hand spreads very efficiently from person to person during the throwing and catching of a baseball, suggesting that a thorough and frequent disinfection of the hands and equipment is important in the prevention of infections that may occur while playing baseball.

Prevalence of SARS-CoV-2 antibodies among university athletic club members: A cross-sectional survey.

School-based coronavirus disease 2019 (COVID-19) testing is an important part of a comprehensive prevention strategy in public health. To assess the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in a university athletic club community with repeated occurrences of SARS-CoV-2 infections, we conducted a cross-sectional survey for asymptomatic antibody prevalence using a SARS-CoV-2 rapid antibody test kit. On January 26, 2021 we administered questionnaires to determine their history of contact with infected individuals and took blood samples from 129 undergraduates. The prevalence of SARS-CoV-2 antibodies among the subjects was 3.9%. Only 6.2% of the participants reported close contact with infected individuals. In this study, we clarified the prevalence of asymptomatic SARS-CoV-2 antibodies in university athletic clubs where SARS-CoV-2 infections had repeatedly occurred, which will be helpful in discussing how to identify and prevent the transmission of infections within university athletic club communities.

Evaluation of Japanese university students' perception of smoking, interest in quitting, and smoking behavior: An examination and public health challenges during the COVID-19 pandemic.

This study examined college students' perceptions of the association between smoking and novel coronavirus disease 2019 (COVID-19), changes in smoking behavior, and interest in quitting categorized by smoking device, to identify public health challenges. A questionnaire survey was conducted among 8,547 students in a Japanese university in March and April 2021. In response to "Awareness of the increased risk of COVID-19 infection due to smoking and the tendency to develop severe disease", current smokers (70.2%) were more aware of the risk than non-smokers (49.8%) (p < 0.001), with no significant difference according to smoking device (p = 0.213). "Interest in quitting smoking" (p = 0.323), and "Changes in smoking behavior during the COVID-19 pandemic" (p = 0.146) did not differ by smoking device. However, approximately 50% of the respondents answered that they were not interested in quitting smoking, while two-thirds reported that the number of cigarettes they smoked did not change during the pandemic. During the COVID-19 pandemic, college students were found to be less interested in quitting and not likely to change their smoking behavior, despite the knowledge of the increased risk of COVID-19 transmission and severity of disease from smoking, regardless of smoking device.

Antibody response of smokers to the COVID-19 vaccination: Evaluation based on cigarette dependence.

Smokers may have lower antibody titers after vaccination with a coronavirus disease 2019 (COVID-19) mRNA vaccine. However, to the best of our knowledge, no study has evaluated antibody titers after COVID-19 vaccination based on the level of smokers' cigarette dependence. In this study, we measured the level of serum anti-severe acute respiratory syndrome coronavirus 2 (anti-SARS-CoV-2) spike protein receptor-binding domain (S-RBD) immunoglobulin-G (IgG) by enzyme linked immunosorbent assay of 55 actively smoking Japanese social workers (firefighters, paramedics, and rescue workers) who had received two doses of the BNT162b2 vaccine. Further, we assessed their cigarette dependence using the Fagerstrom Test for Nicotine Dependence (FTND), measured their serum cotinine levels, and tested for their correlation with anti-RBD IgG levels. Serum anti-SARS-CoV-2 S-RBD protein IgG levels after BNT162b2 vaccination showed a significant negative correlation with FTND (ρ = -0.426, p = 0.001). In addition, serum cotinine level showed a significant positive correlation with FTND (ρ = 0.470, p = 0.000). However, no significant negative correlation was noted between serum cotinine and serum anti-SARS-CoV-2 S-RBD protein IgG levels (ρ = -0.156, p = 0.256). Our results suggest that smokers with strong cigarette dependence have inadequate anti-SARS-CoV-2 S-RBD protein IgG levels after COVID-19 mRNA vaccination.

A Survey of Living Conditions and Psychological Distress in Japanese University Freshmen during the COVID-19 Pandemic.

Since the novel coronavirus disease 2019 (COVID-19) pandemic, educational institutions have implemented measures such as school closures, raising concerns regarding the increase in psychological distress among university students. The purpose of this study is to identify factors that have influenced psychological distress among college freshmen during the COVID-19 pandemic. A questionnaire survey was conducted at the conclusion of the sixth wave of COVID-19 in Japan. Psychological distress was measured using the six-item Kessler Psychological Distress Scale (K6). Factors affecting psychological distress were calculated using regression analysis. Of the 2536 participants, 1841 (72.6%) reported having no psychological distress, while 695 (27.4%) reported having psychological distress. Factors that were identified to contribute to psychological distress were lack of sleep, weight gain or loss, worsening of interpersonal relationships, and physical symptoms and illnesses. A willingness to join an athletic club and having an environment in which it is easy to discuss worries and anxieties with others were factors that were identified to hinder psychological distress. It is necessary for universities to offer enhanced supports for physical and interpersonal activities. Additionally, it is imperative to encourage students to look after their physical health and to actively utilize university-based consultation systems.

Effects of Heat-Not-Burn Cigarette Smoking on the Secretion of Saliva and Its Innate Immune System Components.

Saliva and salivary antimicrobial proteins play important roles in the innate immunity, which prevents infections of orally invading bacteria and viruses. In this study, we compared the secretion rates of salivary lactoferrin (Lac) and lysozyme (Lys) in heat-not-burn (HNB) cigarette smokers and non-smokers. The analysis population for this study included 212 members of the fire department, including 32 HNB cigarette smokers, 17 paper cigarette smokers, 14 combined HNB and paper cigarette smokers, and 149 non-smokers. Salivary Lac and Lys concentrations were assessed using enzyme immunoassay. Saliva secretion was significantly lower among HNB cigarette smokers (p < 0.01) than among non-smokers. Accompanying this result, salivary Lac and Lys secretion rates were significantly lower among smokers, particularly HNB cigarette smokers, than among non-smokers (all p < 0.01). Our findings suggest a possible adverse effect of HNB cigarette on the amount of Lac and Lys released into the oral cavity.

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.

A versatile platform technology for recombinant vaccines using non-propagative human parainfluenza virus type 2 vector.

Ectopic protein with proper steric structure was efficiently loaded onto the envelope of the F gene-defective BC-PIV vector derived from human parainfluenza virus type 2 (hPIV2) by a reverse genetics method of recombinant virus production. Further, ectopic antigenic peptide was successfully loaded either outside, inside, or at both sides of the envelope of the vector. The BC-PIV vector harboring the Ebola virus GP gene was able to elicit neutralizing antibodies in mice. In addition, BC-PIV with antigenic epitopes of both melanoma gp100 and WT1 tumor antigen induced a CD8+ T-cell-mediated response in tumor-transplanted syngeneic mice. Considering the low pathogenicity and recurrent infections of parental hPIV2, BC-PIV can be used as a versatile vector with high safety for recombinant vaccine development, addressing unmet medical needs.

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.

Nucleocytoplasmic shuttling of the human parainfluenza virus type 2 phosphoprotein.

Human parainfluenza virus type 2 phosphoprotein (P) is an essential component of viral polymerase. The P gene encodes both P and accessory V proteins by a specific gene editing mechanism. Therefore, the N-terminal 164 amino acids of P protein are common to V protein. Interestingly, while P protein is located in the cytoplasm, V protein is found mainly in the nucleus. Using deletion mutants, we show the presence of a nuclear localization signal (NLS) in the P/V common domain, and a nuclear export signal (NES) in the C-terminal P specific region. The NLS region makes a complex with importin α5 or 7. In the presence of leptomycin B, P protein is retained in the nucleus, indicating that it contains a CRM1-dependent NES. We identified the NLS (65PVKPRRKK72) and the NES (225IIELLKGLDL234) using ß-galactosidase fusion proteins. Moreover, nucleocytoplasmic shuttling of P protein appears to be important for efficient viral polymerase activity.

The Hemagglutinin-Neuraminidase (HN) Head Domain and the Fusion (F) Protein Stalk Domain of the Parainfluenza Viruses Affect the Specificity of the HN-F Interaction.

Membrane fusion by the parainfluenza viruses is induced by virus-specific functional interaction between the attachment protein (HN) and the fusion (F) protein. This interaction is thought to be mediated by transient contacts between particular amino acids in the HN stalk domain and those in the F head domain. However, we recently reported that replacement of specified amino acids at or around the dimer interface of the HN head domain remarkably affected the F protein specificity. We then intended to further investigate this issue in the present study and revealed that the HPIV2 HN protein can be converted to an SV41 HN-like protein by substituting at least nine amino acids in the HPIV2 HN head domain with the SV41 HN counterparts in addition to the replacement of the stalk domain, indicating that specified amino acids in the HN head domain play very important roles in determining the specificity of the HN-F interaction. On the other hand, we previously reported that the PIV5 F protein can be converted to an SV41 F-like protein by replacing 21 amino acids in the head domain of the PIV5 F protein with those of the SV41 F protein. We then intended to further investigate this issue in the present study and found that replacement of 15 amino acids in the stalk domain in addition to the replacement of the 21 amino acids in the head domain of the PIV5 F protein resulted in creation of a more SV41 F-like protein, indicating that specified amino acids in the F stalk domain play important roles in determining the specificity of the HN-F interaction. These results suggest that the conformations of the HN stalk domain and the F head domain are dependent on the structures of the HN head domain and the F stalk domain, respectively. Presumably, the conformations of the former domains, which are considered directly involved in the HN-F interaction, can be modified by subtle changes in the structure of the latter domains, resulting in an altered specificity for the interacting partners.

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.

Graf1 Controls the Growth of Human Parainfluenza Virus Type 2 through Inactivation of RhoA Signaling.

UNLABELLED: Rho GTPases are involved in a variety of cellular activities and are regulated by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We found that the activation of Rho GTPases by lysophosphatidic acid promotes the growth of human parainfluenza virus type 2 (hPIV-2). Furthermore, hPIV-2 infection causes activation of RhoA, a Rho GTPase. We hypothesized that Graf1 (also known as ARHGAP26), a GAP, regulates hPIV-2 growth by controlling RhoA signaling. Immunofluorescence analysis showed that hPIV-2 infection altered Graf1 localization from a homogenous distribution within the cytoplasm to granules. Graf1 colocalized with hPIV-2 P, NP, and L proteins. Graf1 interacts with P and V proteins via their N-terminal common region, and the C-terminal Src homology 3 domain-containing region of Graf1 is important for these interactions. In HEK293 cells constitutively expressing Graf1, hPIV-2 growth was inhibited, and RhoA activation was not observed during hPIV-2 infection. In contrast, Graf1 knockdown restored hPIV-2 growth and RhoA activation. Overexpression of hPIV-2 P and V proteins enhanced hPIV-2-induced RhoA activation. These results collectively suggested that hPIV-2 P and V proteins enhanced hPIV-2 growth by binding to Graf1 and that Graf1 inhibits hPIV-2 growth through RhoA inactivation. IMPORTANCE: Robust growth of hPIV-2 requires Rho activation. hPIV-2 infection causes RhoA activation, which is suppressed by Graf1. Graf1 colocalizes with viral RNP (vRNP) in hPIV-2-infected cells. We found that Graf1 interacts with hPIV-2 P and V proteins. We also identified regions in these proteins which are important for this interaction. hPIV-2 P and V proteins enhanced the hPIV-2 growth via binding to Graf1, while Graf1 inhibited hPIV-2 growth through RhoA inactivation.

Enhanced growth of influenza A virus by coinfection with human parainfluenza virus type 2.

It has been reported that dual or multiple viruses can coinfect epithelial cells of the respiratory tract. However, little has been reported on in vitro interactions of coinfected viruses. To explore how coinfection of different viruses affects their biological property, we examined growth of influenza A virus (IAV) and human parainfluenza virus type 2 (hPIV2) during coinfection of Vero cells. We found that IAV growth was enhanced by coinfection with hPIV2. The enhanced growth of IAV was not reproduced by coinfection with an hPIV2 mutant with reduced cell fusion activity, or by ectopic expression of the V protein of hPIV2. In contrast, induction of cell fusion by ectopic expression of the hPIV2 HN and F proteins augments IAV growth. hPIV2 coinfection supported IAV growth in cells originated from the respiratory epithelium. The enhancement correlated closely with cell fusion ability of hPIV2 in those cells. These results indicate that cell fusion induced by hPIV2 infection is beneficial to IAV replication and that enhanced viral replication by coinfection with different viruses can modify their pathological consequences.

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.