Quantitative Comparison of Human Parainfluenza Virus Hemagglutinin-Neuraminidase Receptor Binding and Receptor Cleavage

The human parainfluenza virus (hPIV) hemagglutinin-neuraminidase (HN) protein binds (H) oligosaccharide receptors that contain N-acetylneuraminic acid (Neu5Ac) and cleaves (N) Neu5Ac from these oligosaccharides. In order to determine if one of HN′s two functions is predominant, we measured the affinity of H for its ligands by a solid-phase binding assay with two glycoprotein substrates and by surface plasmon resonance with three monovalent glycans. We compared the dissociation constant (K_d) values from these experiments with previously determined Michaelis-Menten constants (K_ms) for the enzyme activity. We found that glycoprotein substrates and monovalent glycans containing Neu5Acα2-3Galβ1-4GlcNAc bind HN with K_d values in the 10 to 100 μM range. K_m values for HN were previously determined to be on the order of 1 mM (M. M. Tappert, D. F. Smith, and G. M. Air, J. Virol. 85:12146–12159, 2011). A K_m value greater than the K_d value indicates that cleavage occurs faster than the dissociation of binding and will dominate under N-permissive conditions. We propose, therefore, that HN is a neuraminidase that can hold its substrate long enough to act as a binding protein. The N activity can therefore regulate binding by reducing virus-receptor interactions when the concentration of receptor is high.     

Human Parainfluenza Virus Infection of the Airway Epithelium: Viral Hemagglutinin-Neuraminidase Regulates Fusion Protein Activation and Modulates Infectivity

Three discrete activities of the paramyxovirus hemagglutinin-neuraminidase (HN) protein, receptor binding, receptor cleaving (neuraminidase), and triggering of the fusion protein, each affect the promotion of viral fusion and entry. For human parainfluenza virus type 3 (HPIV3), the effects of specific mutations that alter these functions of the receptor-binding protein have been well characterized using cultured monolayer cells, which have identified steps that are potentially relevant to pathogenesis. In the present study, proposed mechanisms that are relevant to pathogenesis were tested in natural host cell cultures, a model of the human airway epithelium (HAE) in which primary HAE cells are cultured at an air-liquid interface and retain functional properties. Infection of HAE cells with wild-type HPIV3 and variant viruses closely reflects that seen in an animal model, the cotton rat, suggesting that HAE cells provide an ideal system for assessing the interplay of host cell and viral factors in pathogenesis and for screening for inhibitory molecules that would be effective in vivo. Both HN′s receptor avidity and the function and timing of F activation by HN require a critical balance for the establishment of ongoing infection in the HAE, and these HN functions independently modulate the production of active virions. Alterations in HN′s F-triggering function lead to the release of noninfectious viral particles and a failure of the virus to spread. The finding that the dysregulation of F triggering prohibits successful infection in HAE cells suggests that antiviral strategies targeted to HN′s F-triggering activity may have promise in vivo.Paramyxoviruses are enveloped viruses that enter cells by fusing directly with the cell membrane. During entry, the viral surface glycoproteins hemagglutinin-neuraminidase (HN) (the receptor-binding molecule) and F (the fusion protein) cooperate in a highly specific way to mediate fusion upon receptor binding. To understand these mechanisms, elucidate how paramyxoviruses enter cells, and develop strategies to prevent or treat infection, we study human parainfluenza virus (HPIV), an important cause of croup and bronchiolitis in children. Our results have uncovered fundamental roles of the receptor-binding protein in paramyxovirus fusion and principles of coordinated interaction between the glycoproteins during the viral life cycle.To understand how the diverse functions of the viral glycoproteins are regulated during the viral life cycle, we have used viruses bearing variant HN molecules with mutations at the binding/F-triggering site (and/or the primary receptor-binding site) to study how this molecule works to trigger F (2, 3, 7, 10, 15, 18, 20). The correct timing of F activation (triggering) by HN is essential for entry. For infection to occur, triggering must occur only when F is in proximity to the target cell membrane, and we propose that the regulation of F triggering is essential for the survival of the virus. The outcome of infection is determined by the target cell''s properties and its receptors, and specific mechanisms that are relevant to pathogenesis need to be tested using tissues that reflect the natural host. We therefore tested the hypothesis that a dysregulation of F triggering precludes successful infection in both a cotton rat model and the natural host airway epithelium.For the cotton rat model, previous studies suggested that altered pathogenesis in HPIV infection might be caused by specific HN mutations (24). The present detailed studies of the cotton rat using HN viral variants suggest that the extent of lung infection correlates with the ability of each variant to grow in vivo. The most striking finding is that the ability of the HN variants to grow in vivo is inversely related to their ability to fuse a monolayer of cultured cells. In order to understand the determinants of infection in the natural host, we therefore turned to a model that closely reflects the natural human host tissue, the human airway epithelium (HAE). This model utilizes a recently developed method for culturing primary HAE cells at an air-liquid interface, generating a differentiated, pseudostratified, mucociliary epithelium that faithfully represents the HAE (16). The HAE model was previously used to characterize the polarity and cell specificity of respiratory syncytial virus (26) and HPIV type 3 (HPIV3) (25), confirming that it is suited to studying paramyxovirus-HAE interactions that reflect those in the human lung.We used viruses bearing HNs that are altered in receptor binding or F triggering to reveal the functional relevance of these properties in the HAE and to establish the key role of HN binding site II in infection in the natural host. We propose that an enhanced triggering of F by HN may be a disadvantage in vivo and that the function and timing of F triggering are critical in the target tissue. The correct balance between the three functions of HN (receptor binding, receptor cleaving, and F triggering) resides in the functions of the two binding sites (18), binding and release in site I and F triggering in site II, and both sites of HN play key roles in the natural host.     

Evaluating a Parainfluenza Virus 5-Based Vaccine in a Host with Pre-Existing Immunity against Parainfluenza Virus 5

Parainfluenza virus 5 (PIV5), formerly known as simian virus 5 (SV5), is a paramyxovirus often referred to as canine parainfluenza virus (CPI) in the veterinary field. PIV5 is thought to be a contributing factor to kennel cough. Kennel cough vaccines containing live PIV5 have been used in dogs for many decades. PIV5 is not known to cause any diseases in humans or other animals. PIV5 has been used as a vector for vaccine development for humans and animals. One critical question concerning the use of PIV5 as a vector is whether prior exposure to PIV5 would prevent the use of PIV5-based vaccines. In this work, we have examined immunogenicity of a recombinant PIV5 expressing hemagglutinin (HA) of influenza A virus subtype 3 (rPIV5-H3) in dogs that were immunized against PIV5. We found that vaccination of the dogs containing neutralizing antibodies against PIV5 with rPIV5-H3 generated immunity against influenza A virus, indicting that PIV5-based vaccine is immunogenic in dogs with prior exposure. Furthermore, we have examined exposure of PIV5 in human populations. We have detected neutralizing antibody (nAb) against PIV5 in 13 out of 45 human serum samples (about 29 percent). The nAb titers in humans were lower than that in vaccinated dogs, suggesting that nAb in humans is unlikely to prevent PIV5 from being an efficacious vector in humans.     

On the Stability of Parainfluenza Virus 5 F Proteins

The crystal structure of the F protein (prefusion form) of the paramyxovirus parainfluenza virus 5 (PIV5) WR isolate was determined. We investigated the basis by which point mutations affect fusion in PIV5 isolates W3A and WR, which differ by two residues in the F ectodomain. The P22 stabilizing site acts through a local conformational change and a hydrophobic pocket interaction, whereas the S443 destabilizing site appears sensitive to both conformational effects and amino acid charge/polarity changes.     

PLK1 Down-Regulates Parainfluenza Virus 5 Gene Expression

The paramyxoviruses are a family of negative-sense RNA viruses that includes many important human and animal pathogens. Paramyxovirus RNA synthesis requires the viral phosphoprotein (P) and the large (L) protein. Phosphorylation of P is thought to regulate viral gene expression, though direct proof remains elusive. Recently, we reported that phosphorylation of a specific residue (Ser157) of the P protein of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, correlates with decreased viral gene expression and cytokine expression in infected cells. Here, we show that: Polo-like kinase 1 (PLK1), a serine/theronine kinase that plays a critical role in regulating the cell cycle, interacts with PIV5 P through the S157 residue; PLK1 inhibition increases viral gene expression; PLK1 over-expression inhibits viral gene expression; and PLK1 directly phosphorylates P in vitro, indicating that PLK1 down-regulates viral gene expression by phosphorylating P. Furthermore, we have determined the PLK1 phosphorylation site on P and found that mutant recombinant PIV5 whose P proteins cannot either bind to or be phosphorylated by PLK1 have similar phenotypes. Increased viral gene expression in PIV5 with mutations in the PLK1 binding/phosphorylation sites correlates with increased induction of cell death and cytokine expression, suggesting that PIV5 limits its viral gene expression to avoid these host effects. It is possible that targeting PLK1 will enhance host innate immune responses, leading to a novel strategy of clearing paramyxovirus infections quickly.     

A Mutation in the Stalk of the Newcastle Disease Virus Hemagglutinin-Neuraminidase (HN) Protein Prevents Triggering of the F Protein despite Allowing Efficient HN-F Complex Formation

Newcastle disease virus (NDV)-induced membrane fusion requires formation of a complex between the hemagglutinin-neuraminidase (HN) and fusion (F) proteins. Substitutions for NDV HN stalk residues A89, L90, and L94 block fusion by modulating formation of the HN-F complex. Here, we demonstrate that a nearby L97A substitution, though previously shown to block fusion, allows efficient HN-F complex formation and likely acts by preventing changes in the HN stalk required for triggering of the bound F protein.     

Identification and Characterization of the First Equine Parainfluenza Virus 5

正Dear Editor,Parainfluenza virus 5 (PIV5), known as canine parainfluenza virus in the veterinary field, is a negative-sense,nonsegmented, single-stranded RNA virus belonging to the Paramyxoviridae family (Chen 2018). The virus was first reported in primary monkey kidney cells in 1954 (Hsiung1972), then it has been frequently discovered in various     

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.     

Parainfluenza Virus 5 Expressing the G Protein of Rabies Virus Protects Mice after Rabies Virus Infection

Rabies remains a major public health threat around the world. Once symptoms appear, there is no effective treatment to prevent death. In this work, we tested a recombinant parainfluenza virus 5 (PIV5) strain expressing the glycoprotein (G) of rabies (PIV5-G) as a therapy for rabies virus infection: we have found that PIV5-G protected mice as late as 6 days after rabies virus infection. PIV5-G is a promising vaccine for prevention and treatment of rabies virus infection.     

Phenotypic Mixing of Envelope Proteins of the Parainfluenza Virus SV5 and Vesicular Stomatitis Virus

Cells mixedly infected with parainfluenza virus SV5 and vesicular stomatitis virus (VSV) yield phenotypically mixed virions, in addition to both parental types. Two types of phenotypically mixed virions have been identified: 0.6 to 1.2% of the VSV plaque formers were neutralized by SV5 antiserum, but not by VSV antiserum, suggesting the presence of a VSV genome in an SV5 envelope; 9 to 45% of the VSV plaque formers were neutralized by both antisera, indicating the presence of both SV5 and VSV antigens in their envelopes. The presence of SV5 antigen in virions with the typical bullet-shaped appearance of VSV was confirmed with ferritin-labeled anti-SV5 antibody. In contrast to standard VSV, phenotypically mixed virions adsorbed to and eluted from chicken erythrocytes, indicating that these virions contained in their envelopes SV5 hemagglutinin, and possibly neuraminidase. Thus, the VSV nucleocapsid can interact with membranes which contain SV5 proteins in the manner which leads to virus maturation, and the production of a high yield of phenotypically mixed virions with the morphology of VSV indicates that this process can function efficiently. No evidence of genetic recombination between the two viruses was found. These results raise the possibility of an evolutionary relatedness between the paramyxoviruses and the rhabdoviruses.     

Efficacy of a Parainfluenza Virus 5 (PIV5)-Based H7N9 Vaccine in Mice and Guinea Pigs: Antibody Titer towards HA Was Not a Good Indicator for Protection

H7N9 has caused fatal infections in humans. A safe and effective vaccine is the best way to prevent large-scale outbreaks in the human population. Parainfluenza virus 5 (PIV5), an avirulent paramyxovirus, is a promising vaccine vector. In this work, we generated a recombinant PIV5 expressing the HA gene of H7N9 (PIV5-H7) and tested its efficacy against infection with influenza virus A/Anhui/1/2013 (H7N9) in mice and guinea pigs. PIV5-H7 protected the mice against lethal H7N9 challenge. Interestingly, the protection did not require antibody since PIV5-H7 protected JhD mice that do not produce antibody against lethal H7N9 challenge. Furthermore, transfer of anti-H7 serum did not protect mice against H7N9 challenge. PIV5-H7 generated high HAI titers in guinea pigs, however it did not protect against H7N9 infection or transmission. Intriguingly, immunization of guinea pigs with PIV5-H7 and PIV5 expressing NP of influenza A virus H5N1 (PIV5-NP) conferred protection against H7N9 infection and transmission. Thus, we have obtained a H7N9 vaccine that protected both mice and guinea pigs against lethal H7N9 challenge and infection respectively.     

Structure and Development of Viruses as Observed in the Electron Microscope IX. Entry of Parainfluenza I (Sendai) Virus

After attachment, the uncoating of Sendai virus, which was accompanied by dissolution of the plasma membrane and fusion of virus to cell, proceeded quickly. Nucleoprotein filaments were found at stages of transit from virus to cytoplasm.     

Structure of the Ulster Strain Newcastle Disease Virus Hemagglutinin-Neuraminidase Reveals Auto-Inhibitory Interactions Associated with Low Virulence

Paramyxovirus hemagglutinin-neuraminidase (HN) plays roles in viral entry and maturation, including binding to sialic acid receptors, activation of the F protein to drive membrane fusion, and enabling virion release during virus budding. HN can thereby directly influence virulence and in a subset of avirulent Newcastle disease virus (NDV) strains, such as NDV Ulster, HN must be proteolytically activated to remove a C-terminal extension not found in other NDV HN proteins. Ulster HN is 616 amino acids long and the 45 amino acid C-terminal extension present in its precursor (HN₀) form has to be cleaved to render HN biologically active. Here we show that Ulster HN contains an inter-subunit disulfide bond within the C-terminal extension at residue 596, which regulates HN activities and neuraminidase (NA) domain dimerization. We determined the crystal structure of the dimerized NA domain containing the C-terminal extension, which extends along the outside of the sialidase β-propeller domain and inserts C-terminal residues into the NA domain active site. The C-terminal extension also engages a secondary sialic acid binding site present in NDV HN proteins, which is located at the NA domain dimer interface, that most likely blocks its attachment function. These results clarify how the Ulster HN C-terminal residues lead to an auto-inhibited state of HN, the requirement for proteolytic activation of HN₀ and associated reduced virulence.     

Involvement of the Cytoplasmic Domain of the Hemagglutinin-Neuraminidase Protein in Assembly of the Paramyxovirus Simian Virus 5

Efficient assembly of enveloped viruses at the plasma membranes of virus-infected cells requires coordination between cytosolic viral components and viral integral membrane glycoproteins. As viral glycoprotein cytoplasmic domains may play a role in this coordination, we have investigated the importance of the hemagglutinin-neuraminidase (HN) protein cytoplasmic domain in the assembly of the nonsegmented negative-strand RNA paramyxovirus simian virus 5 (SV5). By using reverse genetics, recombinant viruses which contain HN with truncated cytoplasmic tails were generated. These viruses were shown to be replication impaired, as judged by small plaque size, reduced replication rate, and low maximum titers when compared to those features of wild-type (wt) SV5. Release of progeny virus particles from cells infected with HN cytoplasmic-tail-truncated viruses was inefficient compared to that of wt virus, but syncytium formation was enhanced. Furthermore, accumulation of viral proteins at presumptive budding sites on the plasma membranes of infected cells was prevented by HN cytoplasmic tail truncations. We interpret these data to indicate that formation of budding complexes, from which efficient release of SV5 particles can occur, depends on the presence of an HN cytoplasmic tail.     

Transient Inhibition of Polyoma Virus Synthesis by Sendai Virus (Parainfluenza I) II. Mechanism of the Interference by Inactivated Virus

The mechanism of the transient inhibition of polyoma virus synthesis by betapropiolactone-inactivated Sendai virus was studied. Polyoma virus early functions did not appear to be affected, although deoxyribonucleic acid (DNA) and structural protein synthesis were inhibited 60 and 35% respectively. The inhibition of macromolecular synthesis was not sufficient to account for the 90% inhibition of infectious progeny formation. Encapsidation of polyoma DNA into mature virions appears to be completely inhibited after superinfection by beta-propiolactone-inactivated Sendai virus. Ultraviolet irradiation of live or beta-propiolactone-inactivated Sendai virus preparations abolishes the interfering capacity, indicating that a functional Sendai virus ribonucleic acid molecule is the interfering component.     

A Novel Rabies Vaccine Based on a Recombinant Parainfluenza Virus 5 Expressing Rabies Virus Glycoprotein

Untreated rabies virus (RABV) infection leads to death. Vaccine and postexposure treatment have been effective in preventing RABV infection. However, due to cost, rabies vaccination and treatment have not been widely used in developing countries. There are 55,000 human death caused by rabies annually. An efficacious and cost-effective rabies vaccine is needed. Parainfluenza virus 5 (PIV5) is thought to contribute to kennel cough, and kennel cough vaccines containing live PIV5 have been used in dogs for many years. In this work, a PIV5-vectored rabies vaccine was tested in mice. A recombinant PIV5 encoding RABV glycoprotein (G) (rPIV5-RV-G) was administered to mice via intranasal (i.n.), intramuscular (i.m.), and oral inoculation. The vaccinated mice were challenged with a 50% lethal challenge dose (LD₅₀) of RABV challenge virus standard 24 (CVS-24) intracerebrally. A single dose of 10⁶ PFU of rPIV5-RV-G was sufficient for 100% protection when administered via the i.n. route. The mice vaccinated with a single dose of 10⁸ PFU of rPIV5-RV-G via the i.m. route showed very robust protection (90% to 100%). Intriguingly, the mice vaccinated orally with a single dose of 10⁸ PFU of rPIV5-RV-G showed a 50% survival rate, which is comparable to the 60% survival rate among mice inoculated with an attenuated rabies vaccine strain, recombinant LBNSE. This is first report of an orally effective rabies vaccine candidate in animals based on PIV5 as a vector. These results indicate that rPIV5-RV-G is an excellent candidate for a new generation of recombinant rabies vaccine for humans and animals and PIV5 is a potential vector for oral vaccines.