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Robert A. Lamb - One of the best experts on this subject based on the ideXlab platform.
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structure of the paramyxoVirus Parainfluenza Virus 5 nucleoprotein in complex with an amino terminal peptide of the phosphoprotein
Journal of Virology, 2017Co-Authors: Megha Aggarwal, Christopher A Kors, George P Leser, Robert A. LambAbstract:Parainfluenza Virus 5 (PIV5) belongs to the family Paramyxoviridae, which consists of enveloped Viruses with a nonsegmented negative-strand RNA genome encapsidated by the nucleoprotein (N). ParamyxoVirus replication is regulated by the phosphoprotein (P) through protein-protein interactions with N and the RNA polymerase (L). The chaperone activity of P is essential to maintain the unassembled RNA-free form of N in order to prevent nonspecific RNA binding and premature N oligomerization. Here, we determined the crystal structure of unassembled PIV5 N in complex with a P peptide (N0P) derived from the N terminus of P (P50) at 2.65 A. The PIV5 N0P consists of two domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a hinge region. The cleft at the hinge region of RNA-bound PIV5 N was previously shown to be an RNA binding site. The N0P structure shows that the P peptide binds to the CTD of N and extends toward the RNA binding site to inhibit N oligomerization and, hence, RNA binding. Binding of P peptide also keeps the PIV5 N in the open form. A molecular dynamics (MD) analysis of both the open and closed forms of N shows the flexibility of the CTD and the preference of the N protein to be in an open conformation. The gradual opening of the hinge region, to release the RNA, was also observed. Together, these results advance our knowledge of the conformational swapping of N required for the highly regulated paramyxoVirus replication. IMPORTANCE ParamyxoVirus replication is regulated by the interaction of P with N and L proteins. Here, we report the crystal structure of unassembled Parainfluenza Virus 5 (PIV5) N chaperoned with P peptide. Our results provide a detailed understanding of the binding of P to N. The conformational switching of N between closed and open forms during its initial interaction with P, as well as during RNA release, was analyzed. Our data also show the plasticity of the CTD and the importance of domain movement for conformational switching. The results improve our understanding of the mechanism of interchanging N conformations for RNA replication and release.
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structure of the paramyxoVirus Parainfluenza Virus 5 nucleoprotein rna complex
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Maher Alayyoubi, Christopher A Kors, George P Leser, Robert A. LambAbstract:Parainfluenza Virus 5 (PIV5) is a member of the Paramyxoviridae family of membrane-enveloped Viruses with a negative-sense RNA genome that is packaged and protected by long filamentous nucleocapsid-helix structures (RNPs). These RNPs, consisting of ∼2,600 protomers of nucleocapsid (N) protein, form the template for viral transcription and replication. We have determined the 3D X-ray crystal structure of the nucleoprotein (N)-RNA complex from PIV5 to 3.11-A resolution. The structure reveals a 13-mer nucleocapsid ring whose diameter, cavity, and pitch/height dimensions agree with EM data from early studies on the Paramyxovirinae subfamily of native RNPs, indicating that it closely represents one-turn in the building block of the RNP helices. The PIV5-N nucleocapsid ring encapsidates a nuclease resistant 78-nt RNA strand in its positively charged groove formed between the N-terminal (NTD) and C-terminal (CTD) domains of its successive N protomers. Six nucleotides precisely are associated with each N protomer, with alternating three-base-in three-base-out conformation. The binding of six nucleotides per protomer is consistent with the “rule of six” that governs the genome packaging of the Paramyxovirinae subfamily of Viruses. PIV5-N protomer subdomains are very similar in structure to the previously solved Nipah-N structure, but with a difference in the angle between NTD/CTD at the RNA hinge region. Based on the Nipah-N structure we modeled a PIV5-N open conformation in which the CTD rotates away from the RNA strand into the inner spacious nucleocapsid-ring cavity. This rotation would expose the RNA for the viral polymerase activity without major disruption of the nucleocapsid structure.
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Structure of the paramyxoVirus Parainfluenza Virus 5 nucleoprotein–RNA complex
Proceedings of the National Academy of Sciences, 2015Co-Authors: Maher Alayyoubi, Christopher A Kors, George P Leser, Robert A. LambAbstract:Parainfluenza Virus 5 (PIV5) is a member of the Paramyxoviridae family of membrane-enveloped Viruses with a negative-sense RNA genome that is packaged and protected by long filamentous nucleocapsid-helix structures (RNPs). These RNPs, consisting of ∼2,600 protomers of nucleocapsid (N) protein, form the template for viral transcription and replication. We have determined the 3D X-ray crystal structure of the nucleoprotein (N)-RNA complex from PIV5 to 3.11-A resolution. The structure reveals a 13-mer nucleocapsid ring whose diameter, cavity, and pitch/height dimensions agree with EM data from early studies on the Paramyxovirinae subfamily of native RNPs, indicating that it closely represents one-turn in the building block of the RNP helices. The PIV5-N nucleocapsid ring encapsidates a nuclease resistant 78-nt RNA strand in its positively charged groove formed between the N-terminal (NTD) and C-terminal (CTD) domains of its successive N protomers. Six nucleotides precisely are associated with each N protomer, with alternating three-base-in three-base-out conformation. The binding of six nucleotides per protomer is consistent with the “rule of six” that governs the genome packaging of the Paramyxovirinae subfamily of Viruses. PIV5-N protomer subdomains are very similar in structure to the previously solved Nipah-N structure, but with a difference in the angle between NTD/CTD at the RNA hinge region. Based on the Nipah-N structure we modeled a PIV5-N open conformation in which the CTD rotates away from the RNA strand into the inner spacious nucleocapsid-ring cavity. This rotation would expose the RNA for the viral polymerase activity without major disruption of the nucleocapsid structure.
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on the stability of Parainfluenza Virus 5 f proteins
Journal of Virology, 2015Co-Authors: Taylor A Poor, Albert S Song, Brett D Welch, Christopher A Kors, Theodore S Jardetzky, Robert A. LambAbstract: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.
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Influenza A Virus Uses Intercellular Connections To Spread to Neighboring Cells
Journal of Virology, 2014Co-Authors: Kari L. Roberts, Balaji Manicassamy, Robert A. LambAbstract:In the extracellular environment, cell-free virions seek out naive host cells over long distances and between organisms. This is the primary mechanism of spread for most Viruses. Here we provide evidence for an alternative pathway previously undescribed for orthomyxoViruses, whereby the spread of influenza A Virus (IAV) infectious cores to neighboring cells can occur within intercellular connections. The formation of these connections requires actin dynamics and is enhanced by viral infection. Connected cells have contiguous membranes, and the core infectious viral machinery (RNP and polymerase) was present inside the intercellular connections. A live-cell movie of green fluorescent protein (GFP)-tagged NS1 of IAV shows viral protein moving from one cell to another through an intercellular connection. The movement of tagged protein was saltatory but overall traveled only in one direction. Infectious Virus cores can move from one cell to another without budding and release of cell-free virions, as evidenced by the finding that whereas a neuraminidase inhibitor alone did not inhibit the development of IAV microplaques, the presence of a neuraminidase inhibitor together with drugs inhibiting actin dynamics or the microtubule stabilizer paclitaxel (originally named taxol) precluded microplaque formation. Similar results were also observed with Parainfluenza Virus 5 (PIV5), a paramyxoVirus, when neutralizing antibody was used to block spread by cell-free virions. Intercellular spread of infectious core particles was unaffected or enhanced in the presence of nocodazole for IAV but inhibited for PIV5. The intercellular connections have a core of filamentous actin, which hints toward transport of Virus particles through the use of a myosin motor. IMPORTANCE Here we describe a new method by which influenza A Virus (IAV) spreads from cell to cell: IAV uses intracellular connections. The formation of these connections requires actin dynamics and is enhanced by viral infection and the absence of microtubules. Connected cells appeared to have contiguous membranes, and the core infectious viral machinery (RNP and polymerase) was present inside the intercellular connections. Infectious Virus cores can move from one cell to another without budding and release of cell-free virions. Similar results were also observed with Parainfluenza Virus 5 (PIV5).
Theodore S Jardetzky - One of the best experts on this subject based on the ideXlab platform.
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on the stability of Parainfluenza Virus 5 f proteins
Journal of Virology, 2015Co-Authors: Taylor A Poor, Albert S Song, Brett D Welch, Christopher A Kors, Theodore S Jardetzky, Robert A. LambAbstract: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.
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activation of paramyxoVirus membrane fusion and Virus entry
Current Opinion in Virology, 2014Co-Authors: Theodore S Jardetzky, Robert A. LambAbstract:The paramyxoViruses represent a diverse Virus family responsible for a wide range of human and animal diseases. In contrast to other Viruses, such as HIV and influenza Virus, which use a single glycoprotein to mediate host receptor binding and Virus entry, the paramyxoViruses require two distinct proteins. One of these is an attachment glycoprotein that binds receptor, while the second is a fusion glycoprotein, which undergoes conformational changes that drive Virus-cell membrane fusion and Virus entry. The details of how receptor binding by one protein activates the second to undergo conformational changes have been poorly understood until recently. Over the past couple of years, structural and functional data have accumulated on representative members of this family, including Parainfluenza Virus 5, Newcastle disease Virus, measles Virus, Nipah Virus and others, which suggest a mechanistic convergence of activation models. Here we review the data indicating that paramyxoVirus attachment glycoproteins shield activating residues within their N-terminal stalk domains, which are then exposed upon receptor binding, leading to the activation of the fusion protein by a ‘provocateur’ mechanism.
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mutations in the Parainfluenza Virus 5 fusion protein reveal domains important for fusion triggering and metastability
Journal of Virology, 2013Co-Authors: Sayantan Bose, Maher Alayyoubi, Theodore S Jardetzky, Carissa M Heath, Priya A Shah, Robert A. LambAbstract:ParamyxoVirus membrane glycoproteins F (fusion protein) and HN, H, or G (attachment protein) are critical for Virus entry, which occurs through fusion of viral and cellular envelopes. The F protein folds into a homotrimeric, metastable prefusion form that can be triggered by the attachment protein to undergo a series of structural rearrangements, ultimately folding into a stable postfusion form. In paramyxoVirus-infected cells, the F protein is activated in the Golgi apparatus by cleavage adjacent to a hydrophobic fusion peptide that inserts into the target membrane, eventually bringing the membranes together by F refolding. However, it is not clear how the attachment protein, known as HN in Parainfluenza Virus 5 (PIV5), interacts with F and triggers F to initiate fusion. To understand the roles of various F protein domains in fusion triggering and metastability, single point mutations were introduced into the PIV5 F protein. By extensive study of F protein cleavage activation, surface expression, and energetics of fusion triggering, we found a role for an immunoglobulin-like (Ig-like) domain, where multiple hydrophobic residues on the PIV5 F protein may mediate F-HN interactions. Additionally, destabilizing mutations of PIV5 F that resulted in HN trigger-independent mutant F proteins were identified in a region along the border of F trimer subunits. The positions of the potential HN-interacting region and the region important for F stability in the lower part of the PIV5 F prefusion structure provide clues to the receptor-binding initiated, HN-mediated F trigger.
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structure of the Parainfluenza Virus 5 piv5 hemagglutinin neuraminidase hn ectodomain
PLOS Pathogens, 2013Co-Authors: Brett D Welch, Christopher A Kors, Sayantan Bose, Robert A. Lamb, Ping Yuan, Theodore S JardetzkyAbstract:ParamyxoViruses cause a wide variety of human and animal diseases. They infect host cells using the coordinated action of two surface glycoproteins, the receptor binding protein (HN, H, or G) and the fusion protein (F). HN binds sialic acid on host cells (hemagglutinin activity) and hydrolyzes these receptors during viral egress (neuraminidase activity, NA). Additionally, receptor binding is thought to induce a conformational change in HN that subsequently triggers major refolding in homotypic F, resulting in fusion of Virus and target cell membranes. HN is an oligomeric type II transmembrane protein with a short cytoplasmic domain and a large ectodomain comprising a long helical stalk and large globular head domain containing the enzymatic functions (NA domain). Extensive biochemical characterization has revealed that HN-stalk residues determine F specificity and activation. However, the F/HN interaction and the mechanisms whereby receptor binding regulates F activation are poorly defined. Recently, a structure of Newcastle disease Virus (NDV) HN ectodomain revealed the heads (NA domains) in a “4-heads-down” conformation whereby two of the heads form a symmetrical interaction with two sides of the stalk. The interface includes stalk residues implicated in triggering F, and the heads sterically shield these residues from interaction with F (at least on two sides). Here we report the x-ray crystal structure of Parainfluenza Virus 5 (PIV5) HN ectodomain in a “2-heads-up/2-heads-down” conformation where two heads (covalent dimers) are in the “down position,” forming a similar interface as observed in the NDV HN ectodomain structure, and two heads are in an “up position.” The structure supports a model in which the heads of HN transition from down to up upon receptor binding thereby releasing steric constraints and facilitating the interaction between critical HN-stalk residues and F.
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structure of the cleavage activated prefusion form of the Parainfluenza Virus 5 fusion protein
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Brett D Welch, Christopher A Kors, Theodore S Jardetzky, George P Leser, Robert A. LambAbstract:The paramyxoVirus Parainfluenza Virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and Virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein.
Biao He - One of the best experts on this subject based on the ideXlab platform.
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Parainfluenza Virus 5 Expressing Wild-Type or Prefusion Respiratory Syncytial Virus (RSV) Fusion Protein Protects Mice and Cotton Rats from RSV Challenge.
Journal of Virology, 2017Co-Authors: Shannon Phan, Huiling Wei, James Zengel, Dai Wang, Zhuo Li, Biao HeAbstract:Human respiratory syncytial Virus (RSV) is the leading cause of pediatric bronchiolitis and hospitalizations. RSV can also cause severe complications in elderly and immunocompromised individuals. There is no licensed vaccine. We previously generated a Parainfluenza Virus 5 (PIV5)-vectored vaccine candidate expressing the RSV fusion protein (F) that was immunogenic and protective in mice. In this work, our goal was to improve the original vaccine candidate by modifying the PIV5 vector or by modifying the RSV F antigen. We previously demonstrated that insertion of a foreign gene at the PIV5 small hydrophobic (SH)-hemagglutinin-neuraminidase (HN) junction or deletion of PIV5 SH increased vaccine efficacy. Additionally, other groups have demonstrated that antibodies against the prefusion conformation of RSV F have more potent neutralizing activity than antibodies against the postfusion conformation. Therefore, to improve on our previously developed vaccine candidate, we inserted RSV F at the PIV5 SH-HN gene junction or used RSV F to replace PIV5 SH. We also engineered PIV5 to express a prefusion-stabilized F mutant. The candidates were tested in BALB/c mice via the intranasal route and induced both humoral and cell-mediated immunity. They also protected against RSV infection in the mouse lung. When they were administered intranasally or subcutaneously in cotton rats, the candidates were highly immunogenic and reduced RSV loads in both the upper and lower respiratory tracts. PIV5-RSV F was equally protective when administered intranasally or subcutaneously. In all cases, the prefusion F mutant did not induce higher neutralizing antibody titers than wild-type F. These results show that antibodies against both pre- and postfusion F are important for neutralizing RSV and should be considered when designing a vectored RSV vaccine. The findings also that indicate PIV5-RSV F may be administered subcutaneously, which is the preferred route for vaccinating infants, who may develop nasal congestion as a result of intranasal vaccination.IMPORTANCE Despite decades of research, human respiratory syncytial Virus (RSV) is still a major health concern for which there is no vaccine. A Parainfluenza Virus 5-vectored vaccine expressing the native RSV fusion protein (F) has previously been shown to confer robust immunity against RSV infection in mice, cotton rats, and nonhuman primates. To improve our previous vaccine candidate, we developed four new candidates that incorporate modifications to the PIV5 backbone, replace native RSV F with a prefusion-stabilized RSV F mutant, or combine both RSV F and PIV5 backbone modifications. In this work, we characterized the new vaccine candidates and tested their efficacies in both murine and cotton rat models of RSV infection. Most importantly, we found that PIV5-based RSV vaccine candidates were efficacious in preventing lower respiratory tract infection as well as in reducing the nasal viral load when administered via the subcutaneous route.
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Parainfluenza Virus 5 Expressing Wild-Type or Prefusion Respiratory Syncytial Virus (RSV) Fusion Protein Protects Mice and Cotton Rats from RSV Challenge.
Journal of Virology, 2017Co-Authors: Shannon Phan, Huiling Wei, James Zengel, Dai Wang, Zhuo Li, Biao HeAbstract:Human respiratory syncytial Virus (RSV) is the leading cause of pediatric bronchiolitis and hospitalizations. RSV can also cause severe complications in elderly and immunocompromised individuals. There is no licensed vaccine. We previously generated a Parainfluenza Virus 5 (PIV5)-vectored vaccine candidate expressing the RSV fusion protein (F) that was immunogenic and protective in mice. In this work, our goal was to improve the original vaccine candidate by modifying the PIV5 vector or by modifying the RSV F antigen. We previously demonstrated that insertion of a foreign gene at the PIV5 small hydrophobic (SH)-hemagglutinin-neuraminidase (HN) junction or deletion of PIV5 SH increased vaccine efficacy. Additionally, other groups have demonstrated that antibodies against the prefusion conformation of RSV F have more potent neutralizing activity than antibodies against the postfusion conformation. Therefore, to improve on our previously developed vaccine candidate, we inserted RSV F at the PIV5 SH-HN gene junction or used RSV F to replace PIV5 SH. We also engineered PIV5 to express a prefusion-stabilized F mutant. The candidates were tested in BALB/c mice via the intranasal route and induced both humoral and cell-mediated immunity. They also protected against RSV infection in the mouse lung. When they were administered intranasally or subcutaneously in cotton rats, the candidates were highly immunogenic and reduced RSV loads in both the upper and lower respiratory tracts. PIV5-RSV F was equally protective when administered intranasally or subcutaneously. In all cases, the prefusion F mutant did not induce higher neutralizing antibody titers than wild-type F. These results show that antibodies against both pre- and postfusion F are important for neutralizing RSV and should be considered when designing a vectored RSV vaccine. The findings also that indicate PIV5-RSV F may be administered subcutaneously, which is the preferred route for vaccinating infants, who may develop nasal congestion as a result of intranasal vaccination.IMPORTANCE Despite decades of research, human respiratory syncytial Virus (RSV) is still a major health concern for which there is no vaccine. A Parainfluenza Virus 5-vectored vaccine expressing the native RSV fusion protein (F) has previously been shown to confer robust immunity against RSV infection in mice, cotton rats, and nonhuman primates. To improve our previous vaccine candidate, we developed four new candidates that incorporate modifications to the PIV5 backbone, replace native RSV F with a prefusion-stabilized RSV F mutant, or combine both RSV F and PIV5 backbone modifications. In this work, we characterized the new vaccine candidates and tested their efficacies in both murine and cotton rat models of RSV infection. Most importantly, we found that PIV5-based RSV vaccine candidates were efficacious in preventing lower respiratory tract infection as well as in reducing the nasal viral load when administered via the subcutaneous route.
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Parainfluenza Virus 5 Expressing the G Protein of Rabies Virus Protects Mice after Rabies Virus Infection
Journal of Virology, 2014Co-Authors: Ying Huang, Zhen Fang Fu, Junhua Huang, Zhenhai Chen, Biao HeAbstract: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.
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a novel rabies vaccine based on a recombinant Parainfluenza Virus 5 expressing rabies Virus glycoprotein
Journal of Virology, 2013Co-Authors: Zhenhai Chen, Zhen F. Fu, Ming Zhou, Guoqing Zhang, Clement W Gnanadurai, Biao HeAbstract: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 10 6 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 10 8 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 10 8 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 isfirst 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.
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recombinant Parainfluenza Virus 5 expressing hemagglutinin of influenza a Virus h5n1 protected mice against lethal highly pathogenic avian influenza Virus h5n1 challenge
Journal of Virology, 2013Co-Authors: Zhuo Li, Pei Xu, Alaina J. Mooney, Jon D. Gabbard, Stephen M. Tompkins, Ryan Place, Robert J Hogan, Biao HeAbstract:A safe and effective vaccine is the best way to prevent large-scale highly pathogenic avian influenza Virus (HPAI) H5N1 outbreaks in the human population. The current FDA-approved H5N1 vaccine has serious limitations. A more efficacious H5N1 vaccine is urgently needed. Parainfluenza Virus 5 (PIV5), a paramyxoVirus, is not known to cause any illness in humans. PIV5 is an attractive vaccine vector. In our studies, a single dose of a live recombinant PIV5 expressing a hemagglutinin (HA) gene of H5N1 (rPIV5-H5) from the H5N1 subtype provided sterilizing immunity against lethal doses of HPAI H5N1 infection in mice. Furthermore, we have examined the effect of insertion of H5N1 HA at different locations within the PIV5 genome on the efficacy of a PIV5-based vaccine. Interestingly, insertion of H5N1 HA between the leader sequence, the de facto promoter of PIV5, and the first viral gene, nucleoprotein (NP), did not lead to a viable Virus. Insertion of H5N1 HA between NP and the next gene, V/phosphorprotein (V/P), led to a Virus that was defective in growth. We have found that insertion of H5N1 HA at the junction between the small hydrophobic (SH) gene and the hemagglutinin-neuraminidase (HN) gene gave the best immunity against HPAI H5N1 challenge: a dose as low as 1,000 PFU was sufficient to protect against lethal HPAI H5N1 challenge in mice. The work suggests that recombinant PIV5 expressing H5N1 HA has great potential as an HPAI H5N1 vaccine.
Christopher A Kors - One of the best experts on this subject based on the ideXlab platform.
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structure of the paramyxoVirus Parainfluenza Virus 5 nucleoprotein in complex with an amino terminal peptide of the phosphoprotein
Journal of Virology, 2017Co-Authors: Megha Aggarwal, Christopher A Kors, George P Leser, Robert A. LambAbstract:Parainfluenza Virus 5 (PIV5) belongs to the family Paramyxoviridae, which consists of enveloped Viruses with a nonsegmented negative-strand RNA genome encapsidated by the nucleoprotein (N). ParamyxoVirus replication is regulated by the phosphoprotein (P) through protein-protein interactions with N and the RNA polymerase (L). The chaperone activity of P is essential to maintain the unassembled RNA-free form of N in order to prevent nonspecific RNA binding and premature N oligomerization. Here, we determined the crystal structure of unassembled PIV5 N in complex with a P peptide (N0P) derived from the N terminus of P (P50) at 2.65 A. The PIV5 N0P consists of two domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a hinge region. The cleft at the hinge region of RNA-bound PIV5 N was previously shown to be an RNA binding site. The N0P structure shows that the P peptide binds to the CTD of N and extends toward the RNA binding site to inhibit N oligomerization and, hence, RNA binding. Binding of P peptide also keeps the PIV5 N in the open form. A molecular dynamics (MD) analysis of both the open and closed forms of N shows the flexibility of the CTD and the preference of the N protein to be in an open conformation. The gradual opening of the hinge region, to release the RNA, was also observed. Together, these results advance our knowledge of the conformational swapping of N required for the highly regulated paramyxoVirus replication. IMPORTANCE ParamyxoVirus replication is regulated by the interaction of P with N and L proteins. Here, we report the crystal structure of unassembled Parainfluenza Virus 5 (PIV5) N chaperoned with P peptide. Our results provide a detailed understanding of the binding of P to N. The conformational switching of N between closed and open forms during its initial interaction with P, as well as during RNA release, was analyzed. Our data also show the plasticity of the CTD and the importance of domain movement for conformational switching. The results improve our understanding of the mechanism of interchanging N conformations for RNA replication and release.
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structure of the paramyxoVirus Parainfluenza Virus 5 nucleoprotein rna complex
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Maher Alayyoubi, Christopher A Kors, George P Leser, Robert A. LambAbstract:Parainfluenza Virus 5 (PIV5) is a member of the Paramyxoviridae family of membrane-enveloped Viruses with a negative-sense RNA genome that is packaged and protected by long filamentous nucleocapsid-helix structures (RNPs). These RNPs, consisting of ∼2,600 protomers of nucleocapsid (N) protein, form the template for viral transcription and replication. We have determined the 3D X-ray crystal structure of the nucleoprotein (N)-RNA complex from PIV5 to 3.11-A resolution. The structure reveals a 13-mer nucleocapsid ring whose diameter, cavity, and pitch/height dimensions agree with EM data from early studies on the Paramyxovirinae subfamily of native RNPs, indicating that it closely represents one-turn in the building block of the RNP helices. The PIV5-N nucleocapsid ring encapsidates a nuclease resistant 78-nt RNA strand in its positively charged groove formed between the N-terminal (NTD) and C-terminal (CTD) domains of its successive N protomers. Six nucleotides precisely are associated with each N protomer, with alternating three-base-in three-base-out conformation. The binding of six nucleotides per protomer is consistent with the “rule of six” that governs the genome packaging of the Paramyxovirinae subfamily of Viruses. PIV5-N protomer subdomains are very similar in structure to the previously solved Nipah-N structure, but with a difference in the angle between NTD/CTD at the RNA hinge region. Based on the Nipah-N structure we modeled a PIV5-N open conformation in which the CTD rotates away from the RNA strand into the inner spacious nucleocapsid-ring cavity. This rotation would expose the RNA for the viral polymerase activity without major disruption of the nucleocapsid structure.
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Structure of the paramyxoVirus Parainfluenza Virus 5 nucleoprotein–RNA complex
Proceedings of the National Academy of Sciences, 2015Co-Authors: Maher Alayyoubi, Christopher A Kors, George P Leser, Robert A. LambAbstract:Parainfluenza Virus 5 (PIV5) is a member of the Paramyxoviridae family of membrane-enveloped Viruses with a negative-sense RNA genome that is packaged and protected by long filamentous nucleocapsid-helix structures (RNPs). These RNPs, consisting of ∼2,600 protomers of nucleocapsid (N) protein, form the template for viral transcription and replication. We have determined the 3D X-ray crystal structure of the nucleoprotein (N)-RNA complex from PIV5 to 3.11-A resolution. The structure reveals a 13-mer nucleocapsid ring whose diameter, cavity, and pitch/height dimensions agree with EM data from early studies on the Paramyxovirinae subfamily of native RNPs, indicating that it closely represents one-turn in the building block of the RNP helices. The PIV5-N nucleocapsid ring encapsidates a nuclease resistant 78-nt RNA strand in its positively charged groove formed between the N-terminal (NTD) and C-terminal (CTD) domains of its successive N protomers. Six nucleotides precisely are associated with each N protomer, with alternating three-base-in three-base-out conformation. The binding of six nucleotides per protomer is consistent with the “rule of six” that governs the genome packaging of the Paramyxovirinae subfamily of Viruses. PIV5-N protomer subdomains are very similar in structure to the previously solved Nipah-N structure, but with a difference in the angle between NTD/CTD at the RNA hinge region. Based on the Nipah-N structure we modeled a PIV5-N open conformation in which the CTD rotates away from the RNA strand into the inner spacious nucleocapsid-ring cavity. This rotation would expose the RNA for the viral polymerase activity without major disruption of the nucleocapsid structure.
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on the stability of Parainfluenza Virus 5 f proteins
Journal of Virology, 2015Co-Authors: Taylor A Poor, Albert S Song, Brett D Welch, Christopher A Kors, Theodore S Jardetzky, Robert A. LambAbstract: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.
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structure of the Parainfluenza Virus 5 piv5 hemagglutinin neuraminidase hn ectodomain
PLOS Pathogens, 2013Co-Authors: Brett D Welch, Christopher A Kors, Sayantan Bose, Robert A. Lamb, Ping Yuan, Theodore S JardetzkyAbstract:ParamyxoViruses cause a wide variety of human and animal diseases. They infect host cells using the coordinated action of two surface glycoproteins, the receptor binding protein (HN, H, or G) and the fusion protein (F). HN binds sialic acid on host cells (hemagglutinin activity) and hydrolyzes these receptors during viral egress (neuraminidase activity, NA). Additionally, receptor binding is thought to induce a conformational change in HN that subsequently triggers major refolding in homotypic F, resulting in fusion of Virus and target cell membranes. HN is an oligomeric type II transmembrane protein with a short cytoplasmic domain and a large ectodomain comprising a long helical stalk and large globular head domain containing the enzymatic functions (NA domain). Extensive biochemical characterization has revealed that HN-stalk residues determine F specificity and activation. However, the F/HN interaction and the mechanisms whereby receptor binding regulates F activation are poorly defined. Recently, a structure of Newcastle disease Virus (NDV) HN ectodomain revealed the heads (NA domains) in a “4-heads-down” conformation whereby two of the heads form a symmetrical interaction with two sides of the stalk. The interface includes stalk residues implicated in triggering F, and the heads sterically shield these residues from interaction with F (at least on two sides). Here we report the x-ray crystal structure of Parainfluenza Virus 5 (PIV5) HN ectodomain in a “2-heads-up/2-heads-down” conformation where two heads (covalent dimers) are in the “down position,” forming a similar interface as observed in the NDV HN ectodomain structure, and two heads are in an “up position.” The structure supports a model in which the heads of HN transition from down to up upon receptor binding thereby releasing steric constraints and facilitating the interaction between critical HN-stalk residues and F.
George P Leser - One of the best experts on this subject based on the ideXlab platform.
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structure of the paramyxoVirus Parainfluenza Virus 5 nucleoprotein in complex with an amino terminal peptide of the phosphoprotein
Journal of Virology, 2017Co-Authors: Megha Aggarwal, Christopher A Kors, George P Leser, Robert A. LambAbstract:Parainfluenza Virus 5 (PIV5) belongs to the family Paramyxoviridae, which consists of enveloped Viruses with a nonsegmented negative-strand RNA genome encapsidated by the nucleoprotein (N). ParamyxoVirus replication is regulated by the phosphoprotein (P) through protein-protein interactions with N and the RNA polymerase (L). The chaperone activity of P is essential to maintain the unassembled RNA-free form of N in order to prevent nonspecific RNA binding and premature N oligomerization. Here, we determined the crystal structure of unassembled PIV5 N in complex with a P peptide (N0P) derived from the N terminus of P (P50) at 2.65 A. The PIV5 N0P consists of two domains: an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a hinge region. The cleft at the hinge region of RNA-bound PIV5 N was previously shown to be an RNA binding site. The N0P structure shows that the P peptide binds to the CTD of N and extends toward the RNA binding site to inhibit N oligomerization and, hence, RNA binding. Binding of P peptide also keeps the PIV5 N in the open form. A molecular dynamics (MD) analysis of both the open and closed forms of N shows the flexibility of the CTD and the preference of the N protein to be in an open conformation. The gradual opening of the hinge region, to release the RNA, was also observed. Together, these results advance our knowledge of the conformational swapping of N required for the highly regulated paramyxoVirus replication. IMPORTANCE ParamyxoVirus replication is regulated by the interaction of P with N and L proteins. Here, we report the crystal structure of unassembled Parainfluenza Virus 5 (PIV5) N chaperoned with P peptide. Our results provide a detailed understanding of the binding of P to N. The conformational switching of N between closed and open forms during its initial interaction with P, as well as during RNA release, was analyzed. Our data also show the plasticity of the CTD and the importance of domain movement for conformational switching. The results improve our understanding of the mechanism of interchanging N conformations for RNA replication and release.
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structure of the paramyxoVirus Parainfluenza Virus 5 nucleoprotein rna complex
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: Maher Alayyoubi, Christopher A Kors, George P Leser, Robert A. LambAbstract:Parainfluenza Virus 5 (PIV5) is a member of the Paramyxoviridae family of membrane-enveloped Viruses with a negative-sense RNA genome that is packaged and protected by long filamentous nucleocapsid-helix structures (RNPs). These RNPs, consisting of ∼2,600 protomers of nucleocapsid (N) protein, form the template for viral transcription and replication. We have determined the 3D X-ray crystal structure of the nucleoprotein (N)-RNA complex from PIV5 to 3.11-A resolution. The structure reveals a 13-mer nucleocapsid ring whose diameter, cavity, and pitch/height dimensions agree with EM data from early studies on the Paramyxovirinae subfamily of native RNPs, indicating that it closely represents one-turn in the building block of the RNP helices. The PIV5-N nucleocapsid ring encapsidates a nuclease resistant 78-nt RNA strand in its positively charged groove formed between the N-terminal (NTD) and C-terminal (CTD) domains of its successive N protomers. Six nucleotides precisely are associated with each N protomer, with alternating three-base-in three-base-out conformation. The binding of six nucleotides per protomer is consistent with the “rule of six” that governs the genome packaging of the Paramyxovirinae subfamily of Viruses. PIV5-N protomer subdomains are very similar in structure to the previously solved Nipah-N structure, but with a difference in the angle between NTD/CTD at the RNA hinge region. Based on the Nipah-N structure we modeled a PIV5-N open conformation in which the CTD rotates away from the RNA strand into the inner spacious nucleocapsid-ring cavity. This rotation would expose the RNA for the viral polymerase activity without major disruption of the nucleocapsid structure.
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Structure of the paramyxoVirus Parainfluenza Virus 5 nucleoprotein–RNA complex
Proceedings of the National Academy of Sciences, 2015Co-Authors: Maher Alayyoubi, Christopher A Kors, George P Leser, Robert A. LambAbstract:Parainfluenza Virus 5 (PIV5) is a member of the Paramyxoviridae family of membrane-enveloped Viruses with a negative-sense RNA genome that is packaged and protected by long filamentous nucleocapsid-helix structures (RNPs). These RNPs, consisting of ∼2,600 protomers of nucleocapsid (N) protein, form the template for viral transcription and replication. We have determined the 3D X-ray crystal structure of the nucleoprotein (N)-RNA complex from PIV5 to 3.11-A resolution. The structure reveals a 13-mer nucleocapsid ring whose diameter, cavity, and pitch/height dimensions agree with EM data from early studies on the Paramyxovirinae subfamily of native RNPs, indicating that it closely represents one-turn in the building block of the RNP helices. The PIV5-N nucleocapsid ring encapsidates a nuclease resistant 78-nt RNA strand in its positively charged groove formed between the N-terminal (NTD) and C-terminal (CTD) domains of its successive N protomers. Six nucleotides precisely are associated with each N protomer, with alternating three-base-in three-base-out conformation. The binding of six nucleotides per protomer is consistent with the “rule of six” that governs the genome packaging of the Paramyxovirinae subfamily of Viruses. PIV5-N protomer subdomains are very similar in structure to the previously solved Nipah-N structure, but with a difference in the angle between NTD/CTD at the RNA hinge region. Based on the Nipah-N structure we modeled a PIV5-N open conformation in which the CTD rotates away from the RNA strand into the inner spacious nucleocapsid-ring cavity. This rotation would expose the RNA for the viral polymerase activity without major disruption of the nucleocapsid structure.
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structure of the cleavage activated prefusion form of the Parainfluenza Virus 5 fusion protein
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Brett D Welch, Christopher A Kors, Theodore S Jardetzky, George P Leser, Robert A. LambAbstract:The paramyxoVirus Parainfluenza Virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and Virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein.
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fusion activation by a headless Parainfluenza Virus 5 hemagglutinin neuraminidase stalk suggests a modular mechanism for triggering
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Sayantan Bose, Brett D Welch, Theodore S Jardetzky, Aarohi Zokarkar, George P Leser, Robert A. LambAbstract:The Paramyxoviridae family of enveloped Viruses enters cells through the concerted action of two viral glycoproteins. The receptor-binding protein, hemagglutinin-neuraminidase (HN), H, or G, binds its cellular receptor and activates the fusion protein, F, which, through an extensive refolding event, brings viral and cellular membranes together, mediating Virus–cell fusion. However, the underlying mechanism of F activation on receptor engagement remains unclear. Current hypotheses propose conformational changes in HN, H, or G propagating from the receptor-binding site in the HN, H, or G globular head to the F-interacting stalk region. We provide evidence that the receptor-binding globular head domain of the paramyxoVirus Parainfluenza Virus 5 HN protein is entirely dispensable for F activation. Considering together the crystal structures of HN from different paramyxoViruses, varying energy requirements for fusion activation, F activation involving the Parainfluenza Virus 5 HN stalk domain, and properties of a chimeric paramyxoVirus HN protein, we propose a simple model for the activation of paramyxoVirus fusion.
