Progress 10/01/07 to 09/30/12
Outputs
OUTPUTS: We have made numerous new discoveries over the course of this project that impact the field of paramyxovirus pathogenesis, and these have been published in high impact, peer-reviewed journals. A major goal of the project was to identify host proteins that interact with paramyxovirus matrix (M) proteins. M proteins organize the assembly of virus particles, which spread infections from one host to the next. M proteins manipulate the machinery of the host cell during virus infection. We identified two host proteins that bind with the viral M protein. One protein, AmotL1, localizes to tight junctions of polarized cells and is necessary for efficient viral particle formation. The other protein, 14-3-3, binds to M protein and has the opposite effect, impairing virus particle formation. We propose that these viruses may regulate their budding activity by preferentially binding to one host factor or the other, as the situation dictates. A second major goal of the project was to characterize interactions between viral M and nucleocapsid (NP) proteins. These interactions drive the incorporation of viral genomes into budding particles and are required for particle infectivity. We identified a region near the C-terminal ends of the parainfluenza virus 5 (PIV5) and mumps virus NP proteins that are required for interaction with M protein. Recombinant viruses with mutations in NP protein quickly acquire adaptive mutations in M protein that restore the interaction. Through analysis of these second-site M protein mutations, we have identified clusters of residues on M protein that appear to be involved in its interaction with NP. Finally, we have characterized the way in which viral M protein is modified through monoubiquitination. Lysine residues within M protein that are targets for ubiquitin attachment were identified, and recombinant viruses that lack the ubiquitination sites were constructed. Our results suggest that ubiquitination of M protein is important for proper virus assembly and particle infectivity. Overall, these studies have established a foundation that will benefit future efforts aimed at antiviral drug development. We have identified new interaction interfaces between M protein and host proteins, between M protein and NP protein, and between M protein and ubiquitin. Any of these can now be targeted for disruption in efforts to block paramyxovirus replication PARTICIPANTS: Dr. Anthony Schmitt, Associate Professor, Department of Veterinary and Biomedical Sciences. TARGET AUDIENCES: Stake holders, biotechnology companies, companies interested in antiviral drug development. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Mononegavirales are negative stranded, non-segmented RNA viruses with a lipid membrane. Viruses in the Paramyxoviridae family of Mononegavirales include many important human and animal pathogens such as human parainfluenza viruses, Sendai virus, mumps virus, Newcastle disease virus, measles virus, rinderpest virus, and human respiratory syncytial virus, as well as recently emerged zoonotic viruses Nipah and Hendra virus. Respiratory syncytial virus is the leading cause of bronchiolitis in young children. Measles and mumps viruses have recently caused resurgences of disease, in part due to lack of compliance with vaccination protocols. Newcastle disease virus and rinderpest virus are significant causes of disease in livestock. Hendra virus and Nipah virus have only recently been isolated from Australia and Malaysia. They cause fatal infections in humans. Their genomes are closely related, but they are distinct from other paramyxoviruses. A new genus, Henipavirus, has been created to accommodate the newly discovered viruses. Nipah virus is of particular concern from an agricultural standpoint, as it spreads readily in pigs, which act as amplifying hosts, and most human infections result from direct contact with infected pigs. Parainfluenza virus 5, formerly known as simian virus 5, is a member of the Rubulavirus genus of the family Paramyxoviridae. It is a well-established model virus for the study of paramyxovirus entry, exit, and pathogenesis. Few antiviral drugs exist that are effective at combating paramyxovirus infections. Advances in understanding paramyxovirus replication and biology, including the steps of virus assembly and, will facilitate the development of antiviral drugs that can be used to target these steps of the virus lifecycle
Publications
- Harrison, M. S., P. T. Schmitt, S. Pei, and A. P. Schmitt. 2012. Role of Ubiquitin in PIV5 Particle Formation. J. Virol. 86:3474-3485.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: We have made several new discoveries in the past year that impact the field of paramyxovirus pathogenesis, and these have been published in high impact, peer-reviewed journals. A novel paradigm for the regulation of paramyxovirus matrix (M) proteins was established. M proteins organize paramyxovirus assembly and are among the most abundantly produced proteins in virus-infected cells. For a subset of the paramyxoviruses, including mumps virus and parainfluenza virus 5 (PIV5), we found that a fraction of the viral M protein is phosphorylated and bound by the host protein 14-3-3 during infection. 14-3-3 binding is known to regulate the functions of many cellular proteins. We found that 14-3-3 binding negatively affects the budding functions of paramyxovirus M proteins. Recombinant viruses with mutant M proteins that cannot bind 14-3-3 bud particles more efficiently than wt virus. This suggests that a portion of M protein in virus-infected cells is phosphorylated, bound to 14-3-3, and held away from the sites of virus particle assembly. These results imply that paramyxovirus M proteins are multifunctional, and that the 14-3-3-bound portion of M protein provides a function for the virus that is unrelated to virus assembly. Additional progress was made towards understanding the cellular role of a different M-interacting host factor, angiomotin-like-1 (AmotL1). AmotL1 protein harbors two PPxY motifs. Several retroviruses use PPxY motifs as "late domains" to recruit host machinery for budding. We previously found that PIV5 M protein binds AmotL1, and that this interaction facilitates virus assesmbly. Now we have found that AmotL1 uses its PPxY motifs to bind to the WW domains of the cellular protein YAP, thereby preventing YAP from activating transcription of target genes in the cell nucleus. PARTICIPANTS: Dr. Anthony Schmitt, Associate Professor, Department of Veterinary and Biomedical Sciences. TARGET AUDIENCES: Stake holders, biotechnology companies, and companies interested in antiviral drug development. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Mononegavirales are negative stranded, non-segmented RNA viruses with a lipid membrane. Viruses in the Paramyxoviridae family of Mononegavirales include many important human and animal pathogens such as human parainfluenza viruses, Sendai virus, mumps virus, Newcastle disease virus, measles virus, rinderpest virus, and human respiratory syncytial virus, as well as recently emerged zoonotic viruses Nipah and Hendra virus. Respiratory syncytial virus is the leading cause of bronchiolitis in young children. Measles and mumps viruses have recently caused resurgences of disease, in part due to lack of compliance with vaccination protocols. Newcastle disease virus and rinderpest virus are significant causes of disease in livestock. Hendra virus and Nipah virus have only recently been isolated from Australia and Malaysia. They cause fatal infections in humans. Their genomes are closely related, but they are distinct from other paramyxoviruses. A new genus, Henipavirus, has been created to accommodate the newly discovered viruses. Nipah virus is of particular concern from an agricultural standpoint, as it spreads readily in pigs, which act as amplifying hosts, and most human infections result from direct contact with infected pigs. Parainfluenza virus 5, formerly known as simian virus 5, is a member of the Rubulavirus genus of the family Paramyxoviridae. It is a well-established model virus for the study of paramyxovirus entry, exit, and pathogenesis. Few antiviral drugs exist that are effective at combating paramyxovirus infections. Advances in understanding paramyxovirus replication and biology, including the steps of virus assembly and, will facilitate the development of antiviral drugs that can be used to target these steps of the virus lifecycle.
Publications
- Pei, Z., M. S. Harrison, and A. P. Schmitt. 2011. Parainfluenza virus 5 M protein interaction with host protein 14-3-3 negatively affects virus particle formation. J. Virol. 85:2050-2059.
- Oka, T., A. P. Schmitt, and M. Sudol. 2011. Opposing roles of angiomotin-like-1 and zona occludens-2 on pro-apoptotic function of YAP. Oncogene (In Press).
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: We have made new discoveries in the field of paramyxovirus pathogenesis that have significantly advanced the field, and have been published in high impact peer-reviewed journals. We identified a novel host factor, AmotL1, that binds to the matrix (M) protein of parainfluenza virus 5 (PIV5). This interaction is beneficial to the virus and allows the viral M protein to function optimally during virus particle formation. This interaction is the first involving M protein to be described for any paramyxovirus in the Rubulavirus genus. Our findings have significant implications for translational research. We showed that the binding interface between AmotL1 and M protein can be targeted for disruption, and that this inhibits virus budding. Hence, these findings provide proof-of-principle that paramyxovirus replication can be disrupted through the targeting of M protein:host protein interactions, and lay a framework for future efforts aimed at antiviral drug discovery. Additional progress was made towards understanding the fundamental interactions that allow paramyxovirus ribonucleoprotein (RNP) complexes to be packaged into virus particles. We identified the region of the viral nucleocapsid (NP) protein that is responsible for binding to M protein. Recombinant viruses were generated in which this interaction was disrupted, and second-site adaptive mutations were acquired by the viruses which provide key insights into mechanisms of virus spread. Finally, we have written two comprehensive reviews of paramyxovirus assembly, one targeted to a general scientific audience and another targeted to researchers in the field. These reviews provide a valuable resource that will benefit researchers for years to come. PARTICIPANTS: Dr. Anthony Schmitt, Assistant Professor, Department of Veterinary and Biomedical Sciences. TARGET AUDIENCES: Stake holders, biotechnology companies, companies interested in antiviral drug development. PROJECT MODIFICATIONS: Dr. Biao He has left Penn State University and no longer participates in this project.
Impacts
Mononegavirales are negative stranded, non-segmented RNA viruses with a lipid membrane. Viruses in the Paramyxoviridae family of Mononegavirales include many important human and animal pathogens such as human parainfluenza viruses, Sendai virus, mumps virus, Newcastle disease virus, measles virus, rinderpest virus, and human respiratory syncytial virus, as well as recently emerged zoonotic viruses Nipah and Hendra virus. Respiratory syncytial virus is the leading cause of bronchiolitis in young children. Measles and mumps viruses have recently caused resurgences of disease, in part due to lack of compliance with vaccination protocols. Newcastle disease virus and rinderpest virus are significant causes of disease in livestock. Hendra virus and Nipah virus have only recently been isolated from Australia and Malaysia. They cause fatal infections in humans. Their genomes are closely related, but they are distinct from other paramyxoviruses. A new genus, Henipavirus, has been created to accommodate the newly discovered viruses. Nipah virus is of particular concern from an agricultural standpoint, as it spreads readily in pigs, which act as amplifying hosts, and most human infections result from direct contact with infected pigs. Parainfluenza virus 5, formerly known as simian virus 5, is a member of the Rubulavirus genus of the family Paramyxoviridae. It is a well-established model virus for the study of paramyxovirus entry, exit, and pathogenesis. Few antiviral drugs exist that are effective at combating paramyxovirus infections. Advances in understanding paramyxovirus replication and biology, including the steps of virus assembly and, will facilitate the development of antiviral drugs that can be used to target these steps of the virus lifecycle.
Publications
- Pei, Z., Y. Bai, and A. P. Schmitt. 2010. PIV5 M protein interaction with host protein angiomotin-like 1. Virology 397:155-166.
- Harrison, M. S., T. Sakaguchi, and A. P Schmitt. 2010. Paramyxovirus assembly and budding: building particles that transmit infections. Intl. J. Biochem. Cell Biol. 42:1416-1429.
- Schmitt, P. T., G. Ray, and A. P. Schmitt. 2010. The C-terminal end of PIV5 NP protein is important for virus-like particle production and M-NP protein interaction. J. Virol. (In Press).
- Harrison, M. S., T. Sakaguchi, and A. P. Schmitt. 2010. Paramyxovirus budding mechanisms. In: Luo, M, editor. Negative Strand RNA Virus. World Scientific Publishing (In Press).
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: We have made novel discoveries in the field of paramyxovirus pathogenesis, and they have been published in high impact peer-reviewed journals. Key progress was made in the area of mumps virus particle assembly. Mumps virus is an under-studied paramyxovirus which nonetheless has caused a recent resurgence of disease even in countries where vaccination is widespread. We have found that, unlike many paramyxoviruses, mumps virus particle production cannot occur when the viral matrix (M) protein is expressed by itself in transfected cells. Rather, multiple mumps virus components, including the mumps virus fusion glycoprotein and the mumps virus nucleocapsid protein, must be present in cells together with M protein before efficient particle assembly can take place. This finding demonstrates that different mumps virus proteins cooperate with one another for efficient particle production. Furthermore, a role for host Class E protein machinery in mumps virus budding was demonstrated, as mumps virus budding could be inhibited through expression of dominant-negative Class E proteins. Key progress was also made in understanding how paramyxovirus gene expression is regulated in virus-infected cells. These viruses encode RNA-dependent RNA polymerases, which are composed of large (L) and phospho (P) protein subunits. It has long been thought that the P proteins regulate viral gene expression, perhaps in a manner that involves differential phosphorylation. We showed for the first time that a cellular kinase, polo-like kinase 1 (PLK1) regulates gene expression of the model paramyxovirus parainfluenza virus 5 (PIV5) by binding and phosphorylating the viral P protein at a specific site. Phosphorylation of P protein by PLK1 resulted in down-regulation of viral gene expression. This in turn is likely to benefit the virus, as enhanced viral gene expression triggers cytokine expression and leads to the induction of cell death PARTICIPANTS: Dr. Biao He, Associate Professor, Department of Veterinary and Biomedical Sciences; Dr. Anthony Schmitt, Assistant Professor, Department of Veterinary and Biomedical Sciences. TARGET AUDIENCES: Stake holders, biotechnology companies, companies interested in anti-viral drugs, and companies interested in vaccine development. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Mononegavirales are negative stranded, non-segmented RNA viruses with a lipid membrane. Viruses in the Paramyxoviridae family of Mononegavirales include many important human and animal pathogens such as human parainfluenza viruses, Sendai virus, mumps virus, Newcastle disease virus, measles virus, rinderpest virus, and human respiratory syncytial virus, as well as recently emerged zoonotic viruses Nipah and Hendra virus. Respiratory syncytial virus is the leading cause of bronchiolitis in young children. Measles and mumps viruses have recently caused resurgences of disease, in part due to lack of compliance with vaccination protocols. Newcastle disease virus and rinderpest virus are significant causes of disease in livestock. Hendra virus and Nipah virus have only recently been isolated from Australia and Malaysia. They cause fatal infections in humans. Their genomes are closely related, but they are distinct from other paramyxoviruses. A new genus, Henipavirus, has been created to accommodate the newly discovered viruses. Nipah virus is of particular concern from an agricultural standpoint, as it spreads readily in pigs, which act as amplifying hosts, and most human infections result from direct contact with infected pigs. Parainfluenza virus 5, formerly known as simian virus 5, is a member of the Rubulavirus genus of the family Paramyxoviridae. It is a well-established model virus for the study of paramyxovirus entry, exit, and pathogenesis. Few antiviral drugs exist that are effective at combating paramyxovirus infections. Advances in understanding paramyxovirus replication and biology, including the steps of virus budding and viral gene expression, will facilitate the development of antiviral drugs that can be used to target these steps of the virus lifecycle.
Publications
- Li, M., P. T. Schmitt, Z. Li, T. S. McCrory, B. He, and A. P. Schmitt. 2009. Mumps virus matrix, fusion, and nucleocapsid proteins cooperate for efficient production of virus-like particles. J. Virol. 83:7261-7272.
- Sun, D., P. Luthra, S. Li,and B. He. 2009. PLK1 down-regulates parainfluenza virus 5 gene expression. PLoS Pathog. 5(7):e1000525.
- Cox, R., T. J. Green, S. Qiu, J. Kang, J. Tsao, P. E. Prevelige, B. He and M. Luo, M. 2009. Characterization of a mumps virus nucleocapsidlike particle. J. Virol. 83:11402-11406.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: We have made novel discoveries in our field and they are published in high impact peer-reviewed journals. PARTICIPANTS: Dr. Biao He, Associate Professor, Department of Veterinary and Biomedical Sciences; Dr. Anthony Schmitt, Assistant Professor, Department of Veterinary and Biomedical Sciences. TARGET AUDIENCES: Stake holders, biotechnology companies, companies interested in anti-viral drugs and companies interested in vaccine development. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Mononegavirales are negative stranded, non-segmented RNA viruses (NNSV) with a lipid membrane. Viruses in the Paramyxoviridae family of Mononegavirales include many important human and animal pathogens such as human parainfluenza viruses, Sendai virus (SeV), mumps virus (MuV), Newcastle disease virus (NDV), measles virus (MeV), rinderpest virus and human respiratory syncytial virus (RSV) as well as emerging viruses Nipah and Hendra virus. There are two subfamilies, paramyxovirinae and pneumovirinae, in the Paramyxoviridae family. Until recently, paramyxovirinae contained three genera, rubulavirus, respirovirus and morbillivirus. Hendra virus and Nipah virus, two paramyxoviruses, have been recently isolated from Australia and Malaysia. They cause fatal infection in humans. Their genomes are closely related, but they are different from the rest of the subfamily of paramyxovirinae. A new genus, Henipavirus, has been created to accommodate the newly discovered viruses. Parainfluenza virus 5 (PIV5), formerly known as simian virus 5 (SV5), is a member of the Rubulavirus genus of the family Paramyxoviridae. Its viral RNA-dependent RNA polymerase (RdRp) minimally consists of two proteins, phosphoprotein (P) and the large (L) polymerase protein. This complex transcribes and also replicates the viral RNA genome that is encapsidated with nucleocapsid protein (NP). The RdRp is essential but not sufficient for RNA synthesis and host proteins are required for viral RNA synthesis. Moreover, it is unclear how the processes of transcription and replication are regulated. We have discovered that the V protein of PIV5 inhibits vRNA synthesis, and this activity requires an interaction with a host protein. We have identified a host protein, Akt1, with which V interacts. Furthermore, we have found that Akt1, a serine/threonine kinase, affects phosphorylation status of the P protein in infected cells and phosphorylates the P protein purified from bacteria. This exciting observation has broad relevance to non-segmented negative stranded RNA viruses (NNSVs) because reduction of Akt1 activity with specific siRNA or small molecule inhibitors blocked not only the replication of PIV5 but also that of mumps virus (MuV), measles virus (MeV), Sendai virus (SeV), respiratory syncytial virus (RSV) and vesicular stomatitis virus (VSV).
Publications
- Sun, M., S. Fuentes, K. Timani, C. Murphy, Y. Lin, A. August, M. N. Teng and B. He. 2008. Akt plays a critical role in replication of non-segmented, negative-stranded RNA viruses. J. Virol. 82:105-14.
- Timani, K., D. Sun, M. Sun, C. Keim, Y. Lin, P. Schmitt, A. Schmitt and B. He. 2008. A single amino acid residue change in the P protein of parainfluenza virus 5 (PIV5) elevates viral RNA replication. J. Virol. 82:9123-33.
- Luthra, P. D. Sun, M. Wolfgang and B. He. 2008. Activation of NF-kB Through an AKT1-dependent Pathway by the L protein of parainfluenza virus 5 (PIV5) J. Virol. 82:10887-95.
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