<OL> <LI> Investigate mechanism of inhibition of TNF signaling pathways by the SH protein <BR> It was reported that the SH protein is required to inhibit apoptosis induced by SV5 infection. We have shown the SH protein blocked TNF signaling pathway. However, the mechanism of the inhibition is not known. We propose that the SH protein interdicts TNF induced apoptotic signaling through direct interactions with the proteins in the TNF signaling pathway. We will identify host cell proteins with which SH interacts. We will examine how such interactions result in inhibition of apoptosis and map the regions of the cellular proteins and SH that are important for the interaction to occur. <LI>Investigate mechanism of increased expression of TNF in rSV5?SH infected cells SV5 without the SH gene (rSV5?SH) induced apoptosis in MDBK, L929 and MDCK cells. <BR> Increased levels of TNF-a were detected in the media of rSV5?SH infected L929 cells and found to be responsible for rSV5?SH induced apoptosis. However, it is not clear what caused the increase. We propose that SV5 proteins interact with host cell proteins resulting in increased expression levels of TNF. We will identify SV5 protein(s) that is responsible for the increased expression of TNF and investigate how the viral protein causes the increased levels of TNF. <LI> Study anti-apoptosis functions of SH proteins encoded by other paramyxoviruses <BR> Small hydrophobic proteins are encoded by other paramyxoviruses mump virus and respiratory syncytial (RS) virus, but their functions are not clear. We hypothesize that the SH proteins from them may be functional counterparts of the SH protein of SV5. Thus, the SH protein of SV5 may define a new class of viral proteins with anti-apoptosis function. <LI>Generation of avian influenza virus vaccine based on paramyxovirus simian virus 5 <BR> We plan to study feasibility of using paramyxovirus simian virus 5 (SV5) as a vaccine vector against avian influenza virus (AIV). Previously, we have expressed foreign genes including antigen of human influenza virus using SV5 as a vector. The recombinant SV5 expressing human influenza virus antigen provided immunity to mice against human influenza virus infection. In this proposal, we will generate recombinant SV5 viruses expressing AIV antigens and test their efficacy against avian influenza virus challenge in chicken.
NON-TECHNICAL SUMMARY: A. Paramyxoviruses are important pathogens for human and animals. B. Avian influenza virus epidemic poses major threat to poultry industry. A. This project seeks to understand pathogenesis of paramyxovirus through study of virus and host interaction B. This project seeks to develop a novel vaccine against avian influenza virus based on paramyxovirus SV5.
APPROACH: 1. Investigate mechanism of inhibition of TNF signaling pathway by SH Important amino acid residues of SH required for its inhibitory effect will be identified. Mutant SH will be generated using standard molecular cloning techniques. The abilities to inhibit TNF signaling by mutant SH proteins will be examined. To facilitate the research, antigen epitope tag will be added to the SH protein and tested to ensure that the extra tag does not alter SH?s function. Host proteins in the TNF signaling pathway interacting with SH will be identified using co-immunoprecipitation followed by Western blot with appropriate antibodies. 2. Investigate mechanism of increased expression of TNF in rSV5?SH infected cells Activation of TNF transcription and TNF mRNA stability in virus infected cells will be examined using RNase protection and quantitative RT-PCR; amount of membrane bound TNF will be examined using Western blot. UV-inactivated virus and virus-cell fusion inhibitor will be used to investigate whether virus replication and attachment are sufficient to induce TNF production. SV5 proteins involved in activation of TNF expression will be identified by a reporter gene assay. Mutant rSV5?SH viruses that do not induce apoptosis in L929 cells will be isolated and mutations in the viruses will be identified by RT-PCR sequencing. 3. Study anti-apoptotic activities of SH proteins encoded by mumps virus and RS virus Both mumps virus and RS virus can inhibit apoptosis. However, it is not clear which viral proteins are involved in the inhibition. Functions of the SH proteins encoded by mumps virus and RS virus are not known. The SH proteins may be functional counterparts of the SH protein of SV5. SH proteins of mumps virus and RS virus in L929 cells will be expressed and their abilities to inhibit TNF-a will be examined. The SH gene of SV5 will be replaced with the SH genes from mumps virus and RS virus and the hybrid SV5 viruses will be analyzed. 4. Generation of avian influenza virus vaccine based on SV5 SV5 infects many animals without causing clinical symptoms. It is also known that SV5 grows well in chicken embryonic cells with no significant cytopathic effects. However, SV5?s infectivity in chickens is unknown and immune responses to SV5 infection in chicken is not well studied. We will inoculate SPF chickens with SV5 and examine clinical symptoms of infected chickens. Growth of SV5 in chicken will be monitored by examining SV5 titers in infected chicken at different time points post infection using plaque assay. Chicken?s immune responses such as antibody production and T cell activation to SV5 infection will be studied following inoculation with SV5. We propose to express AIV antigens by inserting AIV genes into SV5 genome and recover recombinant SV5 viruses expressing AIV proteins. We plan to express HA, NA, NP, M1 and M2 proteins of AIV using SV5 as a vector. Abilities of the recombinant SV5 viruses expressing AIV antigens to protect chicken against lethal dosage of AIV challenge will be examined. We will inoculate chickens with recombinant SV5 expressing AIV antigens and then challenge the chickens with AIV.
PROGRESS: 2002/12 TO 2007/09<BR>
OUTPUTS: Parainfluenza virus 5 (PIV5), formerly known as simian virus 5 (SV5) is a prototypical member of the Rubulavirus genus of the family Paramyxoviridae. PIV5 is a non-segmented negative stranded RNA virus (NNSV). The viral RNA-dependent RNA polymerase (vRdRp) minimally consists of two proteins, phosphoprotein (P) and the large (L) polymerase protein, transcribes and replicates the viral RNA genome that is encapsidated with nucleocapsid protein (NP or N). In many paramyxoviruses, it has been demonstrated that the vRdRp is essential for RNA synthesis but not sufficient, indicating host proteins are required for viral RNA synthesis. In our work, we have found that a host protein, Akt1, plays a critical role in the viral RNA synthesis of PIV5. This exciting novel discovery has broad implication since inhibiting Akt1 using siRNA or small molecule inhibitors results in inhibition of replication of PIV5 as well as replications of mumps virus (MuV), measles virus (MeV), Sendai virus (SeV), respiratory syncytial virus (RSV) and vesicular stomatitis virus (VSV), which are non-segmented negative stranded RNA viruses (NNSVs). We hypothesize that Akt1 plays a critical role in replication of NNSVs and Akt is a good target for anti-viral therapy. This novel discovery will not only lead to better understanding of viral pathogenesis, it will also enables novel strategies to combat infections caused by these viruses. In addition. PIV5 is an excellent candidate as a vector for vaccine development. PIV5 does not have a DNA phase in its life cycle and it replicates solely in cytoplasm, therefore using PIV5 as a vector avoids possible unintended consequences of genetic modifications of host cell DNAs. The genome structure of PIV5 is stable and simple. Avian influenza virus (AIV) epidemic is a major threat to domestic poultry in Pennsylvania and United States. Vaccination is the best way to prevent and control AIV outbreaks. However, attenuated or inactivated AIV vaccines are not feasible to use because of their interference with AIV surveillance program. Thus, using a vector to express AIV antigens, especially antigens other than HA and NA of AIV, provides a viable approach for AIV vaccine development. We have demonstrated that PIV5 is a good vector to express influenza A virus antigen HA in mice. <BR> PARTICIPANTS: Biao He, Associate Professor, Department of Veterinary and Biomedical Sciences, Pennsylvania State University, University Park, PA. <BR> TARGET AUDIENCES: Stake holders, bio-technology companies, companies interested in anti-viral drugs and companies interested in vaccine development.
IMPACT: 2002/12 TO 2007/09<BR>
Viruses in the Paramyxoviridae family in the order of Mononegavirales are negative stranded, non-segmented RNA viruses that 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 virus and Ebola virus. Hendra virus and Nipah virus, two paramyxoviruses, have been recently isolated from Australia and Malaysia. They cause fatal infection in humans. At present, there is no anti-viral treatment for infections caused by these viruses. We have identified a host protein that interacted with the V protein of PIV5 that plays a critical role in regulating virus RNA synthesis. Inhibitors targeting the host protein resulted in inhibition of replication of paramyxoviruses. Targeting the host protein for treating infections caused by these viruses will lead to novel strategies to control these infections. Epidemic of avian influenza virus (AIV), such as H5N1, is a major threat to domestic poultry as well as to human health in Pennsylvania and United States. Outbreaks of AIV in Pennsylvania caused tremendous economic losses to the poultry industry. Concern about possible infection of human by avian influenza virus poses perhaps the greatest challenge to human health. Vaccination is the best way to prevent and control AIV outbreaks. We seek to develop an AIV vaccine using recombinant PIV5 as a vaccine vector. Such a novel approach will hopefully prevent AIV outbreaks without interfering with the AIV surveillance programs. Outcomes of this research will bring great benefits to poultry industry and have great potential for AIV control and possible prevention worldwide. Technology developed based on the proposed work can potentially be applied to combat other viral infections as well.