Respiratory virus infection caused by enveloped RNA viruses, such as parainfluenza and respiratory syncytial viruses, is a major public health concern. These viruses infect and replicate in the upper and/or lower respiratory tracts and transmit through airways. These respiratory viruses bud from the apical surface of polarized epithelial cells. Obviously, efficient virus assembly in respiratory epithelial cells is a key factor for virus transmission and pathogenicity. However, the molecular mechanism of virus assembly, especially how viral structural components are transported to the plasma membrane assembly/budding sites is poorly understood, partially due to lack of a method to monitor trafficking of viral components in live infected cells. In this proposal, we will use Sendai virus (SeV), one of the best-characterized paramyxoviruses, to reveal how viral nucleocapsids (vRNPs) are transported to assembly sites. Using the reverse genetics system, we successfully rescued a recombinant SeV (rSeV-LeGFP) whose L protein is tagged with enhanced green fluorescent protein (eGFP). This virus allows us to monitor real time trafficking of the vRNP in live infected cells. Using the virus, we successfully recorded vRNP movement through microtubules in infected cells. Additional data strongly suggest that the intracellular vesicular trafficking pathway regulated by Rab11a is involved in vRNP translocation. Taking full advantage of rSeV-LeGFP and our ability to rescue various SeV mutants, we propose to unveil the cellular and viral factors involved in the process of vRNP trafficking and virus assembly in respiratory epithelial cells. We will identify the cellular machinery and proteins responsible for vRNP transport, as well as the viral proteins that mediate the process. In this project, we will uncover the process of assembly of SeV and related human respiratory paramyxoviruses, especially the role of the intracellular vesicular trafficking pathway in vRNP transport and assembly of virions at the apical surface budding sites of epithelial cells. This research will shed light on the unknown critical process of respiratory virus assembly, which is expected to provide valuable data to develop safe attenuated vaccines or antiviral agents.
Although parainfluenza and respiratory syncytial viruses are the major causes of respiratory viral disease in infants and young children, no vaccines or therapeutic drugs are available. Studies proposed in this grant will unveil the virus-host interactions required for virus assembly and formation, which will provide important implications for the design of novel vaccines and antiviral reagents.
|Stone, Raychel; Hayashi, Tsuyoshi; Bajimaya, Shringkhala et al. (2016) Critical role of Rab11a-mediated recycling endosomes in the assembly of type I parainfluenza viruses. Virology 487:11-8|
|Fukushima, Keijo; Takahashi, Tadanobu; Ueyama, Hiroo et al. (2015) Amino acid substitutions contributing to Î±2,6-sialic acid linkage binding specificity of human parainfluenza virus type 3 hemagglutinin-neuraminidase. FEBS Lett 589:1278-82|
|Zhan, Xiaoyan; Slobod, Karen S; Jones, Bart G et al. (2015) Sendai virus recombinant vaccine expressing a secreted, unconstrained respiratory syncytial virus fusion protein protects against RSV in cotton rats. Int Immunol 27:229-36|
|Fukushima, Keijo; Takahashi, Tadanobu; Ito, Seigo et al. (2014) Terminal sialic acid linkages determine different cell infectivities of human parainfluenza virus type 1 and type 3. Virology 464-465:424-31|
|Kobayashi, Hirofumi; Iwatsuki-Horimoto, Kiyoko; Kiso, Maki et al. (2013) A replication-incompetent influenza virus bearing the HN glycoprotein of human parainfluenza virus as a bivalent vaccine. Vaccine 31:6239-46|
|Stone, Raychel; Takimoto, Toru (2013) Critical role of the fusion protein cytoplasmic tail sequence in parainfluenza virus assembly. PLoS One 8:e61281|
|Jones, Bart G; Sealy, Robert E; Rudraraju, Rajeev et al. (2012) Sendai virus-based RSV vaccine protects African green monkeys from RSV infection. Vaccine 30:959-68|
|Fukushima, Keijo; Takahashi, Tadanobu; Takaguchi, Masahiro et al. (2011) Plaque formation assay for human parainfluenza virus type 1. Biol Pharm Bull 34:996-1000|
|Chambers, Raychel; Takimoto, Toru (2010) Trafficking of Sendai virus nucleocapsids is mediated by intracellular vesicles. PLoS One 5:e10994|