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URMC / Labs / Takimoto Lab / Projects / Molecular Mechanism of Paramyxovirus Assembly

Molecular Mechanism of Paramyxovirus Assembly

Sendai virus (SeV) is a pneumotropic murine pathogen in the Paramyxoviridae family, which includes many clinically important human pathogens. It is an enveloped negative-strand RNA virus and encodes 6 major structural proteins: two glycoproteins (HN and F), the matrix (M) protein, the nucleoprotein (NP), and the phosphoprotein (P) and large (L) proteins, which make up the polymerase complex. The viral RNA (vRNA) is encapsidated by NP, and the P-L polymerase complex associates with the vRNA-NP complex to form ribonucleocapsids (vRNPs). The polymerase complex is responsible for transcription and replication of the viral genome, which occurs completely in the cytoplasm of infected cells. Newly synthesized structural components including vRNPs are transported to the plasma membrane, where progeny viruses are assembled and formed by budding. At the budding sites, vRNPs are wrapped by a lipid bilayer envelope containing an internal M layer and the HN and F spike proteins, which are exposed on the virion surface. Although components of virions must translocate to the budding sites for progeny virion formation, little is known about the mechanism of how viral structural components are transported to the plasma membrane for assembly. A widely used approach for the visualization of viral components is to rescue recombinant viruses which express a structural protein fused to a fluorescent protein, such as enhanced green fluorescent protein (eGFP). This approach has been successfully utilized to track virions or viral proteins in infected cells. To study the translocation process of vRNPs in live cells, we rescued a recombinant SeV, rSeVLeGFP, which expresses the L protein fused to eGFP. Analysis of cells infected with rSeVLeGFP showed vRNPs trafficking on the MT network. Additional study using electron microscopy (EM) revealed close association of vRNPs with intracellular vesicles, which appear to be abundant in virus-infected cells. The vRNPs in cells also co-localized with Rab11a, a known regulator of the recycling endosome pathway. In addition, concomitant movement of vRNPs with transferrin and with Rab11a was observed. Our data suggest that SeV vRNPs traffic through the cytoplasm using intracellular vesicles along the MT network, which is likely to lead to the translocation of vRNP to viral assembly/budding sites at the plasma membrane of infected cells.

Land and Linehan

Tracking LeGFP movement in live cells along MT structures. HeLa cells infected with rSeV-LeGFP were treated with Tubulin Tracker Green. Images were collected by digital video microscopy.

 

Chambers R, Takimoto T. Trafficking of Sendai virus nucleocapsids is mediated by intracellular vesicles. PLoS One 5(6):e10994, 2010.

 

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