Molecular Mechanism of Paramyxovirus Assembly
Human parainfluenza virus type 1 (hPIV1) and Sendai virus (SeV) are enveloped negative-sense RNA viruses and encode 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. Viral RNA (vRNA) is encapsidated with NP, and the P-L polymerase complex associates with the vRNA-NP complex to form ribonucleocapsids (vRNPs). Newly synthesized structural components including vRNPs are transported to the plasma membrane, where progeny viruses are assembled and formed. 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 surface of the virion. 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. 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 microtubule (MT) network. Analysis using electron microscopy revealed close association of vRNPs with intracellular vesicles, which appear to be abundant in virus-infected cells. The vRNPs also co-localized with Rab11a, a known regulator of the recycling endosome pathway. In addition, concomitant movement of vRNPs with transferrin and Rab11a was observed. These data suggest that parainfluenza virus vRNPs traffic through the cytoplasm using intracellular vesicles along the MT network, which likely lead them to the viral assembly/budding sites at the plasma membrane. We also found that cholesterol plays a major role in virion assembly. Cholesterol reducing agents prevented cell surface accumulation of viral glycoproteins and association of parainfluenza virus vRNPs with the raft membranes. Our studies revealed an essential role of recycling endosomes and cholesterol in virus assembly, and suggest that cholesterol could be an attractive target for the control of acute respiratory virus infections.
Chambers R, Takimoto T. Trafficking of Sendai virus nucleocapsids is mediated by intracellular vesicles. PLoS One 5(6):e10994, 2010. PMCID: PMC2881874.
Stone RA and Takimoto T. Critical role of the fusion protein cytoplasmic tail sequence in parainfluenza virus assembly. PLoS ONE 8(4): e61281, 2013. PMCID: PMC3625212.
Stone RA, Hayashi T, Bajimaya S, Hodges E, Takimoto T. Critical role of Rab11a-mediated recycling endosomes in the assembly of type I parainfluenza viruses. Virology 487:11-18. 2016. PMCID: PMC4679493.
Bajimaya S, Hayashi T, Frankl T, Bryk P, Ward B, Takimoto T. Cholesterol reducing agents inhibit assembly of type I parainfluenza viruses. Virology 501:127-35. 2017. PMCID: PMC5201439.
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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