Abstract
BackgroundParamyxoviruses are assembled at the plasma membrane budding sites after synthesis of all the structural components in the cytoplasm. Although viral ribonuclocapsid (vRNP) is an essential component of infectious virions, the process of vRNP translocation to assembly sites is poorly understood.Methodology/Principal FindingsTo analyze real-time trafficking of vRNPs in live infected cells, we created a recombinant Sendai virus (SeV), rSeVLeGFP, which expresses L protein fused to enhanced green fluorescent protein (eGFP). The rSeVLeGFP showed similar growth kinetics compared to wt SeV, and newly synthesized LeGFP could be detected as early as 8 h postinfection. The majority of LeGFP co-localized with other components of vRNPs, NP and P proteins, suggesting the fluorescent signals of LeGFP represent the locations of vRNPs. Analysis of LeGFP movement using time-lapse digital video microscopy revealed directional and saltatory movement of LeGFP along microtubules. Treatment of the cells with nocodazole restricted vRNP movement and reduced progeny virion production without affecting viral protein synthesis, suggesting the role of microtubules in vRNP trafficking and virus assembly. Further study with an electron microscope showed close association of vRNPs with intracellular vesicles present in infected cells. In addition, the vRNPs co-localized with Rab11a protein, which is known to regulate the recycling endocytosis pathway and Golgi-to-plasma membrane trafficking. Simultaneous movement between LeGFP and Rab11a was also observed in infected cells, which constitutively express mRFP-tagged Rab11a. Involvement of recycling endosomes in vRNP translocation was also suggested by the fact that vRNPs move concomitantly with recycling transferrin labeled with Alexa 594.Conclusions/SignificanceCollectively, our results strongly suggest a previously unrecognized involvement of the intracellular vesicular trafficking pathway in vRNP translocation and provide new insights into the transport of viral structural components to the assembly sites of enveloped viruses.
Highlights
Sendai virus (SeV) is a pneumotropic murine pathogen in the Paramyxoviridae family, which includes many clinically important human pathogens [1]
The fluorescent punctae, which appeared in rSeVLeGFP-infected cells were clearly distinguishable from the diffuse signals of enhanced green fluorescent protein (eGFP) observed in cells infected with rSeV-eGFP that expresses non-tagged free eGFP from an additional gene (Figure 1C)
We determined the co-localization of L protein gene with the eGFP gene (LeGFP) with P and NP proteins to confirm that LeGFPs are associated with nucleocapsids
Summary
Sendai virus (SeV) is a pneumotropic murine pathogen in the Paramyxoviridae family, which includes many clinically important human pathogens [1]. 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. Synthesized structural components including vRNPs are transported to the plasma membrane, where progeny viruses are assembled and formed by budding. Paramyxoviruses are assembled at the plasma membrane budding sites after synthesis of all the structural components in the cytoplasm. Viral ribonuclocapsid (vRNP) is an essential component of infectious virions, the process of vRNP translocation to assembly sites is poorly understood
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