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Laboratory evolution of virus-like nucleocapsids from nonviral protein cages.

ABSTRACT: Viruses are remarkable nanomachines that efficiently hijack cellular functions to replicate and self-assemble their components within a complex biological environment. As all steps of the viral life cycle depend on formation of a protective proteinaceous shell that packages the DNA or RNA genome, bottom-up construction of virus-like nucleocapsids from nonviral materials could provide valuable insights into virion assembly and evolution. Such constructs could also serve as safe alternatives to natural viruses for diverse nano- and biotechnological applications. Here we show that artificial virus-like nucleocapsids can be generated-rapidly and surprisingly easily-by engineering and laboratory evolution of a nonviral protein cage formed by Aquifex aeolicus lumazine synthase (AaLS) and its encoding mRNA. Cationic peptides were appended to the engineered capsid proteins to enable specific recognition of packaging signals on cognate mRNAs, and subsequent evolutionary optimization afforded nucleocapsids with expanded spherical structures that encapsulate their own full-length RNA genome in vivo and protect the cargo molecules from nucleases. These findings provide strong experimental support for the hypothesis that subcellular protein-bounded compartments may have facilitated the emergence of ancient viruses.

SUBMITTER: Terasaka N

PROVIDER: S-EPMC6003473 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Laboratory evolution of virus-like nucleocapsids from nonviral protein cages.

Terasaka Naohiro N Azuma Yusuke Y Hilvert Donald D

Proceedings of the National Academy of Sciences of the United States of America 20180507 21

Viruses are remarkable nanomachines that efficiently hijack cellular functions to replicate and self-assemble their components within a complex biological environment. As all steps of the viral life cycle depend on formation of a protective proteinaceous shell that packages the DNA or RNA genome, bottom-up construction of virus-like nucleocapsids from nonviral materials could provide valuable insights into virion assembly and evolution. Such constructs could also serve as safe alternatives to na ...[more]

PMID: 29735682

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Project description: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.

Dataset Information

Laboratory evolution of virus-like nucleocapsids from nonviral protein cages.

Publications

Laboratory evolution of virus-like nucleocapsids from nonviral protein cages.

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