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Biophysical properties of single rotavirus particles account for the functions of protein shells in a multilayered virus.

ABSTRACT: The functions performed by the concentric shells of multilayered dsRNA viruses require specific protein interactions that can be directly explored through their mechanical properties. We studied the stiffness, breaking force, critical strain and mechanical fatigue of individual Triple, Double and Single layered rotavirus (RV) particles. Our results, in combination with Finite Element simulations, demonstrate that the mechanics of the external layer provides the resistance needed to counteract the stringent conditions of extracellular media. Our experiments, in combination with electrostatic analyses, reveal a strong interaction between the two outer layers and how it is suppressed by the removal of calcium ions, a key step for transcription initiation. The intermediate layer presents weak hydrophobic interactions with the inner layer that allow the assembly and favor the conformational dynamics needed for transcription. Our work shows how the biophysical properties of the three shells are finely tuned to produce an infective RV virion.

SUBMITTER: Jimenez-Zaragoza M

PROVIDER: S-EPMC6133545 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Biophysical properties of single rotavirus particles account for the functions of protein shells in a multilayered virus.

Jiménez-Zaragoza Manuel M Yubero Marina Pl MP Martín-Forero Esther E Castón Jose R JR Reguera David D Luque Daniel D de Pablo Pedro J PJ Rodríguez Javier M JM

eLife 20180911

The functions performed by the concentric shells of multilayered dsRNA viruses require specific protein interactions that can be directly explored through their mechanical properties. We studied the stiffness, breaking force, critical strain and mechanical fatigue of individual Triple, Double and Single layered rotavirus (RV) particles. Our results, in combination with Finite Element simulations, demonstrate that the mechanics of the external layer provides the resistance needed to counteract th ...[more]

PMID: 30201094

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Project description:Rotavirus virus-like particles (RV-VLPs) are potential alternative non-live vaccine candidates due to their high immunogenicity. They mimic the natural conformation of native viral proteins but cannot replicate because they do not contain genomic material which makes them safe. To date, most RV-VLPs have been derived from cell culture adapted strains or common G1 and G3 rotaviruses that have been circulating in communities for some time. In this study, chimaeric RV-VLPs were generated from the consensus sequences of African rotaviruses (G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes) characterised directly from human stool samples without prior adaptation of the wild type strains to cell culture. Codon-optimised sequences for insect cell expression of genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBAC vector, which allowed simultaneous expression of up to four genes using the Bac-to-Bac Baculovirus Expression System (BEVS; Invitrogen). Several combinations of the genome segments originating from different field strains were cloned to produce double-layered RV-VLPs (dRV-VLP; VP2/6), triple-layered RV-VLPs (tRV-VLP; VP2/6/7 or VP2/6/7/4) and chimaeric tRV-VLPs. The RV-VLPs were produced by infecting Spodoptera frugiperda 9 and Trichoplusia ni cells with recombinant baculoviruses using multi-cistronic, dual co-infection and stepwise-infection expression strategies. The size and morphology of the RV-VLPs, as determined by transmission electron microscopy, revealed successful production of RV-VLPs. The novel approach of producing tRV-VLPs, by using the consensus insect cell codon-optimised nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed-up vaccine research and development by by-passing the need to adapt rotaviruses to cell culture. Other problems associated with cell culture adaptation, such as possible changes in epitopes, can also be circumvented. Thus, it is now possible to generate tRV-VLPs for evaluation as non-live vaccine candidates for any human or animal field rotavirus strain.

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Biophysical properties of single rotavirus particles account for the functions of protein shells in a multilayered virus.

Publications

Biophysical properties of single rotavirus particles account for the functions of protein shells in a multilayered virus.

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