Structural correlates of rotavirus cell entry.

Aliaa H. Abdelhakim,Tomas Kirchhausen,Mithun Pasham,Eric N. Salgado,Stephen C. Harrison,Daniela Nicastro,Xiaofeng Fu,John T. Patton

doi:10.1371/journal.ppat.1004355

Aliaa H. Abdelhakim, Tomas Kirchhausen + Show 6 more

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https://doi.org/10.1371/journal.ppat.1004355

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Abstract

Cell entry by non-enveloped viruses requires translocation into the cytosol of a macromolecular complex—for double-strand RNA viruses, a complete subviral particle. We have used live-cell fluorescence imaging to follow rotavirus entry and penetration into the cytosol of its ∼700 Å inner capsid particle (“double-layered particle”, DLP). We label with distinct fluorescent tags the DLP and each of the two outer-layer proteins and track the fates of each species as the particles bind and enter BSC-1 cells. Virions attach to their glycolipid receptors in the host cell membrane and rapidly become inaccessible to externally added agents; most particles that release their DLP into the cytosol have done so by ∼10 minutes, as detected by rapid diffusional motion of the DLP away from residual outer-layer proteins. Electron microscopy shows images of particles at various stages of engulfment into tightly fitting membrane invaginations, consistent with the interpretation that rotavirus particles drive their own uptake. Electron cryotomography of membrane-bound virions also shows closely wrapped membrane. Combined with high resolution structural information about the viral components, these observations suggest a molecular model for membrane disruption and DLP penetration.

Highlights

IntroductionVarious groups of viruses have evolved distinct molecular mechanisms to carry out this penetration step, which leads to translocation of the infecting particle from an endocytic vesicle or other intracellular compartment to the surrounding cytosol
Non-enveloped viruses must breach a membrane to enter and infect a cell
The final, stable conformation of VP5* is probably the folded-back structure shown in the last panel of Fig. 1C, which illustrates a model for membrane disruption generated by conformational changes in VP5*

Summary

Introduction

Various groups of viruses have evolved distinct molecular mechanisms to carry out this penetration step, which leads to translocation of the infecting particle from an endocytic vesicle or other intracellular compartment to the surrounding cytosol. Structural and mutational evidence suggests that rotaviruses penetrate by disruption of an endocytic membrane, driven by a conformational change in one of its outer-surface proteins; the transition has some similarities to the fusion-promoting conformational change of certain enveloped-virus glycoproteins [2,3]. The inner two layers, composed of viral proteins 2 and 6 (VP2 and VP6) respectively, remain associated with the RNA as a ‘‘double layered particle’’ (DLP), even after penetration. The final, stable conformation of VP5* is probably the folded-back structure shown in the last panel of Fig. 1C, which illustrates a model for membrane disruption generated by conformational changes in VP5*. The model derives from structural studies of VP4 and its fragments [2,14,15,16]; the experiments we describe here test some of the model’s predictions

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