STRURANA · Structure, maturation and cell entry mechanisms of ranavirus virions

Periodic Report Summary 1 - STRURANA (Structure, maturation and cell entry mechanisms of ranavirus virions)

The StruRANA project aims to elucidate the structural mechanisms for assembly, maturation and infectivity of the nucleocytoplasmic large DNA viruses from the family Iridoviridae (ranaviruses) that infect lower vertebrates. These viruses have caused economic losses to the fish industry and threaten wildlife biodiversity of frogs and snakes worldwide. Ranaviruses are also unique as they exist in two infectious forms - as naked capsids and enveloped virions, each with a different pathway of cell entry and cell egress. We characterized the 3D structure and morphology of the type member of ranaviruses, the Frog virus 3 (FV3) by using cryo-electron microscopy. We found that the naked icosahedral capsid (165-185nm in diameter) contains an inner lipid membrane and a DNA-dense core that is asymmetrically placed inside the inner lipid membrane. We also identified a unique vertex with a short tail that presumably facilitates ejection of the DNA genome into the host cell during infection by the naked capsids. Ongoing reconstructions are aimed to improve resolution of the current reconstruction of the FV3 capsid and identify the assembly and interaction of the major and minor capsid subunits in the assembled icosahedral capsid shell. Recent cryoEM reconstructions of related nucleocytoplasmic large dsDNA viruses show similar morphology and suggest a common evolution of large icosahedral capsids to encapsidate large genomes. However, it has been shown only for ranaviruses that the capsids can be enveloped with an outer membrane. We reconstructed the 3D structure of the enveloped virions by cryo-electron tomography. The capsid is enveloped with a lipid membrane that contains relatively small number of embedded glycoproteins that might be responsible for recognition of the host cell and initiate fusion of the outer viral envelope with the host membrane. We also identified tegument (a layer of viral and host-cell proteins) between the outer viral membrane and the inner capsid. Herpesviruses also contain a tegument that connects the capsid with outer envelope. In contrast to the human herpes virus, the FV3 capsid occupies most of virion volume and the tegument is regularly distributed around the capsid suggesting that the tegument proteins are recruited into the enveloped virion with help of some scaffolding proteins during assembly and budding at the cell membrane. Future studies will examine interaction of the FV3 proteins with the host cell and test the proposed mechanisms based on the structure of FV3 virions. Initial cryoEM reconstructions and results were obtained during guest affiliation of the fellow at the Max Planck Institute of Biochemistry. The fellow was then responsible for installation, starting operations and running the CryoEM facility at his home institution, CEITEC-Masaryk University in the Czech Republic. He has integrated into the Czech community of structural biologists and established new contacts and collaborations with scientists from research institutions in Europe and USA. He received a positive review from the International Advisory Board of CEITEC in 2014.

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