Minna Poranen, University of Helsinki

Xiaoyu Sun, Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland Serban L. Ilca, Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, United Kingdom Gabija Ziedaite-Ojalaa, Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland Juha T. Huiskonen, Division of Structural Biology, Wellcome Centre for Human Genetics, University of Oxford, Oxford, United Kingdom and Helsinki Institute of Life Science, University of Helsinki, Finland Minna M. Poranen, Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland

Structural Biology

Cystovirus phi6 encodes a viral NTPase P4 forming hexameric turret-like structures at the icosahedral five-fold vertices of the inner capsid of the phi6 virion. During the phi6 life cycle, P4 hexamers drive the assembly of empty single-layered particles (SLPs) and act as molecular motors for the translocation of the genomic ssRNA precursors into these particles. In addition, role in the production of viral mRNA molecules has been proposed. Our single-molecule experiments indicate that phi6 genome packaging into empty SLPs proceeds intermittently in slow and fast phases, which likely reflects differences in the unfolding of the RNA secondary structures of the ssRNA being packaged. Using detergent treatment, it was possible to reduce the number of P4 hexamers associated with phi6 SLP. Such particles are active in ssRNA packaging, but deficient in transcription. However, incubation of P4-deficient SLPs with purified P4 hexamers resulted in the incorporation of P4s on the pre-assembled empty SLPs rescuring the transcription. Furthermore, our cryoEM analyses on transcriptionally active phi6 SLPs reveled ssRNA density within the central cavity of some P4 hexamers. Interestingly, only P4 hexamers located at the polar regions of the SLPs are used for ssRNA exit while no RNA density was observed in the equatorial P4s. In conclusion, our results confirm the role of P4 in phi6 transcription and suggest that P4s may have different functional roles depending on their position.