In-depth analysis of the replication cycle of Orpheovirus

Background After the isolation of Acanthamoeba polyphaga mimivirus (APMV), the study and search for new giant viruses has been intensified. Most giant viruses are associated with free-living amoebae of the genus Acanthamoeba; however other giant viruses have been isolated in Vermamoeba vermiformis, such as Faustovirus, Kaumoebavirus and Orpheovirus. These studies have considerably expanded our knowledge about the diversity, structure, genomics, and evolution of giant viruses. Until now, there has been only one Orpheovirus isolate, and many aspects of its life cycle remain to be elucidated. Methods In this study, we performed an in-depth characterization of the replication cycle and particles of Orpheovirus by transmission and scanning electron microscopy, optical microscopy and IF assays. Results We observed, through optical and IF microscopy, morphological changes in V. vermiformis cells during Orpheovirus infection, as well as increased motility at 12 h post infection (h.p.i.). The viral factory formation and viral particle morphogenesis were analysed by transmission electron microscopy, revealing mitochondria and membrane recruitment into and around the electron-lucent viral factories. Membrane traffic inhibitor (Brefeldin A) negatively impacted particle morphogenesis. The first structure observed during particle morphogenesis was crescent-shaped bodies, which extend and are filled by the internal content until the formation of multi-layered mature particles. We also observed the formation of defective particles with different shapes and sizes. Virological assays revealed that viruses are released from the host by exocytosis at 12 h.p.i., which is associated with an increase of particle counts in the supernatant. Conclusions The results presented here contribute to a better understanding of the biology, structures and important steps in the replication cycle of Orpheovirus.

Using iterative fragment assembly and progressive sequence truncation to facilitate phasing and crystal structure determination of distantly related proteins

Molecular replacement (MR) often requires templates with high homology to solve the phase problem in X-ray crystallography.I-TASSER-MRhas been developed to test whether the success rate for structure determination of distant-homology proteins could be improved by a combination of iterative fragmental structure-assembly simulations with progressive sequence truncation designed to trim regions with high variation. The pipeline was tested on two independent protein sets consisting of 61 proteins from CASP8 and 100 high-resolution proteins from the PDB. After excluding homologous templates,I-TASSERgenerated full-length models with an average TM-score of 0.773, which is 12% higher than the best threading templates. Using these as search models,I-TASSER-MRfound correct MR solutions for 95 of 161 targets as judged by having a TFZ of >8 or with the final structure closer to the native than the initial search models. The success rate was 16% higher than when using the best threading templates.I-TASSER-MRwas also applied to 14 protein targets from structure genomics centers. Seven of these were successfully solved byI-TASSER-MR. These results confirm that advanced structure assembly and progressive structural editing can significantly improve the success rate of MR for targets with distant homology to proteins of known structure.