Membrane rupture as an alternative membrane assembly pathway

• Applicant: Professorin Jacomine Krijnse Locker, Ph.D.
• Subject Area: Biochemistry
• Term: from 2013 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 229552073

Nucleo-cytoplasmic large DNA viruses (NCLDVs) are a group of double-stranded DNA viruses with sizes ranging from 150 to 750nm encoding for 150 to 1000 proteins and are thus among the largest viruses known. NCLDVs replicate their DNA in the cellular cytoplasm and share about 60 genes suggesting they might be derived from a common ancestor. Interestingly, a substantial number of NCLDV-proteins show sequence homology to proteins of Archaea, Bacteria and Eukarya and it has been proposed that NCLDVs represent the fourth domain of life providing missing links in evolution. Our group studies vaccinia virus (VACV), the prototype member of the poxvirus family and belonging to the NCLDV-group. We use state of the art microscopy techniques to analyze VACV at the cellular level and use it as a tool to understand cells. Thus, using electron microscopy (EM) and tomography (ET) we recently showed that VACV acquires its membranes in an unconventional way using open membrane intermediates to build an open membrane sphere in the cellular cytoplasm. This single-membraned sphere is closed after uptake of the viral DNA. The basis of the present proposal is preliminary EM and ET results obtained with two other members of the NCLDV-group (African swine fever virus (ASFV) and Mimivirus), showing that these may acquire their membrane in a similar way. We will extent our preliminary data and include two other NCLDVs, Marseillevirus and the recently identified Cafeteria roenbergensis virus that infects marine zooplankton. We will test for a role of individual viral proteins in membrane rupture by studying VACV or ASFV viruses in which genes of interest are placed under inducible promotors. We focus on five genes encoding for membrane proteins involved in virion assembly and that are conserved among all NCLDVs. The data will be complemented by biophysical studies reconstituting proteins of interest in artificial membranes and assay for membrane modifying properties in vitro. Finally, potential candidate (viral) proteins mediating membrane rupture will be characterized computationally regarding their potential origin, homologues, evolution, secondary and tertiary structures as well as interaction partners and multiprotein complex assembly structure. We will use protein structure and architecture at large-scale to go beyond the limits of detection at the sequence level. we will use structural information to detect potential NCLDVs/eukaryotic/bacterial/archaeal proteins displaying a homologous structure. Thus, through our study we wish to ask whether membrane biogenesis using open intermediates is also a cellular mechanism to enclose cytoplasmic structures by a membrane and if it is a general phenomenon conserved in viral or other infection.

• DFG Programme: Research Grants