Project Details
Projekt
Molecular insight into viral infection of Methanoarchaea and their respective viruses
Applicant
Professorin Dr. Ruth Anne Schmitz-Streit
Subject Area
Metabolism, Biochemistry and Genetics of Microorganisms
Virology
Virology
Term
since 2021
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 464460608
The proposal in general addresses viral infection of Methanoarchaea. The first part aims to close the knowledge gaps by studying the interaction between methanoarchaeal virus MetSV and its host Methanosarcina mazei on a molecular, mechanistic, and cellular level. We will particularly address virus replication and the function of virus and host derived proteins in this process, as well as the host response due to virus infection. The establishment of a genetic system for MetSV in the first funding phase now allows us to use genetic manipulation of the virus in combination with biochemical approaches for validating our functional predictions. We will elucidate, whether MetSVORF12 represents a PCNA-like protein involved in virus replication, characterize the proposed MetSV encoded polymerase (MetSVORF07/08) and study whether, MetSVORF06 represents a terminal protein priming DNA replication (goal 1). Further, the proposed role of two host derived proteins in the virus propagation, a ssDNA binding protein and a putative EF-hand protein, will be elucidated (goal 2). Moreover, TEM/SEM analysis of selected host and virus mutants will be performed as well as single particle analysis of the viral replication complex by cryo-electron microscopy in close collaboration with Jan Schuller (goal 3). In the second part we aim to recover and isolate novel methanoarchaeal (MA) viruses from biogas reactor samples (time series over two years, 20 samples), building on the insights gained from the in-depth bioinformatics analysis of the metagenomic and an initial analysis of the complementing metatranscriptomic data sets. We aim to bioinformatically analyze DNA viruses and recover RNA viruses if present infecting MA hosts (goal 4), and isolate the respective predicted hosts by single-cell sorting using a cell printer and the specific F420-dependent fluorescence to identify MA cells in collaboration with Anne Kaster (goal 5). The isolated MA hosts will be used for enrichment and targeted isolation of bioinformatically identified MA viruses (goal 6). In addition to detecting lytic viruses, we will screen for chronically infecting viruses in supernatants of the enrichments by TEM analysis as well as extraction of DNA and RNA followed by sequencing. Finally, novel MA viruses will be initially characterized (genome analysis and infection studies). Overall, we anticipate to get a deeper molecular inside into one MA host-virus example (M. mazei/MetSV), as well as into the diversity of new MA viruses to reinforce the field and increase the collection of MA viruses, which are of potential interest for microbiome studies.
DFG Programme
Priority Programmes
