Assembly and dynamics of a bacterial virus factory – BacVirFactory

We use screening methods for (i) genome-wide identification of host genes necessary for viral multiplication and for (ii) establishment of phage-host protein-protein interaction networks. These studies are complemented by imaging of viral factories that assemble in the cytoplasm of bacteria infected by phages.

Our work showed that bacteriophage SPP1 has a very limited dependence on host non-essential genes. In contrast, it hijacks and uses the host DNA replication machinery to replicate its genome. Real-time imaging of infected bacteria allowed to visualize the assembly and dynamics of SPP1 viral factories in the B subtilis cytoplasm.

Uncovering the molecular mechanisms that drive restructuring of the cell into a viral factory provides a rational framework to control phage infection and will likely lead to identifcation of new functions to cellular proteins.

- Cvirkaite-Krupovic V., Carballido-López, R., and Tavares P. (2015). Virus evolution towards limited dependence on the non-essential functions of the host: the bacteriophage SPP1 case. J. Virol. in press. doi:10.1128/JVI.03540-14.

- Yao Z. and Carballido-López (2014). Fluorescence imaging for bacterial cell biology: from localization to dynamics, from ensembles to single molecules. Annu. Rev. Microbiol. 68:459-476.

During the long co-evolution of viruses and cells, viruses exploited numerous ways to hijack cell machineries for their efficient multiplication and dissemination. In turn, cells developed defensive mechanisms to oppose virus attack. Studies of these encounters aimed to understand infection but also to dissect molecular and cell biology complex processes like signaling pathways, cell architecture and compartmentalization, traffic, protein folding and others. However, in contrast to eukaryotic viruses, the cell biology of bacterial viruses (phages or bacteriophages) infection remains largely uncharacterized. Recent progress that uncovered the bacterium complex architecture created the momentum to study how phages take advantage of the host cell organization to optimize their multiplication.
This research proposal brings together a team specialized in phage biology and another with extensive expertise in bacterial cell biology. We have demonstrated that the preferential binding of phage SPP1 to Bacillus subtilis cell poles determines the position of DNA entry in the cell and the site of viral genome replication that occurs in defined foci. The recent observation that a protein of the host replication machinery and a protein of the SPP1 capsid cluster in defined foci during infection indicates, for the first time in a prokaryote system, that phage multiplication likely occurs in viral factories assembled in the infected bacterium. SPP1 thus provides an excellent model system to dissect in space and time the sequence of molecular events leading to delivery of a tailed phage genome across the envelope of a Gram-positive bacterium followed by establishment of a virus multiplication center. The underlying cell biology mechanisms that will be studied in this project are not known for any virus:
1. Genome delivery to the host cell: to define the phage and host factors required for passage of the viral genome through the cell wall and host membrane. We aim to identify biochemically phage protein(s) that insert on the bacterial membrane and to use genetics for discovering potential host proteins that participate in the process. Real time imaging of these effectors and of DNA entry will provide topological and temporal information on DNA passage across the bacterial membrane.
2. Assembly, organization, and dynamics of the putative viral factory: to define the composition, architecture and dynamics of the factory throughout the viral cycle. Infected cells will be imaged for co-localisation of bacterial and phage proteins with SPP1 DNA. When an effector essential for phage multiplication is inactivated by mutation we anticipate that the viral factory will be “frozen” at a defined state. Characterization of different states arrested using a set of mutants aims to define the sequence of macromolecular interactions in the factory, their correlation with reactions during the major steps of the viral cycle (gene expression, phage genome replication, assembly of viral particles), and, thus, to identify the effectors necessary for assembly and maintenance of the viral factory. Time-lapse and photobleaching recovery experiments will be used to follow the dynamic re-localization of these effectors in the cell. Characterization of interactions between host and phage proteins will provide additional molecular detail to establish a model how the viral factory works. A particular interest will be devoted to key interactions of phage proteins that recruit host machineries to convert the bacterial cell into a highly efficient virus multiplication environment.
The results of this work will provide a significant advance to our understanding of phage multiplication in vivo. The research is expected also to deliver reagents and relevant novel information for bacterial cell biology.