Quemin, E. | Search bioRxiv

bioRxiv2025

Subcellular reorganization upon phage infection reveals stepwise assembly of viral particles from membrane-associated precursors

Summary paragraphViruses are obligate intracellular parasites and viral infections lead to massive host cell rearrangement to support the rapid generation of progeny. Host take-over and remodelling include formation of viral-induced compartments for viral genome replication and/or assembly. While viruses infecting bacteria, bacteriophages or phages, have been extensively characterized in vitro, the molecular mechanisms underlying the viral cycle inside the crowded cytoplasm remain unclear. Here, we investigate the spatial reorganization of SPP1-infected bacteria under near-native conditions by electron cryo tomography. The most prominent feature is the formation of a large viral DNA (vDNA) compartment from which ribosomes are excluded. In SPP1 infection, there is no membrane nor proteinaceous shell surrounding these compartments. Also, we identified novel key intermediates in virus assembly: open precursors of procapsid lattice were found at the cytoplasmic membrane in a process that requires expression of the portal protein. Next, DNA-free procapsids relocate inside the vDNA compartment where vDNA is packed in a stepwise manner. Finally, DNA-filled capsids segregate to the periphery of the compartment for assembly completion and storage. Collectively, we provide comprehensive mechanistic insights into the complete viral assembly pathway of SPP1 directly in cellula and show how specific steps are coordinated inside the reorganized bacterial cell.

cell biology↗

bioRxiv2022

Structural insights into viral genome replication by the Severe fever with thrombocytopenia syndrome virus L protein

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a phenuivirus that has rapidly become endemic in several East Asian countries. The large (L) protein of SFTSV, which includes the RNA-dependent RNA polymerase (RdRp), is responsible for catalysing viral genome replication and transcription. Here, we present 5 cryo-electron microscopy (cryo-EM) structures of the L protein in several states of the genome replication process, from pre-initiation to late-stage elongation, at a resolution of up to 2.6 [A]. We identify how the L protein binds the 5' viral RNA in a hook-like conformation and show how the distal 5' and 3' RNA ends form a duplex positioning the 3' RNA terminus in the RdRp active site ready for initiation. We also observe the L protein stalled in the early- and late-stages of elongation with the RdRp core accommodating a 9-bp product-template duplex. This duplex ultimately splits with the template binding to a designated 3' secondary binding site. The structural data and observations are complemented by in vitro biochemical and cell-based mini-replicon assays. Altogether, our data provide novel key insights into the mechanism of viral genome replication by the SFTSV L protein and will aid drug development against segmented negative-strand RNA viruses.

biochemistry↗