Although vaccinia virus (VACV) is the best studied poxvirus, the structure of the mature virus (MV) remains poorly understood. Its asymmetric shape, size and compactness poses a major challenge for electron microscopy (EM) analysis, including cryoEM. Sub-viral particles, in particular membrane-free viral cores, may overcome these limitations. We compare cores obtained by detergent-stripping MVs with cores in the cellular cytoplasm, early in infection. By combining cryo-electron tomography (cryoET), subtomogram averaging (STA) and AlphaFold2 (AF2), abundant core-structures are analyzed, focusing on the prominent palisade layer on the core surface. On detergent-stripped cores, the palisade is composed of densely packed trimers of the major core protein A10. On the core surface they display a random order and their classification indicate structural flexibility. On cytoplasmic cores A10 is organized in a similar manner, indicating that the structures obtained in vitro are physiologically relevant. CryoET and STA also uncover unexpected details of the layers beneath the palisade both on in vitro and in situ cores, that are compared to AF2 structure predictions of known VACV core-associated proteins. Altogether, our data identify for the first time the structure and molecular composition of the palisade units. The results are discussed in the context of the VACV replicative cycle, the assembly and disassembly of the infectious MV.

Summary paragraph: Viruses 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.

Cryo-electron tomography (cryo-ET) enables the three-dimensional visualization of molecular architectures within complex biological environments in a near-native state. In recent years, major advances in sample preparation, instrumentation, data acquisition, and computational processing have considerably expanded the scope of cryo-ET. Workflows that were once low-throughput and largely restricted to a small number of thin specimens and use cases can now be applied to many different samples from isolated macromolecular assemblies to cells, organoids, or tissues. In parallel, the advent of (i) automatic procedures for cryo-focused ion beam milling used for sample thinning as well as streamlined tilt series acquisition, (ii) improved methods for high-pressure-frozen specimens including lift-out, (iii) together with increasingly integrated reconstruction and processing pipelines are all contributing to improve throughput, reproducibility, and accessibility of the technique. Emerging deep learning tools further facilitate target identification, quality control with feedback loops, tomogram reconstruction, and downstream analysis by sub-tomogram averaging. Here, we discuss recent developments on different aspects of the cryo-ET workflow that are progressively bridging molecular and cellular scales in conjunction with multi-modal imaging. We also highlight current limitations and emerging technologies that may further extend cryo-ET capabilities, particularly in combination with complementary approaches toward integrative structural cell biology.