Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate cytopathic events induced by SARS-CoV-2 with virus replication processes in frozen-hydrated cells, we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. Here we report critical SARS-CoV-2 structural events – e.g. viral RNA transport portals, virus assembly intermediates, virus egress pathway, and native virus spike structures, in the context of whole-cell volumes revealing drastic cytppathic changes. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.

Gag is the HIV structural precursor protein which is cleaved by viral protease to produce mature infectious viruses. Gag is a polyprotein composed of MA (matrix), CA (capsid), SP1, NC (nucleocapsid), SP2 and p6 domains. SP1, together with the last eight residues of CA, have been hypothesized to form a six-helix bundle responsible for the higher-order multimerization of Gag necessary for HIV particle assembly. However, the structure of the complete six-helix bundle has been elusive. Here, we determined the structures of both Gag in vitro assemblies and Gag viral-like particles (VLPs) to 4.2 Å and 4.5 Å resolutions using cryo-electron tomography and subtomogram averaging by emClarity. A single amino acid mutation (T8I) in SP1 stabilizes the six-helix bundle, allowing to discern the entire CA-SP1 helix connecting to the NC domain. These structures provide a blueprint for future development of small molecule inhibitors that can lock SP1 in a stable helical conformation, interfere with virus maturation, and thus block HIV-1 infection.

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, structural and ultrastructural evidence on how the SARS-CoV-2 infection progresses in the frozen-hydrated native cellular context is scarce, and there is a lack of comprehensive knowledge on the SARS-CoV-2 replicative cycle. To correlate the cytopathic events induced by SARS-CoV-2 with virus replication process under the frozen-hydrated condition, here we established a unique multi-modal, multi-scale cryo-correlative platform to image SARS-CoV-2 infection in Vero cells. This platform combines serial cryoFIB/SEM volume imaging and soft X-ray cryo-tomography with cell lamellae-based cryo-electron tomography (cryoET) and subtomogram averaging. The results place critical SARS-CoV-2 structural events – e.g. viral RNA transport portals on double membrane vesicles, virus assembly and budding intermediates, virus egress pathways, and native virus spike structures from intracellular assembled and extracellular released virus - in the context of whole-cell images. The latter revealed numerous heterogeneous cytoplasmic vesicles, the formation of membrane tunnels through which viruses exit, and the drastic cytoplasm invasion into the nucleus. This integrated approach allows a holistic view of SARS-CoV-2 infection, from the whole cell to individual molecules.

Retrovirus immature particle morphology consists of a membrane enclosed, pleomorphic, spherical and incomplete lattice of Gag hexamers. Previously, we demonstrated that human immunodeficiency virus type 2 (HIV-2) immature particles possess a distinct and extensive Gag lattice morphology. To better understand the nature of the continuously curved hexagonal Gag lattice, we used single particle cryo-electron microscopy to determine the HIV-2 Gag lattice structure for immature virions. The reconstruction map revealed a stable, wineglass-shaped Gag hexamer structure with structural features consistent with other lentiviral immature Gag structures. We also solved a 1.98 Å resolution crystal structure of the carboxyl-terminal domain (CTD) of the HIV-2 capsid (CA) protein that identified a structured helix 12 supported via an interaction of helix 10 in the absence of the SP1 region of Gag. Residues at the helix 10-12 interface proved critical in maintaining HIV-2 particle release and infectivity. Taken together, our findings provide evidence for a novel stabilization interface mediated by the HIV-2 CA_CTD that delivers important clues for HIV Gag lattice stabilization as well as important insights into virus maturation. Significance Retrovirus Gag multimerization triggers the particle assembly, resulting in production of pleomorphic spherical immature virus particles. Previous work has been directed towards defining the amino acid residues critical for intermolecular Gag interactions in the incomplete hexagonal Gag lattice. The structure determination of the HIV-2 Gag hexamer presented here has help define the residues in the HIV-2 capsid that provide important insights into particle assembly, lattice stability and virus maturation.

HIV is a lentivirus characterized by its cone shaped mature core. Visualization and structural examination of HIV requires the purification of virions to high concentrations. The yield and integrity of these virions are crucial for ensuring a uniform representation of all viral particles in subsequent analyses. In this study, we present a method for the purification of HIV virions which minimizes the forces applied to virions while maximizing the efficiency of collection. This method, which relies on virion sedimentation simulations, allows us to capture between 1000 and 5000 HIV virions released from individual HEK293 cells after transfection with the NL4.3 HIV backbone. We utilized this approach to investigate HIV core formation from several constructs: pNL4-3(RT:D₁₈₅A&D₁₈₆A) with an inactive reverse transcriptase, NL4.3(IN: V₁₆₅A&R₁₆₆A) with a type-II integrase mutation, and NL4.3(Ψ: Δ(105–278)&Δ(301–332)) featuring an edited Ψ packaging signal. Notably, virions from NL4.3(Ψ: Δ(105–278)&Δ(301–332)) displayed a mixed population, comprising immature virions, empty cores, and cores with detectable internal density. Conversely, virions derived from NL4.3(IN: V₁₆₅A&R₁₆₆A) exhibited a type II integrase mutant phenotype characterized by empty cores and RNP density localized around the cores, consistent with previous studies. In contrast, virions released from pNL4-3(RT:D₁₈₅A&D₁₈₆A) displayed mature cores containing detectable RNP density. We suggest that the sedimentation simulations developed in this study can facilitate the characterization of enveloped viruses.