Biology subjects

Mendonca, L.

Publications and source records attributed to Mendonca, L..

7 recordsLinked to original sources

bioRxiv2026

Cryoelectron tomography of HIV-1 cell-cell transmission conjugates reveals a secluded environment for viral assembly and transfer

Cell-cell transmission of HIV-1 (CCT) is a highly efficient mode of intrahost transmission that provides viral particles broad protection from antiretroviral agents including antiretroviral drugs, host restriction factors, and broadly neutralizing antibodies. Interestingly, the exact factors that grant viral particles increased protection and efficiency during intrahost spread remain unclear. Using a T cell co-culturing system and correlative cryoelectron tomography (cryoET) workflow, we investigated the architecture of HIV-1 CCT sites in a near-native, in situ context to address why CCT is efficient and grants this resistance. Contrary to previous models which suggest large, virus-packed sites, our 3D reconstructions reveal that CCT occurs within small intercellular spaces containing on average 1-5 viral particles. Surrounding these spaces are long, tight membrane interfaces that seclude the viral particles from the remaining extracellular environment. These observations suggest a model where spatial isolation may limit accessibility of antiretroviral agents to the virus-containing spaces thereby conferring protection to viral particles during transmission. To functionally validate these structural insights, we developed a quantitative flow cytometry strategy that decouples CCT conjugate formation from successful CCT infection through the analysis of multicell events. This methodology allows us to systematically compare the impact different molecules such as Env and CD4 have on HIV-1 CCT in a quantitative and population-level manner. Using this approach, we determined that initial HIV-1 CCT conjugate formation is partially dependent on CD4, but the Env involved in conjugate formation may differ from Env used for viral entry. Overall, these findings may prompt a rethinking of intervention strategies for HIV-1 CCT and have further implications with cytoplasmic trafficking, nuclear import, and host restriction factors.

microbiology↗

bioRxiv2024

Enhanced Yield and Gentle Purification of HIV for Cryo-Electron Tomography Analysis of Virion Maturation.

HIV is a lentivirus characterized by the formation of its mature core. Visualization and structural examination of HIV requires 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 purification of HIV virions which minimizes forces applied to virions while maximizing the efficiency of collection. This method allows us to capture between 1,000 and 5,000 HIV virions released from individual HEK293 cells after transfection with the NL4.3 HIV backbone, a 10 fold advantage over other methods. 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(: {Delta}(105-278)&{Delta}(301-332)) featuring an edited packaging signal. Notably, virions from NL4.3(: {Delta}(105-278)&{Delta}(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 purification methods developed in this study can significantly facilitate the characterization of enveloped viruses.

microbiology↗

bioRxiv2023

ChAdOx1 COVID vaccines express RBD open prefusion SARS-CoV-2 spikes on the cell surface

Vaccines against SARS-CoV-2 have been proven to be an effective means of decreasing COVID-19 mortality, hospitalization rates, and transmission. One of the vaccines deployed worldwide is ChAdOx1 nCoV-19, which uses an adenovirus vector to drive the expression of the original SARS-CoV-2 spike on the surface of transduced cells. Using cryo-electron tomography and subtomogram averaging, we determined the native structures of the vaccine product expressed on cell surfaces in situ. We show that ChAdOx1-vectored vaccines expressing the Beta SARS-CoV-2 variant produce abundant native prefusion spikes predominantly in one-RBD-up conformation. Furthermore, the ChAdOx1 vectored HexaPro stabilized spike yields higher cell surface expression, enhanced RBD exposure, and reduced shedding of S1 compared to the wild-type. We demonstrate in situ structure determination as a powerful means for studying antigen design options in future vaccine development against emerging novel SARS-CoV-2 variants and broadly against other infectious viruses.

microbiology↗

bioRxiv2022

HIV-2 Immature Particle Morphology Provides Insights into Gag Lattice Stability and Virus Maturation

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 have used single particle cryo-electron microscopy with a retrovirus to determine the HIV-2 Gag lattice structure for immature virions. The reconstruction map at 5.5 [A] resolution revealed a stable, wineglass-shaped Gag hexamer structure with structural features consistent with other lentiviral immature Gag structures. Cryo-electron tomography provided evidence for nearly complete ordered Gag lattice structures in HIV-2 immature particles. We also solved a 1.98 [A] 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 the first 3D organization of HIV-2 immature Gag lattice and important insights into both HIV Gag lattice stabilization and virus maturation.

microbiology↗

bioRxiv2021

Native-like SARS-CoV-2 spike glycoprotein expressed by ChAdOx1 nCoV-19/AZD1222 vaccine

Vaccine development against the SARS-CoV-2 virus focuses on the principal target of the neutralizing immune response, the spike (S) glycoprotein. Adenovirus-vectored vaccines offer an effective platform for the delivery of viral antigen, but it is important for the generation of neutralizing antibodies that they produce appropriately processed and assembled viral antigen that mimics that observed on the SARS-CoV-2 virus. Here, we describe the structure, conformation and glycosylation of the S protein derived from the adenovirus-vectored ChAdOx1 nCoV-19/AZD1222 vaccine. We demonstrate native-like post-translational processing and assembly, and reveal the expression of S proteins on the surface of cells adopting the trimeric prefusion conformation. The data presented here confirms the use of ChAdOx1 adenovirus vectors as a leading platform technology for SARS-CoV-2 vaccines.

microbiology↗

bioRxiv2020

SARS-CoV-2 Assembly and Egress Pathway Revealed by Correlative Multi-modal Multi-scale Cryo-imaging

Since the outbreak of the SARS-CoV-2 pandemic, there have been intense structural studies on purified recombinant viral components and inactivated viruses. However, investigation of the SARS-CoV-2 infection in the native cellular context is scarce, and there is a lack of comprehensive knowledge on SARS-CoV-2 replicative cycle. Understanding the genome replication, assembly and egress of SARS-CoV-2, a multistage process that involves different cellular compartments and the activity of many viral and cellular proteins, is critically important as it bears the means of medical intervention to stop infection. Here, we investigated SARS-CoV-2 replication in Vero cells under the near-native frozen-hydrated condition using a unique correlative multi-modal, multi-scale cryo-imaging approach combining soft X-ray cryo-tomography and serial cryoFIB/SEM volume imaging of the entire SARS-CoV-2 infected cell with cryo-electron tomography (cryoET) of cellular lamellae and cell periphery, as well as structure determination of viral components by subtomogram averaging. Our results reveal at the whole cell level profound cytopathic effects of SARS-CoV-2 infection, exemplified by a large amount of heterogeneous vesicles in the cytoplasm for RNA synthesis and virus assembly, formation of membrane tunnels through which viruses exit, and drastic cytoplasm invasion into nucleus. Furthermore, cryoET of cell lamellae reveals how viral RNAs are transported from double-membrane vesicles where they are synthesized to viral assembly sites; how viral spikes and RNPs assist in virus assembly and budding; and how fully assembled virus particles exit the cell, thus stablishing a model of SARS-CoV-2 genome replication, virus assembly and egress pathways.

microbiology↗

bioRxiv2020

CryoET structures of immature HIV Gag reveal a complete six-helix bundle and stabilizing small molecules distinct from IP6

Gag is the major HIV-1 structural polyprotein precursor. The Gag SP1 domain with the last residues of CA have been hypothesized to form a six-helix bundle necessary for particle assembly, but this bundle has not been fully resolved. Here, we determined the structures of complete CA-SP1 six-helix bundle connecting to the NC domain, from both in vitro Gag assemblies and viral-like particles (VLPs) carrying a T8I mutation in SP1, to near-atomic resolutions using cryoET and subtomogram averaging. The structures revealed novel densities, however distinct from IP6, inside the six-helix bundle of Gag assemblies, stabilizing the immature lattice. Interestingly, the T8I mutation impaired proteolytic cleavage of Gag at both SP1 boundaries. Our findings signify the involvement of small molecules in immature Gag assembly and provide the structural basis for development of small molecule inhibitors that stabilize SP1 helix, thus interfere with PR-mediated virus maturation.

microbiology↗