Biology subjects

Neufeldt, C. J.

Publications and source records attributed to Neufeldt, C. J..

9 recordsLinked to original sources

bioRxiv2024

Spatiotemporal dispersion of DENV1 genotype V in western Colombia

Dengue virus (DENV) is a significant public health concern in Colombia, with increased transmission of DENV type 1 (DENV-1) in the departments of Risaralda and Valle del Cauca in the Central-West region of the country following a large outbreak in 2019. However, little is known about the source, genetic diversity, and evolution of circulating viruses. We obtained plasma samples from individuals with acute DENV infection and analyzed DENV-1 genetic diversity, phylodynamics, and phylogeography. We found that most viruses belonged to DENV-1 genotype V, and phylogenetic analysis revealed three distinct clades, each of which was most closely related to viruses from neighboring departments of Colombia sampled over the last 5-10 years. Thus, the 2019 outbreak and subsequent DENV-1 circulation was not due to the introduction of a new lineage but rather reflected local DENV-1V dispersion and evolution. We identified amino acid positions under positive selection in structural proteins and NS1, which may have a role in immune evasion and pathogenesis. Overall, our analysis of DENV1 genotype V diversity, evolution and spread within Colombia highlights the important role of genomic surveillance in understanding virus dynamics during endemic circulation and outbreaks.

evolutionary biology↗

bioRxiv2023

Zika virus remodels and hijacks IGF2BP2 ribonucleoprotein complex to promote viral replication organelle biogenesis

Zika virus (ZIKV) infection causes significant human disease that, with no approved treatment or vaccine, constitutes a major public health concern. Its life cycle entirely relies on the cytoplasmic fate of the viral RNA genome (vRNA) through a fine-tuned equilibrium between vRNA translation, replication and packaging into new virions, all within virus-induced replication organelles (vRO). In this study, with an RNAi mini-screening and subsequent functional characterization, we have identified insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) as a new host dependency factor that regulates vRNA synthesis. In infected cells, IGF2BP2 associates with viral NS5 polymerase and redistributes to the perinuclear viral replication compartment. Combined fluorescence in situ hybridization-based confocal imaging, in vitro binding assays, and immunoprecipitation coupled to RT-qPCR, showed that IGF2BP2 directly interacts with ZIKV vRNA 3-nontranslated region. Using ZIKV sub-genomic replicons and a replication-independent vRO induction system, we demonstrated that IGF2BP2 knockdown impairs de novo viral organelle biogenesis and, consistently, vRNA synthesis. Finally, the analysis of immunopurified IGF2BP2 complex using quantitative mass spectrometry and RT-qPCR, revealed that ZIKV infection alters the protein and RNA interactomes of IGF2BP2. Altogether, our data support that ZIKV hijacks and remodels the IGF2BP2 ribonucleoprotein complex to regulate vRO biogenesis and vRNA neosynthesis.

microbiology↗

bioRxiv2021

Convergent use of phosphatidic acid for Hepatitis C virus and SARS-CoV-2 replication organelle formation

Double membrane vesicles (DMVs) are used as replication organelles by phylogenetically and biologically distant pathogenic RNA viruses such as hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Viral DMVs are morphologically analogous to DMVs formed during autophagy, and although the proteins required for DMV formation are extensively studied, the lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV replication and DMV formation. AGPAT1/2 double knockout also impaired SARS-CoV-2 replication and the formation of autophagosome-like structures. By using correlative light and electron microscopy, we observed the relocalization of AGPAT proteins to HCV and SARS-CoV-2 induced DMVs. In addition, an intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways via phosphotidylcholine (PC) and diacylglycerol (DAG). Pharmacological inhibition of these synthesis pathways also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as an important lipid used for replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. In addition, our data argue that host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses. One Sentence SummaryPhosphatidic acid is important for the formation of double membrane vesicles, serving as replication organelles of hepatitis C virus and SARS-CoV-2, and offering a possible host-targeting strategy to treat SARS-CoV-2 infection.

microbiology↗

bioRxiv2021

The FDA-approved drug cobicistat synergizes with remdesivir to inhibit SARS-CoV-2 replication

Combinations of direct-acting antivirals are needed to minimize drug-resistance mutations and stably suppress replication of RNA viruses. Currently, there are limited therapeutic options against the Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) and testing of a number of drug regimens has led to conflicting results. Here we show that cobicistat, which is an-FDA approved drug-booster that blocks the activity of the drug metabolizing proteins Cytochrome P450-3As (CYP3As) and P-glycoprotein (P-gp), inhibits SARS-CoV-2 replication. Cell-to-cell membrane fusion assays indicated that the antiviral effect of cobicistat is exerted through inhibition of spike protein-mediated membrane fusion. In line with this, incubation with low micromolar concentrations of cobicistat decreased viral replication in three different cell lines including cells of lung and gut origin. When cobicistat was used in combination with the putative CYP3A target and nucleoside analog remdesivir, a synergistic effect on the inhibition of viral replication was observed in cell lines and in a primary human colon organoid. The cobicistat/remdesivir combination was able to potently abate viral replication to levels comparable to mock-infected cells leading to an almost complete rescue of infected cell viability. These data highlight cobicistat as a therapeutic candidate for treating SARS-CoV-2 infection and as a potential building block of combination therapies for COVID-19.

microbiology↗

bioRxiv2020

Global analysis of protein-RNA interactions in SARS-CoV-2 infected cells reveals key regulators of infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19. SARS-CoV-2 relies on cellular RNA-binding proteins (RBPs) to replicate and spread, although which RBPs control SARS-CoV-2 infection remains largely unknown. Here, we employ a multi-omic approach to identify systematically and comprehensively which cellular and viral RBPs are involved in SARS-CoV-2 infection. We reveal that the cellular RNA-bound proteome is remodelled upon SARS-CoV-2 infection, having widespread effects on RNA metabolic pathways, non-canonical RBPs and antiviral factors. Moreover, we apply a new method to identify the proteins that directly interact with viral RNA, uncovering dozens of cellular RBPs and six viral proteins. Amongst them, several components of the tRNA ligase complex, which we show regulate SARS-CoV-2 infection. Furthermore, we discover that available drugs targeting host RBPs that interact with SARS-CoV-2 RNA inhibit infection. Collectively, our results uncover a new universe of host-virus interactions with potential for new antiviral therapies against COVID-19.

microbiology↗

bioRxiv2020

A versatile reporter system to monitor virus infected cells and its application to dengue virus and SARS-CoV-2

Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of that are flaviviruses, such as dengue virus and Zika virus that cause millions of yearly infections and spread around the globe, and coronaviruses, such as SARS-CoV-2, which is the cause of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of virology research in determining mechanisms to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective interventions. Here, we describe the generation and characterization of a reporter system to visualize dengue virus and SARS-CoV-2 replication in live cells. The system is based on viral protease activity causing cleavage and nuclear translocation of an engineered fluorescent protein that is expressed in the infected cells. We show the suitability of the system for live cell imaging and visualization of single infected cells as well as for screening and testing of antiviral compounds. Given the modular building blocks, the system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility. IMPORTANCEReporter systems are useful tools for fast and quantitative visualization of viral replication and spread within a host cell population. Here we describe a reporter system that takes advantage of virus-encoded proteases that are expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the fluorescent protein translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.

microbiology↗

bioRxiv2020

SARS-CoV-2 infection induces a pro-inflammatory cytokine response through cGAS-STING and NF-κB

SARS-CoV-2 is a novel virus that has rapidly spread, causing a global pandemic. In the majority of infected patients, SARS-CoV-2 leads to mild disease; however, in a significant proportion of infections, individuals develop severe symptoms that can lead to permanent lung damage or death. These severe cases are often associated with high levels of pro-inflammatory cytokines and low antiviral responses which can lead to systemic complications. We have evaluated transcriptional and cytokine secretion profiles from infected cell cultures and detected a distinct upregulation of inflammatory cytokines that parallels samples taken from infected patients. Building on these observations, we found a specific activation of NF-{kappa}B and a block of IRF3 nuclear translocation in SARS-CoV-2 infected cells. This NF-{kappa}B response is mediated by cGAS-STING activation and could be attenuated through STING targeting drugs. Our results show that SARS-CoV-2 curates a cGAS-STING mediated NF-{kappa}B driven inflammatory immune response in epithelial cells that likely contributes to inflammatory responses seen in patients and might be a target to suppress severe disease symptoms.

microbiology↗

bioRxiv2020

Structures, conformations and distributions of SARS-CoV-2 spike protein trimers on intact virions

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind the ACE2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates the exposure of its receptor binding site and later undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy. The structure and distribution of S on the virion surface, however, has not been characterised. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions, determining the high-resolution structure, conformational flexibility and distributions of S trimers in situ on the virion surface. These results provide a basis for understanding the conformations of S present on the virion, and for studying their interactions with neutralizing antibodies.

microbiology↗

bioRxiv2020

SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID19 pandemic, is a highly pathogenic {beta}-coronavirus. As other coronaviruses, SARS-CoV-2 is enveloped, replicates in the cytoplasm and assembles at intracellular membranes. Here, we structurally characterize the viral replication compartment and report critical insights into the budding mechanism of the virus, and the structure of extracellular virions close to their native state by in situ cryo-electron tomography and subtomogram averaging. We directly visualized RNA filaments inside the double membrane vesicles, compartments associated with viral replication. The RNA filaments show a diameter consistent with double-stranded RNA and frequent branching likely representing RNA secondary structures. We found that assembled S trimers in lumenal cisternae do not alone induce membrane bending but laterally reorganize on the envelope during virion assembly. The viral ribonucleoprotein complexes (vRNPs) are accumulated at the curved membrane characteristic for budding sites suggesting that vRNP recruitment is enhanced by membrane curvature. Subtomogram averaging shows that vRNPs are distinct cylindrical assemblies. We propose that the genome is packaged around multiple separate vRNP complexes, thereby allowing incorporation of the unusually large coronavirus genome into the virion while maintaining high steric flexibility between the vRNPs.

microbiology↗