Biology subjects

Bartenschlager, R.

Publications and source records attributed to Bartenschlager, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Serum IgM crosslinks hepatitis B subviral particles into supramolecular immune complexes

Hepatitis B virus (HBV)-infected cells release hepatitis B surface antigen (HBsAg)-rich subviral particles (SVPs) in large excess over infectious virions. SVPs bind antibodies against HBsAg (anti-HBs), including neutralizing antibodies, but the physical products of antibody capture in human blood are uncharacterized. Here we used cryo-electron tomography (cryo-ET) to image HBsAg-enriched serum from a patient with severe acute Hepatitis B and observed three-dimensional assemblies of intact IgM-SVP immune complexes. These associations persisted at a lower particle density and were validated by a spatial-randomization null model. Subtomogram averaging and single-particle cryo-electron microscopy (cryo-EM) identified three serum IgM Fc-core assemblies, IgM-J-CD5L, IgM-J and IgM-J-SC, with cryo-EM maps reaching 2.8 angstrom resolution. Mapping these Fc core assemblies into tomograms revealed full-length IgM engaging individual SVPs through several Fab arms and bridging neighboring particles into immune complex networks containing multiple IgM molecules and SVPs. Thus, serum IgM uses multivalent Fab interactions to crosslink HBsAg-rich SVPs into supramolecular immune complexes. This observation extends the SVP decoy model from passive antibody sequestration to higher-order particle organization.

molecular biology↗

bioRxiv2026

Circulating apoB-containing lipoproteins constituted blood-borne innate immune barrier limits ZIKV dissemination and congenital infection risk

Following establishment of primary viremia, flaviviruses require prompt penetration through vascular endothelium to achieve systemic dissemination, yet most infections remain asymptomatic or self-limited. Canonical defenses, including complement and phagocyte clearance, cannot fully explain this containment, suggesting an additional blood-borne antiviral barrier. Here, we identify apoB containing lipoproteins as potent restriction factors against Zika virus (ZIKV), a devastating danger of global pregnancy. By displaying surface accessible phosphatidylserine (PS), they competitively occupy host PS receptors and block entry by viruses exploiting apoptotic mimicry, including flaviviruses, Ebola, and chikungunya virus. In vivo, increasing apoB lipoproteins enhances serum antiviral activity and limits systemic ZIKV dissemination. This barrier extends to the maternal fetal interface, where elevated maternal LDL attenuates vertical transmission and fetal abnormalities. Its efficacy is modulated by gestational fluctuations in maternal LDL level and low abundance in fetal circulating LDL. Collectively, apoB containing lipoproteins form a constitutive blood-borne innate immune barrier that restrains apoptotic-mimicry viruses during the intravascular phase, limits viral dissemination and reduces congenital infection risk. This study provides a rationale for developing diet augmentation strategy to strengthen this innate barrier in at-risk individuals.

microbiology↗

bioRxiv2026

Hepatitis B virus proteome analysis identifies apolipoprotein C1 facilitating particle production and virus entry

Background & AimsAntiviral therapies targeting hepatitis B virus (HBV) suppress viral replication, but rarely achieve functional cure. Understanding HBV-host cell interaction is crucial for developing novel therapeutic approaches. Here, we report host cell proteins associated with HBV virions and filamentous subviral particles (fSVPs) and characterize one of them, apolipoprotein C1 (ApoC1), mechanistically. MethodsHighly purified HBV virions and fSVPs were obtained by sequential use of several biophysical methods. Particles were analyzed by mass spectrometry and associated proteins were evaluated phenotypically using an HBV infection model. The top hit, ApoC1 was characterized in detail. ResultsAssociated with virions and fSVPs, we identified in addition to known chaperones such as HSP90AB1 and HSC70, several apolipoprotein-related factors. RNAi-based phenotypic validation identified strongest effects for ApoC1, likely due to two complementary effects. First, ApoC1 depletion reduced intracellular cholesterol level impairing HBV infection and SVP production, which was compensated by exogenous cholesterol substitution. Second, ApoC1 that is mainly enriched in high-density lipoprotein (HDL), associates with HBV virions and fSVPs and increases HBV infectivity. The same was found for hepatitis D virus (HDV), a satellite virus utilizing HBV envelopes. Supplementation of exogenous HDL enhanced infection most likely via scavenger receptor class B type 1 (SR-B1), the natural HDL receptor. Consistently, inhibition of SR-B1 suppressed HBV and HDV infection. ConclusionsWe established a method for obtaining highly purified HBV virions and fSVPs and identified the HDL component ApoC1 to associate with both particle types. ApoC1 promotes HBV and HDV infection most likely via SR-B1 facilitating viral entry.

molecular biology↗

bioRxiv2025

Internally twin-Strep tagged CD63 for rapid and efficient isolation of engineered extracellular vesicles encapsulating functional proteins

Extracellular vesicles (EVs) are important mediators of intercellular communication. Achieving high purity of intact engineered EVs through separation from the overwhelming background of unmodified vesicles and cellular or serum-derived contaminants remains a major challenge, yet is critical for fundamental studies of EV biology and EV-based therapeutics. To address this unmet need, we applied structure-guided engineering to canonical EV tetraspanins. Focusing on CD63 and CD9, we identified sites for internal insertion of the twin-Strep tag via flexible linkers into unstructured regions of their large extracellular loops. This design retains functional properties of the scaffolds while providing a robust molecular handle enabling rapid, efficient, and selective antibody-free affinity-based isolation of intact EVs using advanced Strep technology. Fusing proteins of interest (POIs) to the C-terminus of these scaffolds, either directly or via a photoactivatable protein, allowed POI loading into the lumen of twin-Strep tagged EVs with optional optogenetic control of POI release. Proteomic analysis confirmed the high purity of captured engineered EVs with >96% of contaminants removed, enabling sensitive detection of numerous EV-associated proteins. We also report the efficient removal of contaminating virus particles resembling EVs in size and density. Purified engineered EVs successfully delivered an encapsulated fluorescent reporter into target cells, bypassing dye labeling commonly used to track EVs. We demonstrate endosomal escape of the reporter, facilitated by EV decoration with vesicular stomatitis virus glycoprotein, and cytoplasmic release upon optogenetic activation. Our toolbox may serve as a broadly applicable strategy for the efficient production of intact and highly pure engineered EVs to support potential fundamental or translational EV studies.

molecular biology↗

bioRxiv2024

Identification of Covalent Cyclic Peptide Inhibitors Targeting Protein-Protein Interactions Using Phage Display

Peptide macrocycles are promising therapeutics for a variety of disease indications due to their overall metabolic stability and potential to make highly selective binding interactions with targets. Recent advances in covalent macrocycle peptide discovery, driven by phage and mRNA display methods, have enabled the rapid identification of highly potent and selective molecules from large libraires of diverse macrocycles. However, there are currently limited examples of macrocycles that can be used to disrupt protein-protein interactions and even fewer examples that function by formation of a covalent bond to a target protein. In this work, we describe a directed counter-selection method that enables identification of covalent macrocyclic ligands targeting a protein-protein interaction using a phage display screening platform. This method utilizes binary and ternary screenings of a chemically modified phage display library, employing the stable and weakly reactive aryl fluorosulfate electrophile. We demonstrate the utility of this approach using the SARS-CoV-2 Spike-ACE2 protein-protein interaction and identify multiple covalent macrocyclic inhibitors that disrupt this interaction. The resulting compounds displayed antiviral activity against live virus that was irreversible after washout due to the covalent binding mechanism. These results highlight the potential of this screening platform for developing covalent macrocyclic drugs that disrupt protein-protein interactions with long lasting effects.

microbiology↗

bioRxiv2024

RNA genome expansion up to 64 kb in nidoviruses is host constrained and associated with new modes of replicase expression

Positive-strand RNA viruses of the order Nidovirales with genomes larger than [~]20 kb, including the largest known 36.7 kb RNA genome in vertebrate viruses, encode a proofreading exoribonuclease (ExoN). Here, we assemble 76 genome sequences of invertebrate nidoviruses from >500.000 published transcriptome experiments and triple the number of known nidoviruses with >36 kb genomes, including the largest known 64 kb RNA genome. We classify multi-cistronic ExoN-encoding nidoviruses into five groups, according to canonical and non-canonical modes of viral polymerase expression by ribosomes and genome segmentation. The largest group employing the canonical mode comprises invertebrate and vertebrate nidoviruses, including coronaviruses, with genomes ranging from 20-to-36 kb. Four groups with non-canonical expression modes include giant invertebrate nidoviruses with 31-to-64 kb genomes, some of which utilize dual ribosomal frameshifting that we validate experimentally. Thus, expansion of giant RNA virus genomes, the vertebrate/invertebrate host division, and the control of viral replicase expression are interconnected.

microbiology↗

bioRxiv2023

RBM39 shapes innate immunity through transcriptional and splicing control of IRF3 and other key factors

RNA-binding motif protein 39 (RBM39) is an RNA-binding protein involved in tumorigenesis, cell metabolism, and development. Here, we performed a genome-wide CRISPR/Cas9 screen in two liver-derived cell lines and identified RBM39 as a regulator of cell intrinsic innate immune responses. The knockdown of RBM39 or the treatment with Indisulam, an aryl sulfonamide drug targeting RBM39 for proteasomal degradation, strongly reduced the induction of interferon-stimulated genes (ISGs) in response to double-stranded RNA (dsRNA) or viral infections upon sensing by toll-like receptor 3 (TLR3) or cytosolic RIG-I-like receptors. RNA sequencing (seq) and mass spectrometry identified that transcription and/or splicing of the key pathway components IRF3, RIG-I, and MDA5 were affected by RBM39 depletion. RBM39 knockdown further restrained type I and type III IFN pathways, by reducing expression of the type I IFN receptor subunit interferon alpha and beta receptor subunit 2 (IFNAR2), type III IFN receptor subunit interleukin 10 receptor subunit beta (IL-10RB) and transcription factor signal transducer and activator of transcription (STAT) 1 and 2. RBM39 overall orchestrates innate immunity by regulating basal expression of key factors of the interferon response via transcription and/or alternative splicing. SignificanceThe function of RBM39 in tumorigenesis has been investigated intensively in the last decade, but its immunological role is still largely unknown. In our study, we identified RBM39 as a regulatory factor of cell intrinsic signaling via a CRISPR/Cas9 screen. Depletion of RBM39 impairs TLR3, RIG-I/MDA5, and IFN pathways, and thus attenuates innate immune responses. Our omics analysis revealed that RBM39 governs the basal expression of several key factors within these pathways, such as RNA sensors RIG-I and MDA5, type I/III receptors, transcription factors IRF3, STAT1 and STAT2, via its transcriptional and splicing function. Therefore, RBM39 might be a therapeutic target to modulate innate immunity, e.g. in the context of autoimmune disorders.

immunology↗

bioRxiv2023

Phosphatidylinositol 4-kinase III alpha governs cytoskeletal organization for invasiveness of liver cancer cells

Background and AimsHigh expression of phosphatidylinositol 4-kinase III alpha (PI4KIII) correlates with poor survival rates in patients with hepatocellular carcinoma (HCC). In addition, Hepatitis C virus (HCV) infections activate PI4KIII and contribute to HCC progression. We aimed at mechanistically understanding the impact of PI4KIII on the progression of liver cancer and the potential contribution of HCV in this process. MethodsSeveral hepatic cell culture and mouse models were used to study functional importance of PI4KIII on liver pathogenesis. Antibody arrays, gene silencing and PI4KIII specific inhibitor were applied to identify the involved signaling pathways. The contribution of HCV was examined by using HCV infection or overexpression of its nonstructural protein. ResultsHigh PI4KIII expression and/or activity induced cytoskeletal rearrangements via increased-phosphorylation of paxillin and cofilin. This led to morphological alterations and higher migratory and invasive properties of liver cancer cells. We further identified the liver specific lipid kinase phosphatidylinositol 3-kinase C2 domain-containing subunit gamma (PIK3C2{gamma}) working downstream of PI4KIII in regulation of the cytoskeleton. PIK3C2{gamma} generates plasma membrane (PM) phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2]- enriched, invadopodia-like structures which regulate cytoskeletal reorganization by promoting Akt2 phosphorylation. ConclusionsPI4KIII regulates cytoskeleton organization via PIK3C2{gamma}/Akt2/paxillin-cofilin to favor migration and invasion of liver cancer cells. These findings provide mechanistic insight into the contribution of PI4KIII and HCV to progression of liver cancer and identify promising targets for therapeutic intervention. IMPACT AND IMPLICATIONSUnderstanding mechanistically how high PI4KIII expression are associated with poor clinical outcomes of liver cancer is important to develop pharmaceutical interventions. Our study sheds light on the importance of the two lipid kinases PI4KIII and PIK3C2{gamma} as well as the contribution of HCV on liver cancer progression, unraveling the signaling pathway governing this process. This preclinical study contributes to better understanding the complex connection of phospholipids, cytoskeleton and liver cancer and suggests strategies to improve therapeutic outcomes by targeting important signaling molecules. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/541742v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@16ba717org.highwire.dtl.DTLVardef@a6f681org.highwire.dtl.DTLVardef@181c3cdorg.highwire.dtl.DTLVardef@5df6aa_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Endosomal egress and intercellular transmission of hepatic ApoE-containing lipoproteins and its exploitation by the hepatitis C virus

Liver-generated plasma Apolipoprotein E (ApoE)-containing lipoproteins (LPs) (ApoE-LPs) play central roles in lipid transport and metabolism. Perturbations of ApoE can result in several metabolic disorders and ApoE genotypes have been associated with multiple diseases. ApoE is synthesized at the endoplasmic reticulum and transported to the Golgi apparatus for LP assembly; however, ApoE-LPs transport from there to the plasma membrane is largely unknown. Here, we established an integrative imaging approach based on a fully functional fluorescently tagged ApoE. We found that ApoE-LPs accumulate in CD63-positive endosomes of hepatocytes. In addition, we observed the co-egress of ApoE-LPs and extracellular vesicles (EVs) along the late endosomal trafficking route. Moreover, complexes of ApoE-LPs and CD63-positive EVs were found to be transmitted from cell to cell. Given the important role of ApoE in viral infections, we studied the hepatitis C virus (HCV) and found that the viral replicase protein NS5A is enriched in ApoE-containing intraluminal vesicles. Interaction between NS5A and ApoE is required for the efficient release of EVs containing viral RNA. These vesicles are transported along the endosomal ApoE egress pathway. Taken together, our data argue for endosomal egress and transmission of hepatic ApoE-LPs, a pathway that is hijacked by HCV. Given the more general role of EV-mediated cell-to-cell communication, these insights provide new starting points for research into the pathophysiology of ApoE-related metabolic and infection-related disorders. Author SummaryThe post-Golgi egress pathway of hepatocyte-derived ApoE-containing lipoproteins (ApoE-LPs) is largely unknown. By using integrative imaging analyses, we show that ApoE-LPs are enriched in CD63-positive endosomes suggesting that these endosomes might be a central hub for the storage of ApoE-LPs from which they are released into the circulation. In addition, we provide evidence for the co-egress of ApoE-LPs with extracellular vesicles (EVs) along the late endosomal route and their transfer from cell to cell. This pathway is hijacked by the hepatitis C virus that induces the production of ApoE-associated EVs containing viral RNA. Given the important role of ApoE in multiple metabolic, degenerative and infectious diseases, and the role of EVs in cell-to-cell communication, these results provide important information how perturbations of ApoE might contribute to various pathophysiologies.

microbiology↗

bioRxiv2022

Comparison of HAV and HCV infections in vivo and in vitro reveals distinct patterns of innate immune evasion and activation

ObjectiveHepatitis A virus (HAV) infections are considered not to trigger an innate immune response in vivo, in contrast to hepatitis C virus (HCV). This lack of immune induction has been imputed to strong immune counteraction by HAV proteases 3CD and 3ABC. We aimed at elucidating the mechanisms of innate immune induction and counteraction by HAV and HCV in vivo and in vitro. DesignuPA-SCID mice with humanized liver were infected with HAV and HCV. Hepatic cell culture models were used to assess HAV and HCV sensing by TLR3 and RIG-I/MDA5, respectively. Cleavage of the adaptor proteins TRIF and MAVS was analyzed by transient and stable expression of HAV and HCV proteases and virus infection. ResultsWe detected similar levels of Interferon stimulated genes (ISGs) induction in hepatocytes of HAV and HCV infected human liver chimeric mice. In cell culture, HAV induced ISGs exclusively upon sensing by MDA5 and dependent on LGP2. TRIF and MAVS were only partially cleaved by HAV 3ABC and 3CD, not sufficiently to abrogate signalling. In contrast, HCV NS3-4A efficiently degraded MAVS, as previously reported, whereas TRIF was not cleaved. ConclusionsHAV induces an innate immune response in hepatocytes via MDA5/LGP2, with limited control of both pathways by proteolytic cleavage. HCV activates TLR3 and lacks TRIF cleavage, suggesting that this pathway mainly contributes to HCV induced antiviral response in hepatocytes. Our results shed new light on induction and counteraction of innate immunity by HAV and HCV and their potential contribution to clearance and persistence. SIGNIFICANCE OF THIS STUDYO_ST_ABSWhat is already known on this topic?C_ST_ABS-- Despite sharing biological and molecular similarities, HAV infections are always cleared while HCV infections persist in most cases. -- In infected chimpanzees HAV does not trigger a strong innate immune response, as opposed to HCV. This has been imputed to the action of HAV proteases abrogating the signalling pathways. -- Physiological in vitro and in vivo models, based on human hepatocytes, to assess HAV and HCV mechanisms of induction and interference of innate immunity are still missing. What this study adds-- HAV induces an innate immune response in vitro and in vivo, in systems with intact signalling pathways and devoid of adaptive immunity. -- HAV 3ABC and 3CD proteases do not abolish the host innate immune response. -- HCV NS3-4A protease disrupts the RLRs pathways, but cannot cleave TRIF and has no impact on TLR3 response. How this study might affect research, practice or policy-- This study offers a comprehensive, side-by-side investigation on HAV and HCV infections in physiological models which recapitulate a cytokine response in the human liver, and allows a precise assessment of the viral interference related to the function of the respective signalling pathways. -- Our results elucidate mechanisms, so far controversial or poorly investigated, thus contributing to our understanding of HAV clearance and HCV persistence.

immunology↗

bioRxiv2022

SARS-CoV-2 nsp3-4 suffice to form a pore shaping replication organelles

Coronavirus replication is associated with the remodeling of cellular membranes resulting in the formation of double-membrane vesicles (DMVs). Recently, a pore spanning DMV was identified as a putative portal for viral RNA transcription and replication products providing a novel target for antiviral intervention. However, the exact components and the structure of the SARS-CoV-2 pore remain to be determined. Here, we investigate the structure of DMV pores by in situ cryo-electron tomography combined with subtomogram averaging. We reveal non-structural proteins (nsp) 3 and 4 as minimal components forming a DMV spanning pore and show that nsp3 Ubl1-Ubl2 domains are critical for inducing membrane curvature and DMV formation. Altogether, SARS-CoV-2 nsp3-4 has a dual role by driving the biogenesis of replication organelles and forming DMV-spanning replicopores. One-Sentence SummaryBiogenesis of SARS-CoV-2 replication organelles is driven by nsp3-4 constituting the double-membrane vesicle spanning pore.

microbiology↗

bioRxiv2022

Mathematical modeling of plus-strand RNA virus replication to identify broad-spectrum antiviral treatment strategies

Plus-strand RNA viruses are the largest group of viruses. Many are human pathogens that inflict a socio-economic burden. Interestingly, plus-strand RNA viruses share remarkable similarities in their replication. A hallmark of plus-strand RNA viruses is the remodeling of intracellular membranes to establish replication organelles (so-called "replication factories"), which provide a protected environment for the replicase complex, consisting of the viral genome and proteins necessary for viral RNA synthesis. In the current study, we investigate pan-viral similarities and virus-specific differences in the life cycle of this highly relevant group of viruses. We first measured the kinetics of viral RNA, viral protein, and infectious virus particle production of hepatitis C virus (HCV), dengue virus (DENV), and coxsackievirus B3 (CVB3) in the immuno-compromised Huh7 cell line and thus without perturbations by an intrinsic immune response. Based on these measurements, we developed a detailed mathematical model of the replication of HCV, DENV, and CVB3 and show that only small virus-specific changes in the model were necessary to describe the in vitro dynamics of the different viruses. Our model correctly predicted virus-specific mechanisms such as host cell translation shut off and different kinetics of replication organelles. Further, our model suggests that the ability to suppress or shut down host cell mRNA translation may be a key factor for in vitro replication efficiency which may determine acute self-limited or chronic infection. We further analyzed potential broad-spectrum antiviral treatment options in silico and found that targeting viral RNA translation, especially polyprotein cleavage, and viral RNA synthesis may be the most promising drug targets for all plus-strand RNA viruses. Moreover, we found that targeting only the formation of replicase complexes did not stop the viral replication in vitro early in infection, while inhibiting intracellular trafficking processes may even lead to amplified viral growth. Author summaryPlus-strand RNA viruses comprise a large group of related and medically relevant viruses. The current global pandemic of COVID-19 caused by the SARS-coronavirus-2 as well as the constant spread of diseases such as dengue and chikungunya fever show the necessity of a comprehensive and precise analysis of plus-strand RNA virus infections. Plus-strand RNA viruses share similarities in their life cycle. To understand their within-host replication strategies, we developed a mathematical model that studies pan-viral similarities and virus-specific differences of three plus-strand RNA viruses, namely hepatitis C, dengue, and coxsackievirus. By fitting our model to in vitro data, we found that only small virus-specific variations in the model were required to describe the dynamics of all three viruses. Furthermore, our model predicted that ribosomes involved in viral RNA translation seem to be a key player in plus-strand RNA replication efficiency, which may determine acute or chronic infection outcome. Furthermore, our in-silico drug treatment analysis suggests that targeting viral proteases involved in polyprotein cleavage, in combination with viral RNA replication, may represent promising drug targets with broad-spectrum antiviral activity.

microbiology↗

bioRxiv2022

A Hepatitis C virus genotype 1b post transplant isolate with high replication efficiency in cell culture and its adaptation to infectious virus production in vitro and in vivo

Hepatitis C virus (HCV) is highly diverse and grouped into eight genotypes (gts). Infectious cell culture models are limited to a few subtypes, that do not include the highly prevalent gt1b, hampering the development of prophylactic vaccines. A consensus gt1b genome (termed GLT1) was generated from an HCV infected liver-transplanted patient. GLT1 replicated to an outstanding efficiency in Huh7 cells upon SEC14L2 expression, by use of replication enhancing mutations or with a previously developed inhibitor-based regimen. RNA replication levels almost reached JFH-1, but full-length genomes failed to produce detectable amounts of infectious virus. Long-term passaging led to the adaptation of a genome carrying 21 mutations and concomitant production of high levels of transmissible infectivity (GLT1cc). During the adaptation, GLT1 spread in the culture even in absence of detectable amounts of free virus, but cell-to-cell spreading efficiency was not higher as in other isolates like JFH-1. Mechanistically, genome replication and particle production efficiency were enhanced by adaptation, while cell entry competence of HCV pseudoparticles was not affected. Furthermore, GLT1cc retained the ability to replicate in human liver chimeric mice, which was critically dependent on a mutation in domain 3 of nonstructural protein NS5A. Over the course of infection, only one mutation in the surface glycoprotein E2 consistently reverted to wildtype, facilitating assembly in cell culture but potentially affecting CD81 interaction in vivo. Overall, GLT1cc is the first efficient gt1b infectious cell culture model, paving the road to a rationale-based establishment of new infectious HCV isolates and represents an important novel tool for the development of prophylactic HCV vaccines. Author summaryChronic HCV infections remain an important global health issue, despite the availability of highly efficient therapies. So far no protective vaccine is available, which is in part due to the high divergence of HCV variants and the limited possibly to mirror this genetic diversity in cell culture. It has been proven particularly difficult to grow infectious virus in cell culture, requiring extensive adaptation with multiple mutations, which in turn affect infectivity of the adapted variants in vivo. Here we have isolated a genotype 1b variant from a very high titer serum of a patient after liver transplantation (German Liver Transplant 1, GLT1), showing an outstanding genome replication efficiency in cultured hepatoma cells. We were able to adapt this isolate to production of infectious virus, therefore generating the first efficient full-replication cycle cell culture model for highly prevalent HCV genotype 1b. Despite multiple mutations required, adapted GLT1 was still infectious in vivo. GLT1 therefore is not only an important novel development facilitating future efforts in vaccine development. It also provides novel perspectives towards our understanding how liver transplantation drives the evolution of viral isolates with high replication capacity, which might contribute to direct pathogenesis of HCV infection.

microbiology↗

bioRxiv2022

Temporal control of the integrated stress response by a stochastic molecular switch

Stress granules (SGs) are formed in the cytosol as an acute response to environmental cues and activation of the integrated stress response (ISR), a central signaling pathway controlling protein synthesis. Using chronic virus infection as stress model, we previously uncovered a unique temporal control of the ISR resulting in recurrent phases of SG assembly and disassembly. Here, we elucidate the molecular network generating this fluctuating stress response, by integrating quantitative experiments with mathematical modeling, and find that the ISR operates as a stochastic switch. Key elements controlling this switch are the cooperative activation of the stress-sensing kinase PKR, the ultrasensitive response of SG formation to the phosphorylation of the translation initiation factor eIF2, and negative feedback via GADD34, a stress-induced subunit of protein phosphatase 1. We identify GADD34 mRNA levels as the molecular memory of the ISR that plays a central role in cell adaptation to acute and chronic stress.

cell biology↗

bioRxiv2021

Deep mining of the Sequence Read Archive reveals bipartite coronavirus genomes and inter-family Spike glycoprotein recombination

Genetic variation in RNA viruses is generated by point mutation and recombination as well as reassortment in the case of viruses with segmented genomes. While point mutation concerns only few sites per genome copy, recombination and reassortment can affect large genome regions, possibly facilitating the sudden emergence of novel traits. The contribution of recombination and reassortment to genomic plasticity and their rates remain poorly understood and might be underappreciated because of the lack of a comprehensive description of the virosphere. Here we employed a computational approach that directly queries primary sequencing data in a highly parallelized way and involves a targeted viral genome assembly strategy. By screening more than 213,000 data sets from the Sequence Read Archive repository and using two metrics that quantitatively assess assembly quality we discovered 25 novel nidoviruses from a wide range of vertebrate hosts. These include eight fish coronaviruses with bipartite genomes, a giant 36.1 kilobase coronavirus genome with a duplicated Spike glycoprotein (S) gene, and 16 additional so far undescribed vertebrate nidoviruses. Some of these novel virus genomes encode protein domains that have not been described for nidoviruses. We provide evidence for a possible inter-family homologous recombination event involving S between ancestral bipartite coronaviruses and unsegmented tobaniviruses and report a case example of an individual fish simultaneously infected with members from both virus families. Our results shed light on the evolution and genomic plasticity of coronaviruses and identify recombinants with a possibly improved ability to cross species barriers, which might elevate their pandemic potential.

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↗